Nullspace Sampling with Holonomic Constraints Reveals Molecular Mechanisms of Protein Gαs

PubMed Central

Pachov, Dimitar V.; van den Bedem, Henry

2015-01-01

Proteins perform their function or interact with partners by exchanging between conformational substates on a wide range of spatiotemporal scales. Structurally characterizing these exchanges is challenging, both experimentally and computationally. Large, diffusional motions are often on timescales that are difficult to access with molecular dynamics simulations, especially for large proteins and their complexes. The low frequency modes of normal mode analysis (NMA) report on molecular fluctuations associated with biological activity. However, NMA is limited to a second order expansion about a minimum of the potential energy function, which limits opportunities to observe diffusional motions. By contrast, kino-geometric conformational sampling (KGS) permits large perturbations while maintaining the exact geometry of explicit conformational constraints, such as hydrogen bonds. Here, we extend KGS and show that a conformational ensemble of the α subunit Gαs of heterotrimeric stimulatory protein Gs exhibits structural features implicated in its activation pathway. Activation of protein Gs by G protein-coupled receptors (GPCRs) is associated with GDP release and large conformational changes of its α-helical domain. Our method reveals a coupled α-helical domain opening motion while, simultaneously, Gαs helix α5 samples an activated conformation. These motions are moderated in the activated state. The motion centers on a dynamic hub near the nucleotide-binding site of Gαs, and radiates to helix α4. We find that comparative NMA-based ensembles underestimate the amplitudes of the motion. Additionally, the ensembles fall short in predicting the accepted direction of the full activation pathway. Taken together, our findings suggest that nullspace sampling with explicit, holonomic constraints yields ensembles that illuminate molecular mechanisms involved in GDP release and protein Gs activation, and further establish conformational coupling between key structural elements of Gαs. PMID:26218073

Protein Conformational Entropy is Independent of Solvent

NASA Astrophysics Data System (ADS)

Nucci, Nathaniel; Moorman, Veronica; Gledhill, John; Valentine, Kathleen; Wand, A. Joshua

Proteins exhibit most of their conformational entropy in individual bond vector motions on the ps-ns timescale. These motions can be examined through determination of the Lipari-Szabo generalized squared order parameter (O2) using NMR spin relaxation measurements. It is often argued that most protein motions are intimately dependent on the nature of the solvating environment. Here the solvent dependence of the fast protein dynamics is directly assessed. Using the model protein ubiquitin, the order parameters of the backbone and methyl groups are shown to be generally unaffected by up to a six-fold increase in bulk viscosity or by encapsulation in the nanoscale interior of a reverse micelle. In addition, the reverse micelle condition permits direct comparison of protein dynamics to the mobility of the hydration layer; no correlation is observed. The dynamics of aromatic side chains are also assessed and provide an estimate of the length- and timescale of protein motions where solvent dependence is seen. These data demonstrate the solvent independence of conformational entropy, clarifying a long-held misconception in the fundamental behavior of biological macromolecules. Supported by the National Science Foundation.

Characterizing RNA ensembles from NMR data with kinematic models

PubMed Central

Fonseca, Rasmus; Pachov, Dimitar V.; Bernauer, Julie; van den Bedem, Henry

2014-01-01

Functional mechanisms of biomolecules often manifest themselves precisely in transient conformational substates. Researchers have long sought to structurally characterize dynamic processes in non-coding RNA, combining experimental data with computer algorithms. However, adequate exploration of conformational space for these highly dynamic molecules, starting from static crystal structures, remains challenging. Here, we report a new conformational sampling procedure, KGSrna, which can efficiently probe the native ensemble of RNA molecules in solution. We found that KGSrna ensembles accurately represent the conformational landscapes of 3D RNA encoded by NMR proton chemical shifts. KGSrna resolves motionally averaged NMR data into structural contributions; when coupled with residual dipolar coupling data, a KGSrna ensemble revealed a previously uncharacterized transient excited state of the HIV-1 trans-activation response element stem–loop. Ensemble-based interpretations of averaged data can aid in formulating and testing dynamic, motion-based hypotheses of functional mechanisms in RNAs with broad implications for RNA engineering and therapeutic intervention. PMID:25114056

Analysis of the structure and dynamics of human serum albumin.

PubMed

Guizado, T R Cuya

2014-10-01

Human serum albumin (HSA) is a biologically relevant protein that binds a variety of drugs and other small molecules. No less than 50 structures are deposited in the RCSB Protein Data Bank (PDB). Based on these structures, we first performed a clustering analysis. Despite the diversity of ligands, only two well defined conformations are detected, with a deviation of 0.46 nm between the average structures of the two clusters, while deviations within each cluster are smaller than 0.08 nm. Those two conformations are representative of the apoprotein and the HSA-myristate complex already identified in previous literature. Considering the structures within each cluster as a representative sample of the dynamical states of the corresponding conformation, we scrutinize the structural and dynamical differences between both conformations. Analysis of the fluctuations within each cluster set reveals that domain II is the most rigid one and better matches both structures. Then, taking this domain as reference, we show that the structural difference between both conformations can be expressed in terms of twist and hinge motions of domains I and III, respectively. We also characterize the dynamical difference between conformations by computing correlations and principal components for each set of dynamical states. The two conformations display different collective motions. The results are compared with those obtained from the trajectories of short molecular dynamics simulations, giving consistent outcomes. Let us remark that, beyond the relevance of the results for the structural and dynamical characterization of HAS conformations, the present methodology could be extended to other proteins in the PDB archive.

Nanosecond to submillisecond dynamics in dye-labeled single-stranded DNA, as revealed by ensemble measurements and photon statistics at single-molecule level.

PubMed

Kaji, Takahiro; Ito, Syoji; Iwai, Shigenori; Miyasaka, Hiroshi

2009-10-22

Single-molecule and ensemble time-resolved fluorescence measurements were applied for the investigation of the conformational dynamics of single-stranded DNA, ssDNA, connected with a fluorescein dye by a C6 linker, where the motions both of DNA and the C6 linker affect the geometry of the system. From the ensemble measurement of the fluorescence quenching via photoinduced electron transfer with a guanine base in the DNA sequence, three main conformations were found in aqueous solution: a conformation unaffected by the guanine base in the excited state lifetime of fluorescein, a conformation in which the fluorescence is dynamically quenched in the excited-state lifetime, and a conformation leading to rapid quenching via nonfluorescent complex. The analysis by using the parameters acquired from the ensemble measurements for interphoton time distribution histograms and FCS autocorrelations by the single-molecule measurement revealed that interconversion in these three conformations took place with two characteristic time constants of several hundreds of nanoseconds and tens of microseconds. The advantage of the combination use of the ensemble measurements with the single-molecule detections for rather complex dynamic motions is discussed by integrating the experimental results with those obtained by molecular dynamics simulation.

Force-Manipulation Single-Molecule Spectroscopy Studies of Enzymatic Dynamics

NASA Astrophysics Data System (ADS)

Lu, H. Peter; He, Yufan; Lu, Maolin; Cao, Jin; Guo, Qing

2014-03-01

Subtle conformational changes play a crucial role in protein functions, especially in enzymatic reactions involving complex substrate-enzyme interactions and chemical reactions. We applied AFM-enhanced and magnetic tweezers-correlated single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing. Our results support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.

Slow dynamics of a protein backbone in molecular dynamics simulation revealed by time-structure based independent component analysis

NASA Astrophysics Data System (ADS)

Naritomi, Yusuke; Fuchigami, Sotaro

2013-12-01

We recently proposed the method of time-structure based independent component analysis (tICA) to examine the slow dynamics involved in conformational fluctuations of a protein as estimated by molecular dynamics (MD) simulation [Y. Naritomi and S. Fuchigami, J. Chem. Phys. 134, 065101 (2011)]. Our previous study focused on domain motions of the protein and examined its dynamics by using rigid-body domain analysis and tICA. However, the protein changes its conformation not only through domain motions but also by various types of motions involving its backbone and side chains. Some of these motions might occur on a slow time scale: we hypothesize that if so, we could effectively detect and characterize them using tICA. In the present study, we investigated slow dynamics of the protein backbone using MD simulation and tICA. The selected target protein was lysine-, arginine-, ornithine-binding protein (LAO), which comprises two domains and undergoes large domain motions. MD simulation of LAO in explicit water was performed for 1 μs, and the obtained trajectory of Cα atoms in the backbone was analyzed by tICA. This analysis successfully provided us with slow modes for LAO that represented either domain motions or local movements of the backbone. Further analysis elucidated the atomic details of the suggested local motions and confirmed that these motions truly occurred on the expected slow time scale.

Hierarchical Biomolecular Dynamics: Picosecond Hydrogen Bonding Regulates Microsecond Conformational Transitions.

PubMed

Buchenberg, Sebastian; Schaudinnus, Norbert; Stock, Gerhard

2015-03-10

Biomolecules exhibit structural dynamics on a number of time scales, including picosecond (ps) motions of a few atoms, nanosecond (ns) local conformational transitions, and microsecond (μs) global conformational rearrangements. Despite this substantial separation of time scales, fast and slow degrees of freedom appear to be coupled in a nonlinear manner; for example, there is theoretical and experimental evidence that fast structural fluctuations are required for slow functional motion to happen. To elucidate a microscopic mechanism of this multiscale behavior, Aib peptide is adopted as a simple model system. Combining extensive molecular dynamics simulations with principal component analysis techniques, a hierarchy of (at least) three tiers of the molecule's free energy landscape is discovered. They correspond to chiral left- to right-handed transitions of the entire peptide that happen on a μs time scale, conformational transitions of individual residues that take about 1 ns, and the opening and closing of structure-stabilizing hydrogen bonds that occur within tens of ps and are triggered by sub-ps structural fluctuations. Providing a simple mechanism of hierarchical dynamics, fast hydrogen bond dynamics is found to be a prerequisite for the ns local conformational transitions, which in turn are a prerequisite for the slow global conformational rearrangement of the peptide. As a consequence of the hierarchical coupling, the various processes exhibit a similar temperature behavior which may be interpreted as a dynamic transition.

Management of three-dimensional intrafraction motion through real-time DMLC tracking.

PubMed

Sawant, Amit; Venkat, Raghu; Srivastava, Vikram; Carlson, David; Povzner, Sergey; Cattell, Herb; Keall, Paul

2008-05-01

Tumor tracking using a dynamic multileaf collimator (DMLC) represents a promising approach for intrafraction motion management in thoracic and abdominal cancer radiotherapy. In this work, we develop, empirically demonstrate, and characterize a novel 3D tracking algorithm for real-time, conformal, intensity modulated radiotherapy (IMRT) and volumetric modulated arc therapy (VMAT)-based radiation delivery to targets moving in three dimensions. The algorithm obtains real-time information of target location from an independent position monitoring system and dynamically calculates MLC leaf positions to account for changes in target position. Initial studies were performed to evaluate the geometric accuracy of DMLC tracking of 3D target motion. In addition, dosimetric studies were performed on a clinical linac to evaluate the impact of real-time DMLC tracking for conformal, step-and-shoot (S-IMRT), dynamic (D-IMRT), and VMAT deliveries to a moving target. The efficiency of conformal and IMRT delivery in the presence of tracking was determined. Results show that submillimeter geometric accuracy in all three dimensions is achievable with DMLC tracking. Significant dosimetric improvements were observed in the presence of tracking for conformal and IMRT deliveries to moving targets. A gamma index evaluation with a 3%-3 mm criterion showed that deliveries without DMLC tracking exhibit between 1.7 (S-IMRT) and 4.8 (D-IMRT) times more dose points that fail the evaluation compared to corresponding deliveries with tracking. The efficiency of IMRT delivery, as measured in the lab, was observed to be significantly lower in case of tracking target motion perpendicular to MLC leaf travel compared to motion parallel to leaf travel. Nevertheless, these early results indicate that accurate, real-time DMLC tracking of 3D tumor motion is feasible and can potentially result in significant geometric and dosimetric advantages leading to more effective management of intrafraction motion.

Asymmetric breathing motions of nucleosomal DNA and the role of histone tails

NASA Astrophysics Data System (ADS)

Chakraborty, Kaushik; Loverde, Sharon M.

2017-08-01

The most important packing unit of DNA in the eukaryotic cell is the nucleosome. It undergoes large-scale structural re-arrangements during different cell cycles. For example, the disassembly of the nucleosome is one of the key steps for DNA replication, whereas reassembly occurs after replication. Thus, conformational dynamics of the nucleosome is crucial for different DNA metabolic processes. We perform three different sets of atomistic molecular dynamics simulations of the nucleosome core particle at varying degrees of salt conditions for a total of 0.7 μs simulation time. We find that the conformational dynamics of the nucleosomal DNA tails are oppositely correlated from each other during the initial breathing motions. Furthermore, the strength of the interaction of the nucleosomal DNA tail with the neighboring H2A histone tail modulates the conformational state of the nucleosomal DNA tail. With increasing salt concentration, the degree of asymmetry in the conformation of the nucleosomal DNA tails decreases as both tails tend to unwrap. This direct correlation between the asymmetric breathing motions of the DNA tails and the H2A histone tails, and its decrease at higher salt concentrations, may play a significant role in the molecular pathway of unwrapping.

Long-range tertiary interactions in single hammerhead ribozymes bias motional sampling toward catalytically active conformations

PubMed Central

McDowell, S. Elizabeth; Jun, Jesse M.; Walter, Nils G.

2010-01-01

Enzymes generally are thought to derive their functional activity from conformational motions. The limited chemical variation in RNA suggests that such structural dynamics may play a particularly important role in RNA function. Minimal hammerhead ribozymes are known to cleave efficiently only in ∼10-fold higher than physiologic concentrations of Mg2+ ions. Extended versions containing native loop–loop interactions, however, show greatly enhanced catalytic activity at physiologically relevant Mg2+ concentrations, for reasons that are still ill-understood. Here, we use Mg2+ titrations, activity assays, ensemble, and single molecule fluorescence resonance energy transfer (FRET) approaches, combined with molecular dynamics (MD) simulations, to ask what influence the spatially distant tertiary loop–loop interactions of an extended hammerhead ribozyme have on its structural dynamics. By comparing hammerhead variants with wild-type, partially disrupted, and fully disrupted loop–loop interaction sequences we find that the tertiary interactions lead to a dynamic motional sampling that increasingly populates catalytically active conformations. At the global level the wild-type tertiary interactions lead to more frequent, if transient, encounters of the loop-carrying stems, whereas at the local level they lead to an enrichment in favorable in-line attack angles at the cleavage site. These results invoke a linkage between RNA structural dynamics and function and suggest that loop–loop interactions in extended hammerhead ribozymes—and Mg2+ ions that bind to minimal ribozymes—may generally allow more frequent access to a catalytically relevant conformation(s), rather than simply locking the ribozyme into a single active state. PMID:20921269

Conformational and dynamics changes induced by bile acids binding to chicken liver bile acid binding protein.

PubMed

Eberini, Ivano; Guerini Rocco, Alessandro; Ientile, Anna Rita; Baptista, António M; Gianazza, Elisabetta; Tomaselli, Simona; Molinari, Henriette; Ragona, Laura

2008-06-01

The correlation between protein motions and function is a central problem in protein science. Several studies have demonstrated that ligand binding and protein dynamics are strongly correlated in intracellular lipid binding proteins (iLBPs), in which the high degree of flexibility, principally occurring at the level of helix-II, CD, and EF loops (the so-called portal area), is significantly reduced upon ligand binding. We have recently investigated by NMR the dynamic properties of a member of the iLBP family, chicken liver bile acid binding protein (cL-BABP), in its apo and holo form, as a complex with two bile salts molecules. Binding was found to be regulated by a dynamic process and a conformational rearrangement was associated with this event. We report here the results of molecular dynamics (MD) simulations performed on apo and holo cL-BABP with the aim of further characterizing the protein regions involved in motion propagation and of evaluating the main molecular interactions stabilizing bound ligands. Upon binding, the root mean square fluctuation values substantially decrease for CD and EF loops while increase for the helix-loop-helix region, thus indicating that the portal area is the region mostly affected by complex formation. These results nicely correlate with backbone dynamics data derived from NMR experiments. Essential dynamics analysis of the MD trajectories indicates that the major concerted motions involve the three contiguous structural elements of the portal area, which however are dynamically coupled in different ways whether in the presence or in the absence of the ligands. Motions of the EF loop and of the helical region are part of the essential space of both apo and holo-BABP and sample a much wider conformational space in the apo form. Together with NMR results, these data support the view that, in the apo protein, the flexible EF loop visits many conformational states including those typical of the holo state and that the ligand acts stabilizing one of these pre-existing conformations. The present results, in agreement with data reported for other iLBPs, sharpen our knowledge on the binding mechanism for this protein family. (c) 2008 Wiley-Liss, Inc.

Immunoglobulin G1 Fc domain motions: implications for Fc engineering

PubMed Central

Frank, Martin; Walker, Ross C.; Lanzilotta, William N.; Prestegard, James H.; Barb, Adam W.

2014-01-01

The fragment crystallizable (Fc) region links the key pathogen identification and destruction properties of immunoglobulin G(IgG). Pathogen opsonization positions Fcs to activate pro-inflammatory Fcγ receptors (FcγRs) on immune cells. The cellular response and committal to a damaging, though protective, immune response is tightly controlled at multiple levels. Control mechanisms are diverse and in many cases unclear, but one frequently suggested contribution originates in Fcγ receptor affinity being modulated through shifts in Fc conformational sampling. Here we report a previously unseen IgG1 Fc conformation. This observation motivated an extensive molecular dynamics (MD) investigation of polypeptide and glycan motions that revealed greater amplitude of motion for the N-terminal Cγ2 domains and N-glycan than previously observed. Residues in the Cγ2/Cγ3 interface and disulphide-bonded hinge were identified as influencing the Cγ2 motion. Our results are consistent with a model of Fc that is structurally dynamic. Conformational states that are competent to bind immune-stimulating FcγRs interconverted with Fc conformations distinct from those observed in FcγR complexes, which may represent a transient, nonbinding population. PMID:24522230

Polymer Chain Conformation and Dynamical Confinement in a Model One-Component Nanocomposite

NASA Astrophysics Data System (ADS)

Mark, C.; Holderer, O.; Allgaier, J.; Hübner, E.; Pyckhout-Hintzen, W.; Zamponi, M.; Radulescu, A.; Feoktystov, A.; Monkenbusch, M.; Jalarvo, N.; Richter, D.

2017-07-01

We report a neutron-scattering investigation on the structure and dynamics of a single-component nanocomposite based on SiO2 particles that were grafted with polyisoprene chains at the entanglement limit. By skillful labeling, we access both the monomer density in the corona as well as the conformation of the grafted chains. While the corona profile follows a r-1 power law, the conformation of a grafted chain is identical to that of a chain in a reference melt, implying a high mutual penetration of the coronas from different particles. The brush crowding leads to topological confinement of the chain dynamics: (i) At local scales, the segmental dynamics is unchanged compared to the reference melt, while (ii) at the scale of the chain, the dynamics appears to be slowed down; (iii) by performing a mode analysis in terms of end-fixed Rouse chains, the slower dynamics is tracked to topological confinement within the cone spanned by the adjacent grafts; (iv) by adding 50% matrix chains, the topological confinement sensed by the grafted chain is lifted partially and the apparent chain motion is accelerated. We observe a crossover from pure Rouse motion at short times to topological confined motion beyond the time when the segmental mean squared displacement has reached the distance to the next graft.

Interdomain flexibility and pH-induced conformational changes of AcrA revealed by molecular dynamics simulations.

PubMed

Wang, Beibei; Weng, Jingwei; Fan, Kangnian; Wang, Wenning

2012-03-15

The membrane fusion protein (MFP) AcrA is proposed to link the inner membrane transporter AcrB and outer membrane protein TolC, forming the tripartite AcrAB-TolC efflux pump, and was shown to be functionally indispensible. Structural and EPR studies showed that AcrA has high conformational flexibility and exhibited pH-induced conformational change. In this study, we built the complete structure of AcrA through homology modeling and performed atomistic simulations of AcrA at different pH values. It was shown that the conformational flexibility of AcrA originates from the motions of α-hairpin and MP domains. The conformational dynamics of AcrA is sensitive to specific point mutations and pH values. In agreement with the EPR experiments, the interdomain motions were restrained upon lowering pH from 7.0 to 5.0 in the simulations. It was found that the protonation/deprotonation of His285 underlies the pH-regulated conformational dynamics of AcrA by disturbing the local hydrogen bond interactions, suggesting that the changes of pH in the periplasm accompanying the drug efflux could act as a signal to trigger the action of AcrA, which undergoes reversible conformational rearrangement. © 2012 American Chemical Society

Programming Chemical Reaction Networks Using Intramolecular Conformational Motions of DNA.

PubMed

Lai, Wei; Ren, Lei; Tang, Qian; Qu, Xiangmeng; Li, Jiang; Wang, Lihua; Li, Li; Fan, Chunhai; Pei, Hao

2018-06-22

The programmable regulation of chemical reaction networks (CRNs) represents a major challenge toward the development of complex molecular devices performing sophisticated motions and functions. Nevertheless, regulation of artificial CRNs is generally energy- and time-intensive as compared to natural regulation. Inspired by allosteric regulation in biological CRNs, we herein develop an intramolecular conformational motion strategy (InCMS) for programmable regulation of DNA CRNs. We design a DNA switch as the regulatory element to program the distance between the toehold and branch migration domain. The presence of multiple conformational transitions leads to wide-range kinetic regulation spanning over 4 orders of magnitude. Furthermore, the process of energy-cost-free strand exchange accompanied by conformational change discriminates single base mismatches. Our strategy thus provides a simple yet effective approach for dynamic programming of complex CRNs.

Single-Molecule Spectroscopy and Imaging Studies of Protein Dynamics

NASA Astrophysics Data System (ADS)

Lu, H. Peter

2012-04-01

Enzymatic reactions and protein-protein interactions are traditionally studied at the ensemble level, despite significant static and dynamic inhomogeneities. Subtle conformational changes play a crucial role in protein functions, and these protein conformations are highly dynamic rather than being static. We applied AFM-enhanced single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of T4 lysozyme and HPPK enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing, presenting as an extreme dynamic behavior intrinsically related to the time bunching effect that we have reported previously. Our results of HPPK interaction with substrate support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation. Our new approach is applicable to a wide range of single-molecule FRET measurements for protein conformational changes under enzymatic reactions.

A normal mode-based geometric simulation approach for exploring biologically relevant conformational transitions in proteins.

PubMed

Ahmed, Aqeel; Rippmann, Friedrich; Barnickel, Gerhard; Gohlke, Holger

2011-07-25

A three-step approach for multiscale modeling of protein conformational changes is presented that incorporates information about preferred directions of protein motions into a geometric simulation algorithm. The first two steps are based on a rigid cluster normal-mode analysis (RCNMA). Low-frequency normal modes are used in the third step (NMSim) to extend the recently introduced idea of constrained geometric simulations of diffusive motions in proteins by biasing backbone motions of the protein, whereas side-chain motions are biased toward favorable rotamer states. The generated structures are iteratively corrected regarding steric clashes and stereochemical constraint violations. The approach allows performing three simulation types: unbiased exploration of conformational space; pathway generation by a targeted simulation; and radius of gyration-guided simulation. When applied to a data set of proteins with experimentally observed conformational changes, conformational variabilities are reproduced very well for 4 out of 5 proteins that show domain motions, with correlation coefficients r > 0.70 and as high as r = 0.92 in the case of adenylate kinase. In 7 out of 8 cases, NMSim simulations starting from unbound structures are able to sample conformations that are similar (root-mean-square deviation = 1.0-3.1 Å) to ligand bound conformations. An NMSim generated pathway of conformational change of adenylate kinase correctly describes the sequence of domain closing. The NMSim approach is a computationally efficient alternative to molecular dynamics simulations for conformational sampling of proteins. The generated conformations and pathways of conformational transitions can serve as input to docking approaches or as starting points for more sophisticated sampling techniques.

Structure and Function in Homodimeric Enzymes: Simulations of Cooperative and Independent Functional Motions.

PubMed

Wells, Stephen A; van der Kamp, Marc W; McGeagh, John D; Mulholland, Adrian J

2015-01-01

Large-scale conformational change is a common feature in the catalytic cycles of enzymes. Many enzymes function as homodimers with active sites that contain elements from both chains. Symmetric and anti-symmetric cooperative motions in homodimers can potentially lead to correlated active site opening and/or closure, likely to be important for ligand binding and release. Here, we examine such motions in two different domain-swapped homodimeric enzymes: the DcpS scavenger decapping enzyme and citrate synthase. We use and compare two types of all-atom simulations: conventional molecular dynamics simulations to identify physically meaningful conformational ensembles, and rapid geometric simulations of flexible motion, biased along normal mode directions, to identify relevant motions encoded in the protein structure. The results indicate that the opening/closure motions are intrinsic features of both unliganded enzymes. In DcpS, conformational change is dominated by an anti-symmetric cooperative motion, causing one active site to close as the other opens; however a symmetric motion is also significant. In CS, we identify that both symmetric (suggested by crystallography) and asymmetric motions are features of the protein structure, and as a result the behaviour in solution is largely non-cooperative. The agreement between two modelling approaches using very different levels of theory indicates that the behaviours are indeed intrinsic to the protein structures. Geometric simulations correctly identify and explore large amplitudes of motion, while molecular dynamics simulations indicate the ranges of motion that are energetically feasible. Together, the simulation approaches are able to reveal unexpected functionally relevant motions, and highlight differences between enzymes.

Structure and Function in Homodimeric Enzymes: Simulations of Cooperative and Independent Functional Motions

PubMed Central

McGeagh, John D.; Mulholland, Adrian J.

2015-01-01

Large-scale conformational change is a common feature in the catalytic cycles of enzymes. Many enzymes function as homodimers with active sites that contain elements from both chains. Symmetric and anti-symmetric cooperative motions in homodimers can potentially lead to correlated active site opening and/or closure, likely to be important for ligand binding and release. Here, we examine such motions in two different domain-swapped homodimeric enzymes: the DcpS scavenger decapping enzyme and citrate synthase. We use and compare two types of all-atom simulations: conventional molecular dynamics simulations to identify physically meaningful conformational ensembles, and rapid geometric simulations of flexible motion, biased along normal mode directions, to identify relevant motions encoded in the protein structure. The results indicate that the opening/closure motions are intrinsic features of both unliganded enzymes. In DcpS, conformational change is dominated by an anti-symmetric cooperative motion, causing one active site to close as the other opens; however a symmetric motion is also significant. In CS, we identify that both symmetric (suggested by crystallography) and asymmetric motions are features of the protein structure, and as a result the behaviour in solution is largely non-cooperative. The agreement between two modelling approaches using very different levels of theory indicates that the behaviours are indeed intrinsic to the protein structures. Geometric simulations correctly identify and explore large amplitudes of motion, while molecular dynamics simulations indicate the ranges of motion that are energetically feasible. Together, the simulation approaches are able to reveal unexpected functionally relevant motions, and highlight differences between enzymes. PMID:26241964

Engineered control of enzyme structural dynamics and function.

PubMed

Boehr, David D; D'Amico, Rebecca N; O'Rourke, Kathleen F

2018-04-01

Enzymes undergo a range of internal motions from local, active site fluctuations to large-scale, global conformational changes. These motions are often important for enzyme function, including in ligand binding and dissociation and even preparing the active site for chemical catalysis. Protein engineering efforts have been directed towards manipulating enzyme structural dynamics and conformational changes, including targeting specific amino acid interactions and creation of chimeric enzymes with new regulatory functions. Post-translational covalent modification can provide an additional level of enzyme control. These studies have not only provided insights into the functional role of protein motions, but they offer opportunities to create stimulus-responsive enzymes. These enzymes can be engineered to respond to a number of external stimuli, including light, pH, and the presence of novel allosteric modulators. Altogether, the ability to engineer and control enzyme structural dynamics can provide new tools for biotechnology and medicine. © 2018 The Protein Society.

Frustration-guided motion planning reveals conformational transitions in proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Budday, Dominik; Fonseca, Rasmus; Leyendecker, Sigrid

Proteins exist as conformational ensembles, exchanging between substates to perform their function. Advances in experimental techniques yield unprecedented access to structural snapshots of their conformational landscape. However, computationally modeling how proteins use collective motions to transition between substates is challenging owing to a rugged landscape and large energy barriers. Here in this paper, we present a new, robotics-inspired motion planning procedure called dCCRRT that navigates the rugged landscape between substates by introducing dynamic, interatomic constraints to modulate frustration. The constraints balance non-native contacts and flexibility, and instantaneously redirect the motion towards sterically favorable conformations. On a test set of eightmore » proteins determined in two conformations separated by, on average, 7.5Å root mean square deviation (RMSD), our pathways reduced the Cα atom RMSD to the goal conformation by 78%, outperforming peer methods. Additionally, we then applied dCC-RRT to examine how collective, small-scale motions of four side-chains in the active site of cyclophilin A propagate through the protein. dCC-RRT uncovered a spatially contiguous network of residues linked by steric interactions and collective motion connecting the active site to a recently proposed, non-canonical capsid binding site 25Å away, rationalizing NMR and multi-temperature crystallography experiments. In all, dCC-RRT can reveal detailed, all-atom molecular mechanisms for small and large amplitude motions.Source code and binaries are freely available at https://github.com/ExcitedStates/KGS/.« less

Frustration-guided motion planning reveals conformational transitions in proteins.

PubMed

Budday, Dominik; Fonseca, Rasmus; Leyendecker, Sigrid; van den Bedem, Henry

2017-10-01

Proteins exist as conformational ensembles, exchanging between substates to perform their function. Advances in experimental techniques yield unprecedented access to structural snapshots of their conformational landscape. However, computationally modeling how proteins use collective motions to transition between substates is challenging owing to a rugged landscape and large energy barriers. Here, we present a new, robotics-inspired motion planning procedure called dCC-RRT that navigates the rugged landscape between substates by introducing dynamic, interatomic constraints to modulate frustration. The constraints balance non-native contacts and flexibility, and instantaneously redirect the motion towards sterically favorable conformations. On a test set of eight proteins determined in two conformations separated by, on average, 7.5 Å root mean square deviation (RMSD), our pathways reduced the Cα atom RMSD to the goal conformation by 78%, outperforming peer methods. We then applied dCC-RRT to examine how collective, small-scale motions of four side-chains in the active site of cyclophilin A propagate through the protein. dCC-RRT uncovered a spatially contiguous network of residues linked by steric interactions and collective motion connecting the active site to a recently proposed, non-canonical capsid binding site 25 Å away, rationalizing NMR and multi-temperature crystallography experiments. In all, dCC-RRT can reveal detailed, all-atom molecular mechanisms for small and large amplitude motions. Source code and binaries are freely available at https://github.com/ExcitedStates/KGS/. © 2017 Wiley Periodicals, Inc.

Frustration-guided motion planning reveals conformational transitions in proteins

DOE PAGES

Budday, Dominik; Fonseca, Rasmus; Leyendecker, Sigrid; ...

2017-07-12

Proteins exist as conformational ensembles, exchanging between substates to perform their function. Advances in experimental techniques yield unprecedented access to structural snapshots of their conformational landscape. However, computationally modeling how proteins use collective motions to transition between substates is challenging owing to a rugged landscape and large energy barriers. Here in this paper, we present a new, robotics-inspired motion planning procedure called dCCRRT that navigates the rugged landscape between substates by introducing dynamic, interatomic constraints to modulate frustration. The constraints balance non-native contacts and flexibility, and instantaneously redirect the motion towards sterically favorable conformations. On a test set of eightmore » proteins determined in two conformations separated by, on average, 7.5Å root mean square deviation (RMSD), our pathways reduced the Cα atom RMSD to the goal conformation by 78%, outperforming peer methods. Additionally, we then applied dCC-RRT to examine how collective, small-scale motions of four side-chains in the active site of cyclophilin A propagate through the protein. dCC-RRT uncovered a spatially contiguous network of residues linked by steric interactions and collective motion connecting the active site to a recently proposed, non-canonical capsid binding site 25Å away, rationalizing NMR and multi-temperature crystallography experiments. In all, dCC-RRT can reveal detailed, all-atom molecular mechanisms for small and large amplitude motions.Source code and binaries are freely available at https://github.com/ExcitedStates/KGS/.« less

A gratuitous β-Lactamase inducer uncovers hidden active site dynamics of the Staphylococcus aureus BlaR1 sensor domain.

PubMed

Frederick, Thomas E; Peng, Jeffrey W

2018-01-01

Increasing evidence shows that active sites of proteins have non-trivial conformational dynamics. These dynamics include active site residues sampling different local conformations that allow for multiple, and possibly novel, inhibitor binding poses. Yet, active site dynamics garner only marginal attention in most inhibitor design efforts and exert little influence on synthesis strategies. This is partly because synthesis requires a level of atomic structural detail that is frequently missing in current characterizations of conformational dynamics. In particular, while the identity of the mobile protein residues may be clear, the specific conformations they sample remain obscure. Here, we show how an appropriate choice of ligand can significantly sharpen our abilities to describe the interconverting binding poses (conformations) of protein active sites. Specifically, we show how 2-(2'-carboxyphenyl)-benzoyl-6-aminopenicillanic acid (CBAP) exposes otherwise hidden dynamics of a protein active site that binds β-lactam antibiotics. When CBAP acylates (binds) the active site serine of the β-lactam sensor domain of BlaR1 (BlaRS), it shifts the time scale of the active site dynamics to the slow exchange regime. Slow exchange enables direct characterization of inter-converting protein and bound ligand conformations using NMR methods. These methods include chemical shift analysis, 2-d exchange spectroscopy, off-resonance ROESY of the bound ligand, and reduced spectral density mapping. The active site architecture of BlaRS is shared by many β-lactamases of therapeutic interest, suggesting CBAP could expose functional motions in other β-lactam binding proteins. More broadly, CBAP highlights the utility of identifying chemical probes common to structurally homologous proteins to better expose functional motions of active sites.

Computational engineering of cellulase Cel9A-68 functional motions through mutations in its linker region.

PubMed

Costa, M G S; Silva, Y F; Batista, P R

2018-03-14

Microbial cellulosic degradation by cellulases has become a complementary approach for biofuel production. However, its efficiency is hindered by the recalcitrance of cellulose fibres. In this context, computational protein design methods may offer an efficient way to obtain variants with improved enzymatic activity. Cel9A-68 is a cellulase from Thermobifida fusca that is still active at high temperatures. In a previous work, we described a collective bending motion, which governs the overall cellulase dynamics. This movement promotes the approximation of its CBM and CD structural domains (that are connected by a flexible linker). We have identified two residues (G460 and P461) located at the linker that act as a hinge point. Herein, we applied a new level of protein design, focusing on the modulation of this collective motion to obtain cellulase variants with enhanced functional dynamics. We probed whether specific linker mutations would affect Cel9A-68 dynamics through computational simulations. We assumed that P461G and G460+ (with an extra glycine) constructs would present enhanced interdomain motions, while the G460P mutant would be rigid. From our results, the P461G mutation resulted in a broader exploration of the conformational space, as confirmed by clustering and free energy analyses. The WT enzyme was the most rigid system. However, G460P and P460+ explored distinct conformational states described by opposite directions of low-frequency normal modes; they sampled preferentially closed and open conformations, respectively. Overall, we highlight two significant findings: (i) all mutants explored larger conformational spaces than the WT; (ii) the selection of distinct conformational populations was intimately associated with the mutation considered. Thus, the engineering of Cel9A-68 motions through linker mutations may constitute an efficient way to improve cellulase activity, facilitating the disruption of cellulose fibres.

CAMERRA: An analysis tool for the computation of conformational dynamics by evaluating residue-residue associations.

PubMed

Johnson, Quentin R; Lindsay, Richard J; Shen, Tongye

2018-02-21

A computational method which extracts the dominant motions from an ensemble of biomolecular conformations via a correlation analysis of residue-residue contacts is presented. The algorithm first renders the structural information into contact matrices, then constructs the collective modes based on the correlated dynamics of a selected set of dynamic contacts. Associated programs can bridge the results for further visualization using graphics software. The aim of this method is to provide an analysis of conformations of biopolymers from the contact viewpoint. It may assist a systematical uncovering of conformational switching mechanisms existing in proteins and biopolymer systems in general by statistical analysis of simulation snapshots. In contrast to conventional correlation analyses of Cartesian coordinates (such as distance covariance analysis and Cartesian principal component analysis), this program also provides an alternative way to locate essential collective motions in general. Herein, we detail the algorithm in a stepwise manner and comment on the importance of the method as applied to decoding allosteric mechanisms. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

Functionally relevant protein motions: Extracting basin-specific collective coordinates from molecular dynamics trajectories

NASA Astrophysics Data System (ADS)

Pan, Patricia Wang; Dickson, Russell J.; Gordon, Heather L.; Rothstein, Stuart M.; Tanaka, Shigenori

2005-01-01

Functionally relevant motion of proteins has been associated with a number of atoms moving in a concerted fashion along so-called "collective coordinates." We present an approach to extract collective coordinates from conformations obtained from molecular dynamics simulations. The power of this technique for differentiating local structural fuctuations between classes of conformers obtained by clustering is illustrated by analyzing nanosecond-long trajectories for the response regulator protein Spo0F of Bacillus subtilis, generated both in vacuo and using an implicit-solvent representation. Conformational clustering is performed using automated histogram filtering of the inter-Cα distances. Orthogonal (varimax) rotation of the vectors obtained by principal component analysis of these interresidue distances for the members of individual clusters is key to the interpretation of collective coordinates dominating each conformational class. The rotated loadings plots isolate significant variation in interresidue distances, and these are associated with entire mobile secondary structure elements. From this we infer concerted motions of these structural elements. For the Spo0F simulations employing an implicit-solvent representation, collective coordinates obtained in this fashion are consistent with the location of the protein's known active sites and experimentally determined mobile regions.

Protein Allostery and Conformational Dynamics.

PubMed

Guo, Jingjing; Zhou, Huan-Xiang

2016-06-08

The functions of many proteins are regulated through allostery, whereby effector binding at a distal site changes the functional activity (e.g., substrate binding affinity or catalytic efficiency) at the active site. Most allosteric studies have focused on thermodynamic properties, in particular, substrate binding affinity. Changes in substrate binding affinity by allosteric effectors have generally been thought to be mediated by conformational transitions of the proteins or, alternatively, by changes in the broadness of the free energy basin of the protein conformational state without shifting the basin minimum position. When effector binding changes the free energy landscape of a protein in conformational space, the change affects not only thermodynamic properties but also dynamic properties, including the amplitudes of motions on different time scales and rates of conformational transitions. Here we assess the roles of conformational dynamics in allosteric regulation. Two cases are highlighted where NMR spectroscopy and molecular dynamics simulation have been used as complementary approaches to identify residues possibly involved in allosteric communication. Perspectives on contentious issues, for example, the relationship between picosecond-nanosecond local and microsecond-millisecond conformational exchange dynamics, are presented.

Mapping Conformational Dynamics of Proteins Using Torsional Dynamics Simulations

PubMed Central

Gangupomu, Vamshi K.; Wagner, Jeffrey R.; Park, In-Hee; Jain, Abhinandan; Vaidehi, Nagarajan

2013-01-01

All-atom molecular dynamics simulations are widely used to study the flexibility of protein conformations. However, enhanced sampling techniques are required for simulating protein dynamics that occur on the millisecond timescale. In this work, we show that torsional molecular dynamics simulations enhance protein conformational sampling by performing conformational search in the low-frequency torsional degrees of freedom. In this article, we use our recently developed torsional-dynamics method called Generalized Newton-Euler Inverse Mass Operator (GNEIMO) to study the conformational dynamics of four proteins. We investigate the use of the GNEIMO method in simulations of the conformationally flexible proteins fasciculin and calmodulin, as well as the less flexible crambin and bovine pancreatic trypsin inhibitor. For the latter two proteins, the GNEIMO simulations with an implicit-solvent model reproduced the average protein structural fluctuations and sample conformations similar to those from Cartesian simulations with explicit solvent. The application of GNEIMO with replica exchange to the study of fasciculin conformational dynamics produced sampling of two of this protein’s experimentally established conformational substates. Conformational transition of calmodulin from the Ca2+-bound to the Ca2+-free conformation occurred readily with GNEIMO simulations. Moreover, the GNEIMO method generated an ensemble of conformations that satisfy about half of both short- and long-range interresidue distances obtained from NMR structures of holo to apo transitions in calmodulin. Although unconstrained all-atom Cartesian simulations have failed to sample transitions between the substates of fasciculin and calmodulin, GNEIMO simulations show the transitions in both systems. The relatively short simulation times required to capture these long-timescale conformational dynamics indicate that GNEIMO is a promising molecular-dynamics technique for studying domain motion in proteins. PMID:23663843

Mapping conformational dynamics of proteins using torsional dynamics simulations.

PubMed

Gangupomu, Vamshi K; Wagner, Jeffrey R; Park, In-Hee; Jain, Abhinandan; Vaidehi, Nagarajan

2013-05-07

All-atom molecular dynamics simulations are widely used to study the flexibility of protein conformations. However, enhanced sampling techniques are required for simulating protein dynamics that occur on the millisecond timescale. In this work, we show that torsional molecular dynamics simulations enhance protein conformational sampling by performing conformational search in the low-frequency torsional degrees of freedom. In this article, we use our recently developed torsional-dynamics method called Generalized Newton-Euler Inverse Mass Operator (GNEIMO) to study the conformational dynamics of four proteins. We investigate the use of the GNEIMO method in simulations of the conformationally flexible proteins fasciculin and calmodulin, as well as the less flexible crambin and bovine pancreatic trypsin inhibitor. For the latter two proteins, the GNEIMO simulations with an implicit-solvent model reproduced the average protein structural fluctuations and sample conformations similar to those from Cartesian simulations with explicit solvent. The application of GNEIMO with replica exchange to the study of fasciculin conformational dynamics produced sampling of two of this protein's experimentally established conformational substates. Conformational transition of calmodulin from the Ca(2+)-bound to the Ca(2+)-free conformation occurred readily with GNEIMO simulations. Moreover, the GNEIMO method generated an ensemble of conformations that satisfy about half of both short- and long-range interresidue distances obtained from NMR structures of holo to apo transitions in calmodulin. Although unconstrained all-atom Cartesian simulations have failed to sample transitions between the substates of fasciculin and calmodulin, GNEIMO simulations show the transitions in both systems. The relatively short simulation times required to capture these long-timescale conformational dynamics indicate that GNEIMO is a promising molecular-dynamics technique for studying domain motion in proteins. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Protein Conformational Populations and Functionally Relevant Sub-states

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agarwal, Pratul K; Burger, Virginia; Savol, Andrej

2013-01-01

Functioning proteins do not remain fixed in a unique structure, but instead they sample a range of conformations facilitated by motions within the protein. Even in the native state, a protein exists as a collection of interconverting conformations driven by thermodynamic fluctuations. Motions on the fast time scale allow a protein to sample conformations in the nearby area of its conformational landscape, while motions on slower time scales give it access to conformations in distal areas of the landscape. Emerging evidence indicates that protein landscapes contain conformational substates with dynamic and structural features that support the designated function of themore » protein. Nuclear magnetic resonance (NMR) experiments provide information about conformational ensembles of proteins. X-ray crystallography allows researchers to identify the most populated states along the landscape, and computational simulations give atom-level information about the conformational substates of different proteins. This ability to characterize and obtain quantitative information about the conformational substates and the populations of proteins within them is allowing researchers to better understand the relationship between protein structure and dynamics and the mechanisms of protein function. In this Account, we discuss recent developments and challenges in the characterization of functionally relevant conformational populations and substates of proteins. In some enzymes, the sampling of functionally relevant conformational substates is connected to promoting the overall mechanism of catalysis. For example, the conformational landscape of the enzyme dihydrofolate reductase has multiple substates, which facilitate the binding and the release of the cofactor and substrate and catalyze the hydride transfer. For the enzyme cyclophilin A, computational simulations reveal that the long time scale conformational fluctuations enable the enzyme to access conformational substates that allow it to attain the transition state, therefore promoting the reaction mechanism. In the long term, this emerging view of proteins with conformational substates has broad implications for improving our understanding of enzymes, enzyme engineering, and better drug design. Researchers have already used photoactivation to modulate protein conformations as a strategy to develop a hypercatalytic enzyme. In addition, the alteration of the conformational substates through binding of ligands at locations other than the active site provides the basis for the design of new medicines through allosteric modulation.« less

Automated identification of functional dynamic networks from X-ray crystallography

PubMed Central

van den Bedem, Henry; Bhabha, Gira; Yang, Kun; Wright, Peter E.; Fraser, James S.

2013-01-01

Protein function often depends on the exchange between conformational substates. Allosteric ligand binding or distal mutations can stabilize specific active site conformations and consequently alter protein function. In addition to comparing independently determined X-ray crystal structures, alternative conformations observed at low levels of electron density have the potential to provide mechanistic insights into conformational dynamics. Here, we report a new multi-conformer contact network algorithm (CONTACT) that identifies networks of conformationally heterogeneous residues directly from high-resolution X-ray crystallography data. Contact networks in Escherichia coli dihydrofolate reductase (ecDHFR) predict the long-range pattern of NMR chemical shift perturbations of an allosteric mutation. A comparison of contact networks in wild type and mutant ecDHFR suggests how mutations that alter optimized networks of coordinated motions can impair catalytic function. Thus, CONTACT-guided mutagenesis will allow the structure-dynamics-function relationship to be exploited in protein engineering and design. PMID:23913260

Computational prediction of hinge axes in proteins

PubMed Central

2014-01-01

Background A protein's function is determined by the wide range of motions exhibited by its 3D structure. However, current experimental techniques are not able to reliably provide the level of detail required for elucidating the exact mechanisms of protein motion essential for effective drug screening and design. Computational tools are instrumental in the study of the underlying structure-function relationship. We focus on a special type of proteins called "hinge proteins" which exhibit a motion that can be interpreted as a rotation of one domain relative to another. Results This work proposes a computational approach that uses the geometric structure of a single conformation to predict the feasible motions of the protein and is founded in recent work from rigidity theory, an area of mathematics that studies flexibility properties of general structures. Given a single conformational state, our analysis predicts a relative axis of motion between two specified domains. We analyze a dataset of 19 structures known to exhibit this hinge-like behavior. For 15, the predicted axis is consistent with a motion to a second, known conformation. We present a detailed case study for three proteins whose dynamics have been well-studied in the literature: calmodulin, the LAO binding protein and the Bence-Jones protein. Conclusions Our results show that incorporating rigidity-theoretic analyses can lead to effective computational methods for understanding hinge motions in macromolecules. This initial investigation is the first step towards a new tool for probing the structure-dynamics relationship in proteins. PMID:25080829

Long range dynamic effects of point-mutations trap a response regulator in an active conformation

PubMed Central

Bobay, Benjamin G.; Thompson, Richele J.; Hoch, James A.; Cavanagh, John

2010-01-01

When a point-mutation in a protein elicits a functional change, it is most common to assign this change to local structural perturbations. Here we show that point-mutations, distant from an essential highly dynamic kinase recognition loop in the response regulator Spo0F, lock this loop in an active conformation. This ‘conformational trapping’ results in functionally hyperactive Spo0F. Consequently, point-mutations are seen to affect functionally critical motions both close to and far from the mutational site. PMID:20828564

Direct Determination of Site-Specific Noncovalent Interaction Strengths of Proteins from NMR-Derived Fast Side Chain Motional Parameters.

PubMed

Rajeshwar T, Rajitha; Krishnan, Marimuthu

2017-05-25

A novel approach to accurately determine residue-specific noncovalent interaction strengths (ξ) of proteins from NMR-measured fast side chain motional parameters (O axis 2 ) is presented. By probing the environmental sensitivity of side chain conformational energy surfaces of individual residues of a diverse set of proteins, the microscopic connections between ξ, O axis 2 , conformational entropy (S conf ), conformational barriers, and rotamer stabilities established here are found to be universal among proteins. The results reveal that side chain flexibility and conformational entropy of each residue decrease with increasing ξ and that for each residue type there exists a critical range of ξ, determined primarily by the mean side chain conformational barriers, within which flexibility of any residue can be reversibly tuned from highly flexible (with O axis 2 ∼ 0) to highly restricted (with O axis 2 ∼ 1) by increasing ξ by ∼3 kcal/mol. Beyond this critical range of ξ, both side chain flexibility and conformational entropy are insensitive to ξ. The interrelationships between conformational dynamics, conformational entropy, and noncovalent interactions of protein side chains established here open up new avenues to probe perturbation-induced (for example, ligand-binding, temperature, pressure) changes in fast side chain dynamics and thermodynamics of proteins by comparing their conformational energy surfaces in the native and perturbed states.

Leap-dynamics: efficient sampling of conformational space of proteins and peptides in solution.

PubMed

Kleinjung, J; Bayley, P; Fraternali, F

2000-03-31

A molecular simulation scheme, called Leap-dynamics, that provides efficient sampling of protein conformational space in solution is presented. The scheme is a combined approach using a fast sampling method, imposing conformational 'leaps' to force the system over energy barriers, and molecular dynamics (MD) for refinement. The presence of solvent is approximated by a potential of mean force depending on the solvent accessible surface area. The method has been successfully applied to N-acetyl-L-alanine-N-methylamide (alanine dipeptide), sampling experimentally observed conformations inaccessible to MD alone under the chosen conditions. The method predicts correctly the increased partial flexibility of the mutant Y35G compared to native bovine pancreatic trypsin inhibitor. In particular, the improvement over MD consists of the detection of conformational flexibility that corresponds closely to slow motions identified by nuclear magnetic resonance techniques.

An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis*

PubMed Central

Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F.; Cohen, Itay; Henin, Rachel D.; Hockla, Alexandra; Soares, Alexei S.; Papo, Niv; Caulfield, Thomas R.; Radisky, Evette S.

2016-01-01

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis. PMID:27810896

An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis

DOE PAGES

Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F.; ...

2016-11-03

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. While considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here in this paper, we examine the importance of substrate dynamics in the cleavage of Kunitz-BPTI protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4 Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals amore » dramatic conformational change in the substrate upon proteolysis. Using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning three orders of magnitude, we identify global and local dynamic features of substrates on the ns-μs timescale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substratelike and product-like states, linking substrate dynamics on the ns-μs timescale with large collective substrate motions on the much slower timescale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis.« less

The Role of Protein Loops and Linkers in Conformational Dynamics and Allostery.

PubMed

Papaleo, Elena; Saladino, Giorgio; Lambrughi, Matteo; Lindorff-Larsen, Kresten; Gervasio, Francesco Luigi; Nussinov, Ruth

2016-06-08

Proteins are dynamic entities that undergo a plethora of conformational changes that may take place on a wide range of time scales. These changes can be as small as the rotation of one or a few side-chain dihedral angles or involve concerted motions in larger portions of the three-dimensional structure; both kinds of motions can be important for biological function and allostery. It is becoming increasingly evident that "connector regions" are important components of the dynamic personality of protein structures. These regions may be either disordered loops, i.e., poorly structured regions connecting secondary structural elements, or linkers that connect entire protein domains. Experimental and computational studies have, however, revealed that these regions are not mere connectors, and their role in allostery and conformational changes has been emerging in the last few decades. Here we provide a detailed overview of the structural properties and classification of loops and linkers, as well as a discussion of the main computational methods employed to investigate their function and dynamical properties. We also describe their importance for protein dynamics and allostery using as examples key proteins in cellular biology and human diseases such as kinases, ubiquitinating enzymes, and transcription factors.

Shortening the HIV-1 TAR RNA Bulge by a Single Nucleotide Preserves Motional Modes over a Broad Range of Time Scales.

PubMed

Merriman, Dawn K; Xue, Yi; Yang, Shan; Kimsey, Isaac J; Shakya, Anisha; Clay, Mary; Al-Hashimi, Hashim M

2016-08-16

Helix-junction-helix (HJH) motifs are flexible building blocks of RNA architecture that help define the orientation and dynamics of helical domains. They are also frequently involved in adaptive recognition of proteins and small molecules and in the formation of tertiary contacts. Here, we use a battery of nuclear magnetic resonance techniques to examine how deleting a single bulge residue (C24) from the human immunodeficiency virus type 1 (HIV-1) transactivation response element (TAR) trinucleotide bulge (U23-C24-U25) affects dynamics over a broad range of time scales. Shortening the bulge has an effect on picosecond-to-nanosecond interhelical and local bulge dynamics similar to that casued by increasing the Mg(2+) and Na(+) concentration, whereby a preexisting two-state equilibrium in TAR is shifted away from a bent flexible conformation toward a coaxial conformation, in which all three bulge residues are flipped out and flexible. Surprisingly, the point deletion minimally affects microsecond-to-millisecond conformational exchange directed toward two low-populated and short-lived excited conformational states that form through reshuffling of bases pairs throughout TAR. The mutant does, however, adopt a slightly different excited conformational state on the millisecond time scale, in which U23 is intrahelical, mimicking the expected conformation of residue C24 in the excited conformational state of wild-type TAR. Thus, minor changes in HJH topology preserve motional modes in RNA occurring over the picosecond-to-millisecond time scales but alter the relative populations of the sampled states or cause subtle changes in their conformational features.

The solution structures of the cucumber mosaic virus and tomato aspermy virus coat proteins explored with molecular dynamics simulations.

PubMed

Gellért, Akos; Balázs, Ervin

2010-02-26

The three-dimensional structures of two cucumovirus coat proteins (CP), namely Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV), were explored by molecular dynamics (MD) simulations. The N-terminal domain and the C-terminal tail of the CPs proved to be intrinsically unstructured protein regions in aqueous solution. The N-terminal alpha-helix had a partially unrolled conformation. The thermal factor analysis of the CP loop regions demonstrated that the CMV CP had more flexible loop regions than the TAV CP. The principal component analysis (PCA) of the MD trajectories showed that the first three eigenvectors represented the three main conformational motions in the CPs. The first motion components with the highest variance contribution described an opening movement between the hinge and the N-terminal domain of both CPs. The second eigenvector showed a closing motion, while the third eigenvector represented crosswise conformational fluctuations. These new findings, together with previous results, suggest that the hinge region of CPs plays a central role in the recognition and binding of viral RNA. Copyright 2009 Elsevier Inc. All rights reserved.

Dynamics of essential collective motions in proteins: Theory

NASA Astrophysics Data System (ADS)

Stepanova, Maria

2007-11-01

A general theoretical background is introduced for characterization of conformational motions in protein molecules, and for building reduced coarse-grained models of proteins, based on the statistical analysis of their phase trajectories. Using the projection operator technique, a system of coupled generalized Langevin equations is derived for essential collective coordinates, which are generated by principal component analysis of molecular dynamic trajectories. The number of essential degrees of freedom is not limited in the theory. An explicit analytic relation is established between the generalized Langevin equation for essential collective coordinates and that for the all-atom phase trajectory projected onto the subspace of essential collective degrees of freedom. The theory introduced is applied to identify correlated dynamic domains in a macromolecule and to construct coarse-grained models representing the conformational motions in a protein through a few interacting domains embedded in a dissipative medium. A rigorous theoretical background is provided for identification of dynamic correlated domains in a macromolecule. Examples of domain identification in protein G are given and employed to interpret NMR experiments. Challenges and potential outcomes of the theory are discussed.

Ultrafast spectroscopy reveals subnanosecond peptide conformational dynamics and validates molecular dynamics simulation

NASA Astrophysics Data System (ADS)

Spörlein, Sebastian; Carstens, Heiko; Satzger, Helmut; Renner, Christian; Behrendt, Raymond; Moroder, Luis; Tavan, Paul; Zinth, Wolfgang; Wachtveitl, Josef

2002-06-01

Femtosecond time-resolved spectroscopy on model peptides with built-in light switches combined with computer simulation of light-triggered motions offers an attractive integrated approach toward the understanding of peptide conformational dynamics. It was applied to monitor the light-induced relaxation dynamics occurring on subnanosecond time scales in a peptide that was backbone-cyclized with an azobenzene derivative as optical switch and spectroscopic probe. The femtosecond spectra permit the clear distinguishing and characterization of the subpicosecond photoisomerization of the chromophore, the subsequent dissipation of vibrational energy, and the subnanosecond conformational relaxation of the peptide. The photochemical cis/trans-isomerization of the chromophore and the resulting peptide relaxations have been simulated with molecular dynamics calculations. The calculated reaction kinetics, as monitored by the energy content of the peptide, were found to match the spectroscopic data. Thus we verify that all-atom molecular dynamics simulations can quantitatively describe the subnanosecond conformational dynamics of peptides, strengthening confidence in corresponding predictions for longer time scales.

Domain Motion Enhanced (DoME) Model for Efficient Conformational Sampling of Multidomain Proteins.

PubMed

Kobayashi, Chigusa; Matsunaga, Yasuhiro; Koike, Ryotaro; Ota, Motonori; Sugita, Yuji

2015-11-19

Large conformational changes of multidomain proteins are difficult to simulate using all-atom molecular dynamics (MD) due to the slow time scale. We show that a simple modification of the structure-based coarse-grained (CG) model enables a stable and efficient MD simulation of those proteins. "Motion Tree", a tree diagram that describes conformational changes between two structures in a protein, provides information on rigid structural units (domains) and the magnitudes of domain motions. In our new CG model, which we call the DoME (domain motion enhanced) model, interdomain interactions are defined as being inversely proportional to the magnitude of the domain motions in the diagram, whereas intradomain interactions are kept constant. We applied the DoME model in combination with the Go model to simulations of adenylate kinase (AdK). The results of the DoME-Go simulation are consistent with an all-atom MD simulation for 10 μs as well as known experimental data. Unlike the conventional Go model, the DoME-Go model yields stable simulation trajectories against temperature changes and conformational transitions are easily sampled despite domain rigidity. Evidently, identification of domains and their interfaces is useful approach for CG modeling of multidomain proteins.

Characterization of Protein Flexibility Using Small-Angle X-Ray Scattering and Amplified Collective Motion Simulations

PubMed Central

Wen, Bin; Peng, Junhui; Zuo, Xiaobing; Gong, Qingguo; Zhang, Zhiyong

2014-01-01

Large-scale flexibility within a multidomain protein often plays an important role in its biological function. Despite its inherent low resolution, small-angle x-ray scattering (SAXS) is well suited to investigate protein flexibility and determine, with the help of computational modeling, what kinds of protein conformations would coexist in solution. In this article, we develop a tool that combines SAXS data with a previously developed sampling technique called amplified collective motions (ACM) to elucidate structures of highly dynamic multidomain proteins in solution. We demonstrate the use of this tool in two proteins, bacteriophage T4 lysozyme and tandem WW domains of the formin-binding protein 21. The ACM simulations can sample the conformational space of proteins much more extensively than standard molecular dynamics (MD) simulations. Therefore, conformations generated by ACM are significantly better at reproducing the SAXS data than are those from MD simulations. PMID:25140431

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F.

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. While considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here in this paper, we examine the importance of substrate dynamics in the cleavage of Kunitz-BPTI protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4 Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals amore » dramatic conformational change in the substrate upon proteolysis. Using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning three orders of magnitude, we identify global and local dynamic features of substrates on the ns-μs timescale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substratelike and product-like states, linking substrate dynamics on the ns-μs timescale with large collective substrate motions on the much slower timescale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis.« less

Discovering Conformational Sub-States Relevant to Protein Function

PubMed Central

Ramanathan, Arvind; Savol, Andrej J.; Langmead, Christopher J.; Agarwal, Pratul K.; Chennubhotla, Chakra S.

2011-01-01

Background Internal motions enable proteins to explore a range of conformations, even in the vicinity of native state. The role of conformational fluctuations in the designated function of a protein is widely debated. Emerging evidence suggests that sub-groups within the range of conformations (or sub-states) contain properties that may be functionally relevant. However, low populations in these sub-states and the transient nature of conformational transitions between these sub-states present significant challenges for their identification and characterization. Methods and Findings To overcome these challenges we have developed a new computational technique, quasi-anharmonic analysis (QAA). QAA utilizes higher-order statistics of protein motions to identify sub-states in the conformational landscape. Further, the focus on anharmonicity allows identification of conformational fluctuations that enable transitions between sub-states. QAA applied to equilibrium simulations of human ubiquitin and T4 lysozyme reveals functionally relevant sub-states and protein motions involved in molecular recognition. In combination with a reaction pathway sampling method, QAA characterizes conformational sub-states associated with cis/trans peptidyl-prolyl isomerization catalyzed by the enzyme cyclophilin A. In these three proteins, QAA allows identification of conformational sub-states, with critical structural and dynamical features relevant to protein function. Conclusions Overall, QAA provides a novel framework to intuitively understand the biophysical basis of conformational diversity and its relevance to protein function. PMID:21297978

Conformational Sub-states and Populations in Enzyme Catalysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agarwal, Pratul K; Doucet, Nicholas; Chennubholta, C

reactants in the active site, chemical turnover, and release of products. In addition to formation of crucial structural interactions between enzyme and substrate(s), coordinated motions within the enzyme substrate complex allow reaction to proceed at a much faster rate, compared to the reaction in solution and in the absence of enzyme. An increasing number of enzyme systems show the presence of conserved protein motions that are important for function. A wide variety of motions are naturally sampled (over femtosecond to millisecond time-scales) as the enzyme complex moves along the energetic landscape, driven by temperature and dynamical events from the surroundingmore » environment. Areas of low energy along the landscape form conformational sub-states, which show higher conformational populations than surrounding areas. A small number of these protein conformational sub-states contain functionally important structural and dynamical features, which assist the enzyme mechanism along the catalytic cycle. Identification and characterization of these higher-energy (also called excited) sub-states and the associated populations are challenging, as these sub-states are very short-lived and therefore rarely populated. Specialized techniques based on computer simulations, theoretical modeling, and nuclear magnetic resonance have been developed for quantitative characterization of these sub-states and populations. This chapter discusses these techniques and provides examples of their applications to enzyme systems.« less

Substrate binding interferes with active site conformational dynamics in endoglucanase Cel5A from Thermobifida fusca.

PubMed

Jiang, Xukai; Wang, Yuying; Xu, Limei; Chen, Guanjun; Wang, Lushan

2017-09-09

The role of protein dynamics in enzyme catalysis is one of the most active areas in current enzymological research. Here, using endoglucanase Cel5A from Thermobifida fusca (TfCel5A) as a model, we applied molecular dynamics simulations to explore the dynamic behavior of the enzyme upon substrate binding. The collective motions of the active site revealed that the mechanism of TfCel5A substrate binding can likely be described by the conformational-selection model; however, we observed that the conformations of active site residues changed differently along with substrate binding. Although most active site residues retained their native conformational ensemble, some (Tyr163 and Glu355) generated newly induced conformations, whereas others (Phe162 and Tyr189) exhibited shifts in the equilibration of their conformational distributions. These results showed that TfCel5A substrate binding relied on a hybrid mechanism involving induced fit and conformational selection. Interestingly, we found that TfCel5A active site could only partly rebalance its conformational dynamics upon substrate dissociation within the same simulation time, which implies that the conformational rebalance upon substrate dissociation is likely more difficult than the conformational selection upon substrate binding at least in the view of the time required. Our findings offer new insight into enzyme catalysis and potential applications for future protein engineering. Copyright © 2017 Elsevier Inc. All rights reserved.

Control of articulated snake robot under dynamic active constraints.

PubMed

Kwok, Ka-Wai; Vitiello, Valentina; Yang, Guang-Zhong

2010-01-01

Flexible, ergonomically enhanced surgical robots have important applications to transluminal endoscopic surgery, for which path-following and dynamic shape conformance are essential. In this paper, kinematic control of a snake robot for motion stabilisation under dynamic active constraints is addressed. The main objective is to enable the robot to track the visual target accurately and steadily on deforming tissue whilst conforming to pre-defined anatomical constraints. The motion tracking can also be augmented with manual control. By taking into account the physical limits in terms of maximum frequency response of the system (manifested as a delay between the input of the manipulator and the movement of the end-effector), we show the importance of visual-motor synchronisation for performing accurate smooth pursuit movements. Detailed user experiments are performed to demonstrate the practical value of the proposed control mechanism.

Anharmonic longitudinal motion of bases and dynamics of nonlinear excitation in DNA.

PubMed

Di Garbo, Angelo

2016-01-01

The dynamics of the transcription bubble in DNA is studied by using a nonlinear model in which torsional and longitudinal conformations of the biomolecule are coupled. In the absence of forcing and dissipation the torsional dynamics is described by a perturbed kink of the Sine-Gordon DNA model, while the longitudinal conformational energy propagate as phonons. It was found that for random initial conditions of the longitudinal conformational field the presence of the kink promotes the creation of phonons propagating along the chain axis. Moreover, the presence of forcing, describing the active role of RNA polymerase, determines in agreement to the experimental data a modulation of the velocity of the transcription bubble. Lastly, it was shown that the presence of dissipation impacts the dynamic of the phonon by reducing the amplitude of the corresponding conformational field. On the contrary, dissipation and forcing modulate the velocity of the transcription bubble alone.

The role of protein dynamics in the evolution of new enzyme function.

PubMed

Campbell, Eleanor; Kaltenbach, Miriam; Correy, Galen J; Carr, Paul D; Porebski, Benjamin T; Livingstone, Emma K; Afriat-Jurnou, Livnat; Buckle, Ashley M; Weik, Martin; Hollfelder, Florian; Tokuriki, Nobuhiko; Jackson, Colin J

2016-11-01

Enzymes must be ordered to allow the stabilization of transition states by their active sites, yet dynamic enough to adopt alternative conformations suited to other steps in their catalytic cycles. The biophysical principles that determine how specific protein dynamics evolve and how remote mutations affect catalytic activity are poorly understood. Here we examine a 'molecular fossil record' that was recently obtained during the laboratory evolution of a phosphotriesterase from Pseudomonas diminuta to an arylesterase. Analysis of the structures and dynamics of nine protein variants along this trajectory, and three rationally designed variants, reveals cycles of structural destabilization and repair, evolutionary pressure to 'freeze out' unproductive motions and sampling of distinct conformations with specific catalytic properties in bi-functional intermediates. This work establishes that changes to the conformational landscapes of proteins are an essential aspect of molecular evolution and that change in function can be achieved through enrichment of preexisting conformational sub-states.

Picosecond to nanosecond dynamics provide a source of conformational entropy for protein folding.

PubMed

Stadler, Andreas M; Demmel, Franz; Ollivier, Jacques; Seydel, Tilo

2016-08-03

Myoglobin can be trapped in fully folded structures, partially folded molten globules, and unfolded states under stable equilibrium conditions. Here, we report an experimental study on the conformational dynamics of different folded conformational states of apo- and holomyoglobin in solution. Global protein diffusion and internal molecular motions were probed by neutron time-of-flight and neutron backscattering spectroscopy on the picosecond and nanosecond time scales. Global protein diffusion was found to depend on the α-helical content of the protein suggesting that charges on the macromolecule increase the short-time diffusion of protein. With regard to the molten globules, a gel-like phase due to protein entanglement and interactions with neighbouring macromolecules was visible due to a reduction of the global diffusion coefficients on the nanosecond time scale. Diffusion coefficients, residence and relaxation times of internal protein dynamics and root mean square displacements of localised internal motions were determined for the investigated structural states. The difference in conformational entropy ΔSconf of the protein between the unfolded and the partially or fully folded conformations was extracted from the measured root mean square displacements. Using thermodynamic parameters from the literature and the experimentally determined ΔSconf values we could identify the entropic contribution of the hydration shell ΔShydr of the different folded states. Our results point out the relevance of conformational entropy of the protein and the hydration shell for stability and folding of myoglobin.

Conformational diversity and computational enzyme design

PubMed Central

Lassila, Jonathan K.

2010-01-01

The application of computational protein design methods to the design of enzyme active sites offers potential routes to new catalysts and new reaction specificities. Computational design methods have typically treated the protein backbone as a rigid structure for the sake of computational tractability. However, this fixed-backbone approximation introduces its own special challenges for enzyme design and it contrasts with an emerging picture of natural enzymes as dynamic ensembles with multiple conformations and motions throughout a reaction cycle. This review considers the impact of conformational variation and dynamics on computational enzyme design and it highlights new approaches to addressing protein conformational diversity in enzyme design including recent advances in multistate design, backbone flexibility, and computational library design. PMID:20829099

An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis.

PubMed

Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F; Cohen, Itay; Henin, Rachel D; Hockla, Alexandra; Soares, Alexei S; Papo, Niv; Caulfield, Thomas R; Radisky, Evette S

2016-12-16

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Large-Scale Conformational Changes of Trypanosoma cruzi Proline Racemase Predicted by Accelerated Molecular Dynamics Simulation

PubMed Central

McCammon, J. Andrew

2011-01-01

Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is a life-threatening illness affecting 11–18 million people. Currently available treatments are limited, with unacceptable efficacy and safety profiles. Recent studies have revealed an essential T. cruzi proline racemase enzyme (TcPR) as an attractive candidate for improved chemotherapeutic intervention. Conformational changes associated with substrate binding to TcPR are believed to expose critical residues that elicit a host mitogenic B-cell response, a process contributing to parasite persistence and immune system evasion. Characterization of the conformational states of TcPR requires access to long-time-scale motions that are currently inaccessible by standard molecular dynamics simulations. Here we describe advanced accelerated molecular dynamics that extend the effective simulation time and capture large-scale motions of functional relevance. Conservation and fragment mapping analyses identified potential conformational epitopes located in the vicinity of newly identified transient binding pockets. The newly identified open TcPR conformations revealed by this study along with knowledge of the closed to open interconversion mechanism advances our understanding of TcPR function. The results and the strategy adopted in this work constitute an important step toward the rationalization of the molecular basis behind the mitogenic B-cell response of TcPR and provide new insights for future structure-based drug discovery. PMID:22022240

Large-scale conformational changes of Trypanosoma cruzi proline racemase predicted by accelerated molecular dynamics simulation.

PubMed

de Oliveira, César Augusto F; Grant, Barry J; Zhou, Michelle; McCammon, J Andrew

2011-10-01

Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is a life-threatening illness affecting 11-18 million people. Currently available treatments are limited, with unacceptable efficacy and safety profiles. Recent studies have revealed an essential T. cruzi proline racemase enzyme (TcPR) as an attractive candidate for improved chemotherapeutic intervention. Conformational changes associated with substrate binding to TcPR are believed to expose critical residues that elicit a host mitogenic B-cell response, a process contributing to parasite persistence and immune system evasion. Characterization of the conformational states of TcPR requires access to long-time-scale motions that are currently inaccessible by standard molecular dynamics simulations. Here we describe advanced accelerated molecular dynamics that extend the effective simulation time and capture large-scale motions of functional relevance. Conservation and fragment mapping analyses identified potential conformational epitopes located in the vicinity of newly identified transient binding pockets. The newly identified open TcPR conformations revealed by this study along with knowledge of the closed to open interconversion mechanism advances our understanding of TcPR function. The results and the strategy adopted in this work constitute an important step toward the rationalization of the molecular basis behind the mitogenic B-cell response of TcPR and provide new insights for future structure-based drug discovery.

Conformational exchange in pseudoazurin: different kinds of microsecond to millisecond dynamics characterized by their pH and buffer dependence using 15N NMR relaxation.

PubMed

Hass, Mathias A S; Vlasie, Monica D; Ubbink, Marcellus; Led, Jens J

2009-01-13

The dynamics of the reduced form of the blue copper protein pseudoazurin from Alcaligenes faecalis S-6 was investigated using (15)N relaxation measurements with a focus on the dynamics of the micro- to millisecond time scale. Different types of conformational exchange processes are observed in the protein on this time scale. At low pH, the protonation of the C-terminal copper-ligated histidine, His81, is observed. A comparison of the exchange rates in the presence and absence of added buffers shows that the protonation is the rate-limiting step at low buffer concentrations. This finding agrees with previous observations for other blue copper proteins, e.g., amicyanin and plastocyanin. However, in contrast to plastocyanin but similar to amicyanin, a second conformational exchange between different conformations of the protonated copper site is observed at low pH, most likely triggered by the protonation of His81. This process has been further characterized using CPMG dispersion methods and is found to occur with a rate of a few thousands per second. Finally, micro- to millisecond motions are observed in one of the loop regions and in the alpha-helical regions. These motions are unaffected by pH and are unrelated to the conformational changes in the active site of pseudoazurin.

A molecular dynamics investigation of CDK8/CycC and ligand binding: conformational flexibility and implication in drug discovery

NASA Astrophysics Data System (ADS)

Cholko, Timothy; Chen, Wei; Tang, Zhiye; Chang, Chia-en A.

2018-05-01

Abnormal activity of cyclin-dependent kinase 8 (CDK8) along with its partner protein cyclin C (CycC) is a common feature of many diseases including colorectal cancer. Using molecular dynamics (MD) simulations, this study determined the dynamics of the CDK8-CycC system and we obtained detailed breakdowns of binding energy contributions for four type-I and five type-II CDK8 inhibitors. We revealed system motions and conformational changes that will affect ligand binding, confirmed the essentialness of CycC for inclusion in future computational studies, and provide guidance in development of CDK8 binders. We employed unbiased all-atom MD simulations for 500 ns on twelve CDK8-CycC systems, including apoproteins and protein-ligand complexes, then performed principal component analysis (PCA) and measured the RMSF of key regions to identify protein dynamics. Binding pocket volume analysis identified conformational changes that accompany ligand binding. Next, H-bond analysis, residue-wise interaction calculations, and MM/PBSA were performed to characterize protein-ligand interactions and find the binding energy. We discovered that CycC is vital for maintaining a proper conformation of CDK8 to facilitate ligand binding and that the system exhibits motion that should be carefully considered in future computational work. Surprisingly, we found that motion of the activation loop did not affect ligand binding. Type-I and type-II ligand binding is driven by van der Waals interactions, but electrostatic energy and entropic penalties affect type-II binding as well. Binding of both ligand types affects protein flexibility. Based on this we provide suggestions for development of tighter-binding CDK8 inhibitors and offer insight that can aid future computational studies.

Molecular-dynamics simulation of polymethylene chain confined in cylindrical potentials. I. Nature of the conformational defects

NASA Astrophysics Data System (ADS)

Yamamoto, Takashi; Kimikawa, Yuichi

1992-10-01

The conformational motion of a polymethylene molecule constrained by a cylindrical potential is simulated up to 100 ps. The molecule consists of 60 CH2 groups and has variable bond lengths, bond angles, and dihedral angles. Our main concern here is the excitation and the dynamics of the conformational defects: kinks, jogs, etc. Under weaker constraint a number of gauche bonds are excited; they mostly form pairs such as gtḡ kinks or gtttḡ jogs. These conformational defects show no continuous drift in space. Instead they often annihilate and then recreate at different sites showing apparently random positional changes. The conformational defects produce characteristic strain fields around them. It seems that the conformational defects interact attractively through these strain fields. This is evidenced by remarkably correlated spatial distributions of the gauche bonds.

Elucidating Molecular Motion through Structural and Dynamic Filters of Energy-Minimized Conformer Ensembles

PubMed Central

2015-01-01

Complex RNA structures are constructed from helical segments connected by flexible loops that move spontaneously and in response to binding of small molecule ligands and proteins. Understanding the conformational variability of RNA requires the characterization of the coupled time evolution of interconnected flexible domains. To elucidate the collective molecular motions and explore the conformational landscape of the HIV-1 TAR RNA, we describe a new methodology that utilizes energy-minimized structures generated by the program “Fragment Assembly of RNA with Full-Atom Refinement (FARFAR)”. We apply structural filters in the form of experimental residual dipolar couplings (RDCs) to select a subset of discrete energy-minimized conformers and carry out principal component analyses (PCA) to corroborate the choice of the filtered subset. We use this subset of structures to calculate solution T1 and T1ρ relaxation times for 13C spins in multiple residues in different domains of the molecule using two simulation protocols that we previously published. We match the experimental T1 times to within 2% and the T1ρ times to within less than 10% for helical residues. These results introduce a protocol to construct viable dynamic trajectories for RNA molecules that accord well with experimental NMR data and support the notion that the motions of the helical portions of this small RNA can be described by a relatively small number of discrete conformations exchanging over time scales longer than 1 μs. PMID:24479561

Coupled binding-bending-folding: The complex conformational dynamics of protein-DNA binding studied by atomistic molecular dynamics simulations.

PubMed

van der Vaart, Arjan

2015-05-01

Protein-DNA binding often involves dramatic conformational changes such as protein folding and DNA bending. While thermodynamic aspects of this behavior are understood, and its biological function is often known, the mechanism by which the conformational changes occur is generally unclear. By providing detailed structural and energetic data, molecular dynamics simulations have been helpful in elucidating and rationalizing protein-DNA binding. This review will summarize recent atomistic molecular dynamics simulations of the conformational dynamics of DNA and protein-DNA binding. A brief overview of recent developments in DNA force fields is given as well. Simulations have been crucial in rationalizing the intrinsic flexibility of DNA, and have been instrumental in identifying the sequence of binding events, the triggers for the conformational motion, and the mechanism of binding for a number of important DNA-binding proteins. Molecular dynamics simulations are an important tool for understanding the complex binding behavior of DNA-binding proteins. With recent advances in force fields and rapid increases in simulation time scales, simulations will become even more important for future studies. This article is part of a Special Issue entitled Recent developments of molecular dynamics. Copyright © 2014. Published by Elsevier B.V.

Measuring Conformational Dynamics of Single Biomolecules Using Nanoscale Electronic Devices

NASA Astrophysics Data System (ADS)

Akhterov, Maxim V.; Choi, Yongki; Sims, Patrick C.; Olsen, Tivoli J.; Gul, O. Tolga; Corso, Brad L.; Weiss, Gregory A.; Collins, Philip G.

2014-03-01

Molecular motion can be a rate-limiting step of enzyme catalysis, but motions are typically too quick to resolve with fluorescent single molecule techniques. Recently, we demonstrated a label-free technique that replaced fluorophores with nano-electronic circuits to monitor protein motions. The solid-state electronic technique used single-walled carbon nanotube (SWNT) transistors to monitor conformational motions of a single molecule of T4 lysozyme while processing its substrate, peptidoglycan. As lysozyme catalyzes the hydrolysis of glycosidic bonds, two protein domains undergo 8 Å hinge bending motion that generates an electronic signal in the SWNT transistor. We describe improvements to the system that have extended our temporal resolution to 2 μs . Electronic recordings at this level of detail directly resolve not just transitions between open and closed conformations but also the durations for those transition events. Statistical analysis of many events determines transition timescales characteristic of enzyme activity and shows a high degree of variability within nominally identical chemical events. The high resolution technique can be readily applied to other complex biomolecules to gain insights into their kinetic parameters and catalytic function.

Sequence-dependent nanometer-scale conformational dynamics of individual RecBCD–DNA complexes

PubMed Central

Carter, Ashley R.; Seaberg, Maasa H.; Fan, Hsiu-Fang; Sun, Gang; Wilds, Christopher J.; Li, Hung-Wen; Perkins, Thomas T.

2016-01-01

RecBCD is a multifunctional enzyme that possesses both helicase and nuclease activities. To gain insight into the mechanism of its helicase function, RecBCD unwinding at low adenosine triphosphate (ATP) (2–4 μM) was measured using an optical-trapping assay featuring 1 base-pair (bp) precision. Instead of uniformly sized steps, we observed forward motion convolved with rapid, large-scale (∼4 bp) variations in DNA length. We interpret this motion as conformational dynamics of the RecBCD–DNA complex in an unwinding-competent state, arising, in part, by an enzyme-induced, back-and-forth motion relative to the dsDNA that opens and closes the duplex. Five observations support this interpretation. First, these dynamics were present in the absence of ATP. Second, the onset of the dynamics was coupled to RecBCD entering into an unwinding-competent state that required a sufficiently long 5′ strand to engage the RecD helicase. Third, the dynamics were modulated by the GC-content of the dsDNA. Fourth, the dynamics were suppressed by an engineered interstrand cross-link in the dsDNA that prevented unwinding. Finally, these dynamics were suppressed by binding of a specific non-hydrolyzable ATP analog. Collectively, these observations show that during unwinding, RecBCD binds to DNA in a dynamic mode that is modulated by the nucleotide state of the ATP-binding pocket. PMID:27220465

The HAMP Signal Relay Domain Adopts Multiple Conformational States through Collective Piston and Tilt Motions

PubMed Central

Zhu, Lizhe; Bolhuis, Peter G.; Vreede, Jocelyne

2013-01-01

The HAMP domain is a linker region in prokaryotic sensor proteins and relays input signals to the transmitter domain and vice versa. Functional as a dimer, the structure of HAMP shows a parallel coiled-coil motif comprising four helices. To date, it is unclear how HAMP can relay signals from one domain to another, although several models exist. In this work, we use molecular simulation to test the hypothesis that HAMP adopts different conformations, one of which represents an active, signal-relaying configuration, and another an inactive, resting state. We first performed molecular dynamics simulation on the prototype HAMP domain Af1503 from Archaeoglobus fulgidus. We explored its conformational space by taking the structure of the A291F mutant disabling HAMP activity as a starting point. These simulations revealed additional conformational states that differ in the tilt angles between the helices as well as the relative piston shifts of the helices relative to each other. By enhancing the sampling in a metadynamics set up, we investigated three mechanistic models for HAMP signal transduction. Our results indicate that HAMP can access additional conformational states characterized by piston motion. Furthermore, the piston motion of the N-terminal helix of one monomer is directly correlated with the opposite piston motion of the C-terminal helix of the other monomer. The change in piston motion is accompanied by a change in tilt angle between the monomers, thus revealing that HAMP exhibits a collective motion, i.e. a combination of changes in tilt angles and a piston-like displacement. Our results provide insights into the conformational changes that underlie the signaling mechanism involving HAMP. PMID:23468603

Hidden regularity and universal classification of fast side chain motions in proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rajeshwar, Rajitha; Smith, Jeremy C.; Krishnam, Marimuthu

Proteins display characteristic dynamical signatures that appear to be universal across all proteins regardless of topology and size. Here, we systematically characterize the universal features of fast side chain motions in proteins by examining the conformational energy surfaces of individual residues obtained using enhanced sampling molecular dynamics simulation (618 free energy surfaces obtained from 0.94 s MD simulation). The side chain conformational free energy surfaces obtained using the adaptive biasing force (ABF) method for a set of eight proteins with different molecular weights and secondary structures are used to determine the methyl axial NMR order parameters (O axis 2), populationsmore » of side chain rotamer states (ρ), conformational entropies (S conf), probability fluxes, and activation energies for side chain inter-rotameric transitions. The free energy barriers separating side chain rotamer states range from 0.3 to 12 kcal/mol in all proteins and follow a trimodal distribution with an intense peak at ~5 kcal/mol and two shoulders at ~3 and ~7.5 kcal/mol, indicating that some barriers are more favored than others by proteins to maintain a balance between their conformational stability and flexibility. The origin and the influences of the trimodal barrier distribution on the distribution of O axis 2 and the side chain conformational entropy are discussed. A hierarchical grading of rotamer states based on the conformational free energy barriers, entropy, and probability flux reveals three distinct classes of side chains in proteins. A unique nonlinear correlation is established between O axis 2 and the side chain rotamer populations (ρ). In conclusion, the apparent universality in O axis 2 versus correlation, trimodal barrier distribution, and distinct characteristics of three classes of side chains observed among all proteins indicates a hidden regularity (or commonality) in the dynamical heterogeneity of fast side chain motions in proteins.« less

Examinations of tRNA Range of Motion Using Simulations of Cryo-EM Microscopy and X-Ray Data.

PubMed

Caulfield, Thomas R; Devkota, Batsal; Rollins, Geoffrey C

2011-01-01

We examined tRNA flexibility using a combination of steered and unbiased molecular dynamics simulations. Using Maxwell's demon algorithm, molecular dynamics was used to steer X-ray structure data toward that from an alternative state obtained from cryogenic-electron microscopy density maps. Thus, we were able to fit X-ray structures of tRNA onto cryogenic-electron microscopy density maps for hybrid states of tRNA. Additionally, we employed both Maxwell's demon molecular dynamics simulations and unbiased simulation methods to identify possible ribosome-tRNA contact areas where the ribosome may discriminate tRNAs during translation. Herein, we collected >500 ns of simulation data to assess the global range of motion for tRNAs. Biased simulations can be used to steer between known conformational stop points, while unbiased simulations allow for a general testing of conformational space previously unexplored. The unbiased molecular dynamics data describes the global conformational changes of tRNA on a sub-microsecond time scale for comparison with steered data. Additionally, the unbiased molecular dynamics data was used to identify putative contacts between tRNA and the ribosome during the accommodation step of translation. We found that the primary contact regions were H71 and H92 of the 50S subunit and ribosomal proteins L14 and L16.

Integrated description of protein dynamics from room-temperature X-ray crystallography and NMR

PubMed Central

Fenwick, R. Bryn; van den Bedem, Henry; Fraser, James S.; Wright, Peter E.

2014-01-01

Detailed descriptions of atomic coordinates and motions are required for an understanding of protein dynamics and their relation to molecular recognition, catalytic function, and allostery. Historically, NMR relaxation measurements have played a dominant role in the determination of the amplitudes and timescales (picosecond–nanosecond) of bond vector fluctuations, whereas high-resolution X-ray diffraction experiments can reveal the presence of and provide atomic coordinates for multiple, weakly populated substates in the protein conformational ensemble. Here we report a hybrid NMR and X-ray crystallography analysis that provides a more complete dynamic picture and a more quantitative description of the timescale and amplitude of fluctuations in atomic coordinates than is obtainable from the individual methods alone. Order parameters (S2) were calculated from single-conformer and multiconformer models fitted to room temperature and cryogenic X-ray diffraction data for dihydrofolate reductase. Backbone and side-chain order parameters derived from NMR relaxation experiments are in excellent agreement with those calculated from the room-temperature single-conformer and multiconformer models, showing that the picosecond timescale motions observed in solution occur also in the crystalline state. These motions are quenched in the crystal at cryogenic temperatures. The combination of NMR and X-ray crystallography in iterative refinement promises to provide an atomic resolution description of the alternate conformational substates that are sampled through picosecond to nanosecond timescale fluctuations of the protein structure. The method also provides insights into the structural heterogeneity of nonmethyl side chains, aromatic residues, and ligands, which are less commonly analyzed by NMR relaxation measurements. PMID:24474795

Conformational dynamics of bacterial trigger factor in apo and ribosome-bound states.

PubMed

Can, Mehmet Tarik; Kurkcuoglu, Zeynep; Ezeroglu, Gokce; Uyar, Arzu; Kurkcuoglu, Ozge; Doruker, Pemra

2017-01-01

The chaperone trigger factor (TF) binds to the ribosome exit tunnel and helps cotranslational folding of nascent chains (NC) in bacterial cells and chloroplasts. In this study, we aim to investigate the functional dynamics of fully-atomistic apo TF and its complex with 50S. As TF accomodates a high percentage of charged residues on its surface, the effect of ionic strength on TF dynamics is assessed here by performing five independent molecular dynamics (MD) simulations (total of 1.3 micro-second duration) at 29 mM and 150 mM ionic strengths. At both concentrations, TF exhibits high inter- and intra-domain flexibility related to its binding (BD), core (CD), and head (HD) domains. Even though large oscillations in gyration radius exist during each run, we do not detect the so-called 'fully collapsed' state with both HD and BD collapsed upon the core. In fact, the extended conformers with relatively open HD and BD are highly populated at the physiological concentration of 150 mM. More importantly, extended TF snapshots stand out in terms of favorable docking onto the 50S subunit. Elastic network modeling (ENM) indicates significant changes in TF's functional dynamics and domain decomposition depending on its conformation and positioning on the 50S. The most dominant slow motions are the lateral sweeping and vertical opening/closing of HD relative to 50S. Finally, our ENM-based efficient technique -ClustENM- is used to sample atomistic conformers starting with an extended TF-50S complex. Specifically, BD and CD motions are restricted near the tunnel exit, while HD is highly mobile. The atomistic conformers generated without an NC are in agreement with the cryo-EM maps available for TF-ribosome-NC complex.

Monitoring equilibrium reaction dynamics of a nearly barrierless molecular rotor using ultrafast vibrational echoes

NASA Astrophysics Data System (ADS)

Nilsen, Ian A.; Osborne, Derek G.; White, Aaron M.; Anna, Jessica M.; Kubarych, Kevin J.

2014-10-01

Using rapidly acquired spectral diffusion, a recently developed variation of heterodyne detected infrared photon echo spectroscopy, we observe ˜3 ps solvent independent spectral diffusion of benzene chromium tricarbonyl (C6H6Cr(CO)3, BCT) in a series of nonpolar linear alkane solvents. The spectral dynamics is attributed to low-barrier internal torsional motion. This tripod complex has two stable minima corresponding to staggered and eclipsed conformations, which differ in energy by roughly half of kBT. The solvent independence is due to the relative size of the rotor compared with the solvent molecules, which create a solvent cage in which torsional motion occurs largely free from solvent damping. Since the one-dimensional transition state is computed to be only 0.03 kBT above the higher energy eclipsed conformation, this model system offers an unusual, nearly barrierless reaction, which nevertheless is characterized by torsional coordinate dependent vibrational frequencies. Hence, by studying the spectral diffusion of the tripod carbonyls, it is possible to gain insight into the fundamental dynamics of internal rotational motion, and we find some evidence for the importance of non-diffusive ballistic motion even in the room-temperature liquid environment. Using several different approaches to describe equilibrium kinetics, as well as the influence of reactive dynamics on spectroscopic observables, we provide evidence that the low-barrier torsional motion of BCT provides an excellent test case for detailed studies of the links between chemical exchange and linear and nonlinear vibrational spectroscopy.

Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography

DOE PAGES

Keedy, Daniel A.; Kenner, Lillian R.; Warkentin, Matthew; ...

2015-09-30

Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences ofmore » these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Altogether, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.« less

Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keedy, Daniel A.; Kenner, Lillian R.; Warkentin, Matthew

Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences ofmore » these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.« less

Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keedy, Daniel A.; Kenner, Lillian R.; Warkentin, Matthew

Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences ofmore » these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Altogether, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.« less

Dynamics of polydots: Soft luminescent polymeric nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maskey, Sabina; Osti, Naresh C.; Grest, Gary S.

The conformation and dynamics of luminescent polymers collapsed into nanoparticles or polydots were studied using fully atomistic molecular dynamics (MD) simulations, providing a first insight into their internal dynamics. Controlling the conformation and dynamics of confined polymers is essential for realization of the full potential of polydots in nanomedicine and biotechnology. Specifically, the shape and internal dynamics of polydots that consist of highly rigid dialkyl p-phenylene ethynylene (PPE) are probed as a function of temperature. At room temperature, the polydots are spherical without any correlations between the aromatic rings on the PPE backbone. With increasing temperature, they expand and becomemore » slightly aspherical; however, the polymers remain confined. The coherent dynamic structure factor reveals that the internal motion of the polymer backbone is arrested, and the side chains dominate the internal dynamics of the polydots. Lastly, these new soft nanoparticles retain their overall shape and dynamics over an extended temperature range, and their conformation is tunable via their degree of expansion.« less

Dynamics of polydots: Soft luminescent polymeric nanoparticles

DOE PAGES

Maskey, Sabina; Osti, Naresh C.; Grest, Gary S.; ...

2016-03-04

The conformation and dynamics of luminescent polymers collapsed into nanoparticles or polydots were studied using fully atomistic molecular dynamics (MD) simulations, providing a first insight into their internal dynamics. Controlling the conformation and dynamics of confined polymers is essential for realization of the full potential of polydots in nanomedicine and biotechnology. Specifically, the shape and internal dynamics of polydots that consist of highly rigid dialkyl p-phenylene ethynylene (PPE) are probed as a function of temperature. At room temperature, the polydots are spherical without any correlations between the aromatic rings on the PPE backbone. With increasing temperature, they expand and becomemore » slightly aspherical; however, the polymers remain confined. The coherent dynamic structure factor reveals that the internal motion of the polymer backbone is arrested, and the side chains dominate the internal dynamics of the polydots. Lastly, these new soft nanoparticles retain their overall shape and dynamics over an extended temperature range, and their conformation is tunable via their degree of expansion.« less

Partial cooperative unfolding in proteins as observed by hydrogen exchange mass spectrometry

PubMed Central

Engen, John R.; Wales, Thomas E.; Chen, Shugui; Marzluff, Elaine M.; Hassell, Kerry M.; Weis, David D.; Smithgall, Thomas E.

2013-01-01

Many proteins do not exist in a single rigid conformation. Protein motions, or dynamics, exist and in many cases are important for protein function. The analysis of protein dynamics relies on biophysical techniques that can distinguish simultaneously existing populations of molecules and their rates of interconversion. Hydrogen exchange (HX) detected by mass spectrometry (MS) is contributing to our understanding of protein motions by revealing unfolding and dynamics on a wide timescale, ranging from seconds to hours to days. In this review we discuss HX MS-based analyses of protein dynamics, using our studies of multi-domain kinases as examples. Using HX MS, we have successfully probed protein dynamics and unfolding in the isolated SH3, SH2 and kinase domains of the c-Src and Abl kinase families, as well as the role of inter- and intra-molecular interactions in the global control of kinase function. Coupled with high-resolution structural information, HX MS has proved to be a powerful and versatile tool for the analysis of the conformational dynamics in these kinase systems, and has provided fresh insight regarding the regulatory control of these important signaling proteins. HX MS studies of dynamics are applicable not only to the proteins we illustrate here, but to a very wide range of proteins and protein systems, and should play a role in both classification of and greater understanding of the prevalence of protein motion. PMID:23682200

Molecular dynamics simulations of domain motions of substrate-free S-adenosyl- L-homocysteine hydrolase in solution.

PubMed

Hu, Chen; Fang, Jianwen; Borchardt, Ronald T; Schowen, Richard L; Kuczera, Krzysztof

2008-04-01

S-Adenosyl-L-homocysteine hydrolase (SAHH) is an enzyme regulating intracellular methylation reactions. The homotetrameric SAHH exists in an open conformation in absence of substrate, while enzyme:inhibitor complexes crystallize in the closed conformation, in which the ligands are engulfed by the protein due to an 18 degrees domain reorientation within each of the four subunits. We present a microscopic description of the structure and dynamics of the substrate-free, NAD(+)-bound SAHH in solution, based on a 15-ns molecular dynamics simulation in explicit solvent. In the trajectory, the four cofactor-binding domains formed a relatively rigid core with structure very similar to the crystal conformation. The four substrate-binding domains, located at the protein exterior, also retained internal structures similar to the crystal, while undergoing large amplitude rigid-body reorientations. The trajectory domain motions exhibited two interesting properties. First, within each subunit the domains fluctuated between open and closed conformations, while at the tetramer level 80% of the domain motions were perpendicular to the direction of the open-to-closed structural transition. Second, the domain reorientations in solution could be represented as a sum of two components, faster, with 20-50 ps correlation time and 3-4 degrees amplitude, and slower, with 8-23 ns correlation time and amplitude of 14-22 degrees . The faster motion is similar to the 1.5 cm(-1) frequency hinge-bending vibrations found in our recent normal mode analysis (Wang et al., Biochemistry 2005;44:7228-7239). The slower motion agrees with fluorescence anisotropy decay measurements, which detected a 10-20 ns domain reorientation of ca. 26 degrees amplitude in the substrate-free enzyme (Wang et al., Biochemistry 2006;45:7778-7786). Our simulations are thus in excellent agreement with experimental data. The simulations allow us to assign the observed nanosecond fluorescence anisotropy signal to fluctuations in domain orientations, and indicate that the microscopic mechanism of the motion involves rotational diffusion within a cone of 10-20 degrees . Overall, our simulation results complement the existing experimental data and provide important new insights into SAHH domain motions in solution, which play a crucial role in the catalytic mechanism of SAHH. (c) 2007 Wiley-Liss, Inc.

Adaptability of Protein Structures to Enable Functional Interactions and Evolutionary Implications

PubMed Central

Haliloglu, Turkan; Bahar, Ivet

2015-01-01

Several studies in recent years have drawn attention to the ability of proteins to adapt to intermolecular interactions by conformational changes along structure-encoded collective modes of motions. These so-called soft modes, primarily driven by entropic effects, facilitate, if not enable, functional interactions. They represent excursions on the conformational space along principal low-ascent directions/paths away from the original free energy minimum, and they are accessible to the protein even prior to protein-protein/ligand interactions. An emerging concept from these studies is the evolution of structures or modular domains to favor such modes of motion that will be recruited or integrated for enabling functional interactions. Structural dynamics, including the allosteric switches in conformation that are often stabilized upon formation of complexes and multimeric assemblies, emerge as key properties that are evolutionarily maintained to accomplish biological activities, consistent with the paradigm sequence → structure → dynamics → function where ‘dynamics’ bridges structure and function. PMID:26254902

Relationship between femtosecond-picosecond dynamics to enzyme catalyzed H-transfer

PubMed Central

Cheatum, Christopher M.; Kohen, Amnon

2015-01-01

At physiological temperatures, enzymes exhibit a broad spectrum of conformations, which interchange via thermally activated dynamics. These conformations are sampled differently in different complexes of the protein and its ligands, and the dynamics of exchange between these conformers depends on the mass of the group that is moving and the length scale of the motion, as well as restrictions imposed by the globular fold of the enzymatic complex. Many of these motions have been examined and their role in the enzyme function illuminated, yet most experimental tools applied so far have identified dynamics at time scales of seconds to nanoseconds, which are much slower than the time scale for H-transfer between two heavy atoms. This chemical conversion and other processes involving cleavage of covalent bonds occur on picosecond to femtosecond time scales, where slower processes mask both the kinetics and dynamics. Here we present a combination of kinetic and spectroscopic methods that may enable closer examination of the relationship between enzymatic C-H→C transfer and the dynamics of the active site environment at the chemically relevant time scale. These methods include kinetic isotope effects and their temperature dependence, which are used to study the kinetic nature of the H-transfer, and 2D IR spectroscopy, which is used to study the dynamics of transition-state- and ground-state-analog complexes. The combination of these tools is likely to provide a new approach to examine the protein dynamics that directly influence the chemical conversion catalyzed by enzymes. PMID:23539379

Intermolecular correlations are necessary to explain diffuse scattering from protein crystals

DOE PAGES

Peck, Ariana; Poitevin, Frederic; Lane, Thomas Joseph

2018-02-21

Conformational changes drive protein function, including catalysis, allostery, and signaling. X-ray diffuse scattering from protein crystals has frequently been cited as a probe of these correlated motions, with significant potential to advance our understanding of biological dynamics. However, recent work challenged this prevailing view, suggesting instead that diffuse scattering primarily originates from rigid body motions and could therefore be applied to improve structure determination. To investigate the nature of the disorder giving rise to diffuse scattering, and thus the potential applications of this signal, a diverse repertoire of disorder models was assessed for its ability to reproduce the diffuse signalmore » reconstructed from three protein crystals. This comparison revealed that multiple models of intramolecular conformational dynamics, including ensemble models inferred from the Bragg data, could not explain the signal. Models of rigid body or short-range liquid-like motions, in which dynamics are confined to the biological unit, showed modest agreement with the diffuse maps, but were unable to reproduce experimental features indicative of long-range correlations. Extending a model of liquid-like motions to include disorder across neighboring proteins in the crystal significantly improved agreement with all three systems and highlighted the contribution of intermolecular correlations to the observed signal. These findings anticipate a need to account for intermolecular disorder in order to advance the interpretation of diffuse scattering to either extract biological motions or aid structural inference.« less

Intermolecular correlations are necessary to explain diffuse scattering from protein crystals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peck, Ariana; Poitevin, Frederic; Lane, Thomas Joseph

Conformational changes drive protein function, including catalysis, allostery, and signaling. X-ray diffuse scattering from protein crystals has frequently been cited as a probe of these correlated motions, with significant potential to advance our understanding of biological dynamics. However, recent work challenged this prevailing view, suggesting instead that diffuse scattering primarily originates from rigid body motions and could therefore be applied to improve structure determination. To investigate the nature of the disorder giving rise to diffuse scattering, and thus the potential applications of this signal, a diverse repertoire of disorder models was assessed for its ability to reproduce the diffuse signalmore » reconstructed from three protein crystals. This comparison revealed that multiple models of intramolecular conformational dynamics, including ensemble models inferred from the Bragg data, could not explain the signal. Models of rigid body or short-range liquid-like motions, in which dynamics are confined to the biological unit, showed modest agreement with the diffuse maps, but were unable to reproduce experimental features indicative of long-range correlations. Extending a model of liquid-like motions to include disorder across neighboring proteins in the crystal significantly improved agreement with all three systems and highlighted the contribution of intermolecular correlations to the observed signal. These findings anticipate a need to account for intermolecular disorder in order to advance the interpretation of diffuse scattering to either extract biological motions or aid structural inference.« less

The principal motions involved in the coupling mechanism of the recovery stroke of the myosin motor.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mesentean, Sidonia; Koppole, Sampath; Smith, Jeremy C

2007-03-01

Muscle contraction is driven by a cycle of conformational changes in the myosin II head. After myosin binds ATP and releases from the actin fibril, myosin prepares for the next power stroke by rotating back the converter domain that carries the lever arm by 60{sup o}. This recovery stroke is coupled to the activation of myosin ATPase by a mechanism that is essential for an efficient motor cycle. The mechanics of this coupling have been proposed to occur via two distinct and successive motions of the two helices that hold the converter domain: in a first phase a seesaw motionmore » of the relay helix, followed by a piston-like motion of the SH1 helix in a second phase. To test this model, we have determined the principal motions of these structural elements during equilibrium molecular dynamics simulations of the crystallographic end states of the recovery-stroke by using principal component analysis. This reveals that the only principal motions of these two helices that make a large-amplitude contribution towards the conformational change of the recovery stroke are indeed the predicted seesaw and piston motions. Moreover, the results demonstrate that the seesaw motion of the relay helix dominates in the dynamics of the pre-recovery stroke structure, but not in the dynamics of the post-recovery stroke structure, and vice versa for the piston motion of the SH1 helix. This is consistent with the order of the proposed two-phase model for the coupling mechanism of the recovery stroke. Molecular movies of these principal motions are available at http://www.iwr.uni-heidelberg.de/groups/biocomp/fischer.« less

Couplings between hierarchical conformational dynamics from multi-time correlation functions and two-dimensional lifetime spectra: Application to adenylate kinase

NASA Astrophysics Data System (ADS)

Ono, Junichi; Takada, Shoji; Saito, Shinji

2015-06-01

An analytical method based on a three-time correlation function and the corresponding two-dimensional (2D) lifetime spectrum is developed to elucidate the time-dependent couplings between the multi-timescale (i.e., hierarchical) conformational dynamics in heterogeneous systems such as proteins. In analogy with 2D NMR, IR, electronic, and fluorescence spectroscopies, the waiting-time dependence of the off-diagonal peaks in the 2D lifetime spectra can provide a quantitative description of the dynamical correlations between the conformational motions with different lifetimes. The present method is applied to intrinsic conformational changes of substrate-free adenylate kinase (AKE) using long-time coarse-grained molecular dynamics simulations. It is found that the hierarchical conformational dynamics arise from the intra-domain structural transitions among conformational substates of AKE by analyzing the one-time correlation functions and one-dimensional lifetime spectra for the donor-acceptor distances corresponding to single-molecule Förster resonance energy transfer experiments with the use of the principal component analysis. In addition, the complicated waiting-time dependence of the off-diagonal peaks in the 2D lifetime spectra for the donor-acceptor distances is attributed to the fact that the time evolution of the couplings between the conformational dynamics depends upon both the spatial and temporal characters of the system. The present method is expected to shed light on the biological relationship among the structure, dynamics, and function.

Couplings between hierarchical conformational dynamics from multi-time correlation functions and two-dimensional lifetime spectra: Application to adenylate kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ono, Junichi; Takada, Shoji; Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502

2015-06-07

An analytical method based on a three-time correlation function and the corresponding two-dimensional (2D) lifetime spectrum is developed to elucidate the time-dependent couplings between the multi-timescale (i.e., hierarchical) conformational dynamics in heterogeneous systems such as proteins. In analogy with 2D NMR, IR, electronic, and fluorescence spectroscopies, the waiting-time dependence of the off-diagonal peaks in the 2D lifetime spectra can provide a quantitative description of the dynamical correlations between the conformational motions with different lifetimes. The present method is applied to intrinsic conformational changes of substrate-free adenylate kinase (AKE) using long-time coarse-grained molecular dynamics simulations. It is found that the hierarchicalmore » conformational dynamics arise from the intra-domain structural transitions among conformational substates of AKE by analyzing the one-time correlation functions and one-dimensional lifetime spectra for the donor-acceptor distances corresponding to single-molecule Förster resonance energy transfer experiments with the use of the principal component analysis. In addition, the complicated waiting-time dependence of the off-diagonal peaks in the 2D lifetime spectra for the donor-acceptor distances is attributed to the fact that the time evolution of the couplings between the conformational dynamics depends upon both the spatial and temporal characters of the system. The present method is expected to shed light on the biological relationship among the structure, dynamics, and function.« less

Conformational Changes in the Epidermal Growth Factor Receptor: Role of the Transmembrane Domain Investigated by Coarse-Grained MetaDynamics Free Energy Calculations

PubMed Central

2016-01-01

The epidermal growth factor receptor (EGFR) is a dimeric membrane protein that regulates key aspects of cellular function. Activation of the EGFR is linked to changes in the conformation of the transmembrane (TM) domain, brought about by changes in interactions of the TM helices of the membrane lipid bilayer. Using an advanced computational approach that combines Coarse-Grained molecular dynamics and well-tempered MetaDynamics (CG-MetaD), we characterize the large-scale motions of the TM helices, simulating multiple association and dissociation events between the helices in membrane, thus leading to a free energy landscape of the dimerization process. The lowest energy state of the TM domain is a right-handed dimer structure in which the TM helices interact through the N-terminal small-X3-small sequence motif. In addition to this state, which is thought to correspond to the active form of the receptor, we have identified further low-energy states that allow us to integrate with a high level of detail a range of previous experimental observations. These conformations may lead to the active state via two possible activation pathways, which involve pivoting and rotational motions of the helices, respectively. Molecular dynamics also reveals correlation between the conformational changes of the TM domains and of the intracellular juxtamembrane domains, paving the way for a comprehensive understanding of EGFR signaling at the cell membrane. PMID:27459426

Conformational Changes in the Epidermal Growth Factor Receptor: Role of the Transmembrane Domain Investigated by Coarse-Grained MetaDynamics Free Energy Calculations.

PubMed

Lelimousin, Mickaël; Limongelli, Vittorio; Sansom, Mark S P

2016-08-24

The epidermal growth factor receptor (EGFR) is a dimeric membrane protein that regulates key aspects of cellular function. Activation of the EGFR is linked to changes in the conformation of the transmembrane (TM) domain, brought about by changes in interactions of the TM helices of the membrane lipid bilayer. Using an advanced computational approach that combines Coarse-Grained molecular dynamics and well-tempered MetaDynamics (CG-MetaD), we characterize the large-scale motions of the TM helices, simulating multiple association and dissociation events between the helices in membrane, thus leading to a free energy landscape of the dimerization process. The lowest energy state of the TM domain is a right-handed dimer structure in which the TM helices interact through the N-terminal small-X3-small sequence motif. In addition to this state, which is thought to correspond to the active form of the receptor, we have identified further low-energy states that allow us to integrate with a high level of detail a range of previous experimental observations. These conformations may lead to the active state via two possible activation pathways, which involve pivoting and rotational motions of the helices, respectively. Molecular dynamics also reveals correlation between the conformational changes of the TM domains and of the intracellular juxtamembrane domains, paving the way for a comprehensive understanding of EGFR signaling at the cell membrane.

Hamiltonian replica exchange combined with elastic network analysis to enhance global domain motions in atomistic molecular dynamics simulations.

PubMed

Ostermeir, Katja; Zacharias, Martin

2014-12-01

Coarse-grained elastic network models (ENM) of proteins offer a low-resolution representation of protein dynamics and directions of global mobility. A Hamiltonian-replica exchange molecular dynamics (H-REMD) approach has been developed that combines information extracted from an ENM analysis with atomistic explicit solvent MD simulations. Based on a set of centers representing rigid segments (centroids) of a protein, a distance-dependent biasing potential is constructed by means of an ENM analysis to promote and guide centroid/domain rearrangements. The biasing potentials are added with different magnitude to the force field description of the MD simulation along the replicas with one reference replica under the control of the original force field. The magnitude and the form of the biasing potentials are adapted during the simulation based on the average sampled conformation to reach a near constant biasing in each replica after equilibration. This allows for canonical sampling of conformational states in each replica. The application of the methodology to a two-domain segment of the glycoprotein 130 and to the protein cyanovirin-N indicates significantly enhanced global domain motions and improved conformational sampling compared with conventional MD simulations. © 2014 Wiley Periodicals, Inc.

Photoinduced electron transfer and fluorophore motion as a probe of the conformational dynamics of membrane proteins: application to the influenza a M2 proton channel.

PubMed

Rogers, Julie M G; Polishchuk, Alexei L; Guo, Lin; Wang, Jun; DeGrado, William F; Gai, Feng

2011-04-05

The structure and function of the influenza A M2 proton channel have been the subject of intensive investigations in recent years because of their critical role in the life cycle of the influenza virus. Using a truncated version of the M2 proton channel (i.e., M2TM) as a model, here we show that fluctuations in the fluorescence intensity of a dye reporter that arise from both fluorescence quenching via the mechanism of photoinduced electron transfer (PET) by an adjacent tryptophan (Trp) residue and local motions of the dye molecule can be used to probe the conformational dynamics of membrane proteins. Specifically, we find that the dynamics of the conformational transition between the N-terminal open and C-terminal open states of the M2TM channel occur on a timescale of about 500 μs and that the binding of either amantadine or rimantadine does not inhibit the pH-induced structural equilibrium of the channel. These results are consistent with the direct occluding mechanism of inhibition which suggests that the antiviral drugs act by sterically occluding the channel pore.

Opening mechanism of adenylate kinase can vary according to selected molecular dynamics force field

NASA Astrophysics Data System (ADS)

Unan, Hulya; Yildirim, Ahmet; Tekpinar, Mustafa

2015-07-01

Adenylate kinase is a widely used test case for many conformational transition studies. It performs a large conformational transition between closed and open conformations while performing its catalytic function. To understand conformational transition mechanism and impact of force field choice on E. Coli adenylate kinase, we performed all-atom explicit solvent classical molecular dynamics simulations starting from the closed conformation with four commonly used force fields, namely, Amber99, Charmm27, Gromos53a6, Opls-aa. We carried out 40 simulations, each one 200 ns. We analyzed completely 12 of them that show full conformational transition from the closed state to the open one. Our study shows that different force fields can have a bias toward different transition pathways. Transition time scales, frequency of conformational transitions, order of domain motions and free energy landscapes of each force field may also vary. In general, Amber99 and Charmm27 behave similarly while Gromos53a6 results have a resemblance to the Opls-aa force field results.

Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography

PubMed Central

Keedy, Daniel A; Kenner, Lillian R; Warkentin, Matthew; Woldeyes, Rahel A; Hopkins, Jesse B; Thompson, Michael C; Brewster, Aaron S; Van Benschoten, Andrew H; Baxter, Elizabeth L; Uervirojnangkoorn, Monarin; McPhillips, Scott E; Song, Jinhu; Alonso-Mori, Roberto; Holton, James M; Weis, William I; Brunger, Axel T; Soltis, S Michael; Lemke, Henrik; Gonzalez, Ana; Sauter, Nicholas K; Cohen, Aina E; van den Bedem, Henry; Thorne, Robert E; Fraser, James S

2015-01-01

Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function. DOI: http://dx.doi.org/10.7554/eLife.07574.001 PMID:26422513

Examinations of tRNA Range of Motion Using Simulations of Cryo-EM Microscopy and X-Ray Data

PubMed Central

Caulfield, Thomas R.; Devkota, Batsal; Rollins, Geoffrey C.

2011-01-01

We examined tRNA flexibility using a combination of steered and unbiased molecular dynamics simulations. Using Maxwell's demon algorithm, molecular dynamics was used to steer X-ray structure data toward that from an alternative state obtained from cryogenic-electron microscopy density maps. Thus, we were able to fit X-ray structures of tRNA onto cryogenic-electron microscopy density maps for hybrid states of tRNA. Additionally, we employed both Maxwell's demon molecular dynamics simulations and unbiased simulation methods to identify possible ribosome-tRNA contact areas where the ribosome may discriminate tRNAs during translation. Herein, we collected >500 ns of simulation data to assess the global range of motion for tRNAs. Biased simulations can be used to steer between known conformational stop points, while unbiased simulations allow for a general testing of conformational space previously unexplored. The unbiased molecular dynamics data describes the global conformational changes of tRNA on a sub-microsecond time scale for comparison with steered data. Additionally, the unbiased molecular dynamics data was used to identify putative contacts between tRNA and the ribosome during the accommodation step of translation. We found that the primary contact regions were H71 and H92 of the 50S subunit and ribosomal proteins L14 and L16. PMID:21716650

Structure-Preserving Variational Multiscale Modeling of Turbulent Incompressible Flow with Subgrid Vortices

NASA Astrophysics Data System (ADS)

Evans, John; Coley, Christopher; Aronson, Ryan; Nelson, Corey

2017-11-01

In this talk, a large eddy simulation methodology for turbulent incompressible flow will be presented which combines the best features of divergence-conforming discretizations and the residual-based variational multiscale approach to large eddy simulation. In this method, the resolved motion is represented using a divergence-conforming discretization, that is, a discretization that preserves the incompressibility constraint in a pointwise manner, and the unresolved fluid motion is explicitly modeled by subgrid vortices that lie within individual grid cells. The evolution of the subgrid vortices is governed by dynamical model equations driven by the residual of the resolved motion. Consequently, the subgrid vortices appropriately vanish for laminar flow and fully resolved turbulent flow. As the resolved velocity field and subgrid vortices are both divergence-free, the methodology conserves mass in a pointwise sense and admits discrete balance laws for energy, enstrophy, and helicity. Numerical results demonstrate the methodology yields improved results versus state-of-the-art eddy viscosity models in the context of transitional, wall-bounded, and rotational flow when a divergence-conforming B-spline discretization is utilized to represent the resolved motion.

Impact of distal mutations on the network of coupled motions correlated to hydride transfer in dihydrofolate reductase.

PubMed

Wong, Kim F; Selzer, Tzvia; Benkovic, Stephen J; Hammes-Schiffer, Sharon

2005-05-10

A comprehensive analysis of the network of coupled motions correlated to hydride transfer in dihydrofolate reductase is presented. Hybrid quantum/classical molecular dynamics simulations are combined with a rank correlation analysis method to extract thermally averaged properties that vary along the collective reaction coordinate according to a prescribed target model. Coupled motions correlated to hydride transfer are identified throughout the enzyme. Calculations for wild-type dihydrofolate reductase and a triple mutant, along with the associated single and double mutants, indicate that each enzyme system samples a unique distribution of coupled motions correlated to hydride transfer. These coupled motions provide an explanation for the experimentally measured nonadditivity effects in the hydride transfer rates for these mutants. This analysis illustrates that mutations distal to the active site can introduce nonlocal structural perturbations and significantly impact the catalytic rate by altering the conformational motions of the entire enzyme and the probability of sampling conformations conducive to the catalyzed reaction.

Conserved linear dynamics of single-molecule Brownian motion.

PubMed

Serag, Maged F; Habuchi, Satoshi

2017-06-06

Macromolecular diffusion in homogeneous fluid at length scales greater than the size of the molecule is regarded as a random process. The mean-squared displacement (MSD) of molecules in this regime increases linearly with time. Here we show that non-random motion of DNA molecules in this regime that is undetectable by the MSD analysis can be quantified by characterizing the molecular motion relative to a latticed frame of reference. Our lattice occupancy analysis reveals unexpected sub-modes of motion of DNA that deviate from expected random motion in the linear, diffusive regime. We demonstrate that a subtle interplay between these sub-modes causes the overall diffusive motion of DNA to appear to conform to the linear regime. Our results show that apparently random motion of macromolecules could be governed by non-random dynamics that are detectable only by their relative motion. Our analytical approach should advance broad understanding of diffusion processes of fundamental relevance.

Conserved linear dynamics of single-molecule Brownian motion

PubMed Central

Serag, Maged F.; Habuchi, Satoshi

2017-01-01

Macromolecular diffusion in homogeneous fluid at length scales greater than the size of the molecule is regarded as a random process. The mean-squared displacement (MSD) of molecules in this regime increases linearly with time. Here we show that non-random motion of DNA molecules in this regime that is undetectable by the MSD analysis can be quantified by characterizing the molecular motion relative to a latticed frame of reference. Our lattice occupancy analysis reveals unexpected sub-modes of motion of DNA that deviate from expected random motion in the linear, diffusive regime. We demonstrate that a subtle interplay between these sub-modes causes the overall diffusive motion of DNA to appear to conform to the linear regime. Our results show that apparently random motion of macromolecules could be governed by non-random dynamics that are detectable only by their relative motion. Our analytical approach should advance broad understanding of diffusion processes of fundamental relevance. PMID:28585925

Conserved linear dynamics of single-molecule Brownian motion

NASA Astrophysics Data System (ADS)

Serag, Maged F.; Habuchi, Satoshi

2017-06-01

Macromolecular diffusion in homogeneous fluid at length scales greater than the size of the molecule is regarded as a random process. The mean-squared displacement (MSD) of molecules in this regime increases linearly with time. Here we show that non-random motion of DNA molecules in this regime that is undetectable by the MSD analysis can be quantified by characterizing the molecular motion relative to a latticed frame of reference. Our lattice occupancy analysis reveals unexpected sub-modes of motion of DNA that deviate from expected random motion in the linear, diffusive regime. We demonstrate that a subtle interplay between these sub-modes causes the overall diffusive motion of DNA to appear to conform to the linear regime. Our results show that apparently random motion of macromolecules could be governed by non-random dynamics that are detectable only by their relative motion. Our analytical approach should advance broad understanding of diffusion processes of fundamental relevance.

The dynamics, structure, and conformational free energy of proline-containing antifreeze glycoprotein.

PubMed Central

Nguyen, Dat H; Colvin, Michael E; Yeh, Yin; Feeney, Robert E; Fink, William H

2002-01-01

Recent NMR studies of the solution structure of the 14-amino acid antifreeze glycoprotein AFGP-8 have concluded that the molecule lacks long-range order. The implication that an apparently unstructured molecule can still have a very precise function as a freezing inhibitor seems startling at first consideration. To gain insight into the nature of conformations and motions in AFGP-8, we have undertaken molecular dynamics simulations augmented with free energy calculations using a continuum solvation model. Starting from 10 different NMR structures, 20 ns of dynamics of AFGP were explored. The dynamics show that AFGP structure is composed of four segments, joined by very flexible pivots positioned at alanine 5, 8, and 11. The dynamics also show that the presence of prolines in this small AFGP structure facilitates the adoption of the poly-proline II structure as its overall conformation, although AFGP does adopt other conformations during the course of dynamics as well. The free energies calculated using a continuum solvation model show that the lowest free energy conformations, while being energetically equal, are drastically different in conformations. In other words, this AFGP molecule has many structurally distinct and energetically equal minima in its energy landscape. In addition, conformational, energetic, and hydrogen bond analyses suggest that the intramolecular hydrogen bonds between the N-acetyl group and the protein backbone are an important integral part of the overall stability of the AFGP molecule. The relevance of these findings to the mechanism of freezing inhibition is discussed. PMID:12023212

The dynamics, structure, and conformational free energy of proline-containing antifreeze glycoprotein.

PubMed

Nguyen, Dat H; Colvin, Michael E; Yeh, Yin; Feeney, Robert E; Fink, William H

2002-06-01

Recent NMR studies of the solution structure of the 14-amino acid antifreeze glycoprotein AFGP-8 have concluded that the molecule lacks long-range order. The implication that an apparently unstructured molecule can still have a very precise function as a freezing inhibitor seems startling at first consideration. To gain insight into the nature of conformations and motions in AFGP-8, we have undertaken molecular dynamics simulations augmented with free energy calculations using a continuum solvation model. Starting from 10 different NMR structures, 20 ns of dynamics of AFGP were explored. The dynamics show that AFGP structure is composed of four segments, joined by very flexible pivots positioned at alanine 5, 8, and 11. The dynamics also show that the presence of prolines in this small AFGP structure facilitates the adoption of the poly-proline II structure as its overall conformation, although AFGP does adopt other conformations during the course of dynamics as well. The free energies calculated using a continuum solvation model show that the lowest free energy conformations, while being energetically equal, are drastically different in conformations. In other words, this AFGP molecule has many structurally distinct and energetically equal minima in its energy landscape. In addition, conformational, energetic, and hydrogen bond analyses suggest that the intramolecular hydrogen bonds between the N-acetyl group and the protein backbone are an important integral part of the overall stability of the AFGP molecule. The relevance of these findings to the mechanism of freezing inhibition is discussed.

Conformational Changes of Trialanine in Water Induced by Vibrational Relaxation of the Amide I Mode.

PubMed

Bastida, Adolfo; Zúñiga, José; Requena, Alberto; Miguel, Beatriz; Candela, María Emilia; Soler, Miguel Angel

2016-01-21

Most of the protein-based diseases are caused by anomalies in the functionality and stability of these molecules. Experimental and theoretical studies of the conformational dynamics of proteins are becoming in this respect essential to understand the origin of these anomalies. However, a description of the conformational dynamics of proteins based on mechano-energetic principles still remains elusive because of the intrinsic high flexibility of the peptide chains, the participation of weak noncovalent interactions, and the role of the ubiquitous water solvent. In this work, the conformational dynamics of trialanine dissolved in water (D2O) is investigated through Molecular Dynamics (MD) simulations combined with instantaneous normal modes (INMs) analysis both at equilibrium and after the vibrational excitation of the C-terminal amide I mode. The conformational equilibrium between α and pPII conformers is found to be altered by the intramolecular relaxation of the amide I mode as a consequence of the different relaxation pathways of each conformer which modify the amount of vibrational energy stored in the torsional motions of the tripeptide, so the α → pPII and pPII → α conversion rates are increased differently. The selectivity of the process comes from the shifts of the vibrational frequencies with the conformational changes that modify the resonance conditions driving the intramolecular energy flows.

Glutamine Hydrolysis by Imidazole Glycerol Phosphate Synthase Displays Temperature Dependent Allosteric Activation

PubMed Central

Lisi, George P.; Currier, Allen A.; Loria, J. Patrick

2018-01-01

The enzyme imidazole glycerol phosphate synthase (IGPS) is a model for studies of long-range allosteric regulation in enzymes. Binding of the allosteric effector ligand N'-[5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) stimulates millisecond (ms) timescale motions in IGPS that enhance its catalytic function. We studied the effect of temperature on these critical conformational motions and the catalytic mechanism of IGPS from the hyperthermophile Thermatoga maritima in an effort to understand temperature-dependent allostery. Enzyme kinetic and NMR dynamics measurements show that apo and PRFAR-activated IGPS respond differently to changes in temperature. Multiple-quantum Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments performed at 303, 323, and 343 K (30, 50, and 70°C) reveal that millisecond flexibility is enhanced to a higher degree in apo IGPS than in the PRFAR-bound enzyme as the sample temperature is raised. We find that the flexibility of the apo enzyme is nearly identical to that of its PRFAR activated state at 343 K, whereas conformational motions are considerably different between these two forms of the enzyme at room temperature. Arrhenius analyses of these flexible sites show a varied range of activation energies that loosely correlate to allosteric communities identified by computational methods and reflect local changes in dynamics that may facilitate conformational sampling of the active conformation. In addition, kinetic assays indicate that allosteric activation by PRFAR decreases to 65-fold at 343 K, compared to 4,200-fold at 303 K, which mirrors the decreased effect of PRFAR on ms motions relative to the unactivated enzyme. These studies indicate that at the growth temperature of T. maritima, PFRAR is a weaker allosteric activator than it is at room temperature and illustrate that the allosteric mechanism of IGPS is temperature dependent. PMID:29468164

Measurement of energy landscape roughness of folded and unfolded proteins

PubMed Central

Milanesi, Lilia; Waltho, Jonathan P.; Hunter, Christopher A.; Shaw, Daniel J.; Beddard, Godfrey S.; Reid, Gavin D.; Dev, Sagarika; Volk, Martin

2012-01-01

The dynamics of protein conformational changes, from protein folding to smaller changes, such as those involved in ligand binding, are governed by the properties of the conformational energy landscape. Different techniques have been used to follow the motion of a protein over this landscape and thus quantify its properties. However, these techniques often are limited to short timescales and low-energy conformations. Here, we describe a general approach that overcomes these limitations. Starting from a nonnative conformation held by an aromatic disulfide bond, we use time-resolved spectroscopy to observe nonequilibrium backbone dynamics over nine orders of magnitude in time, from picoseconds to milliseconds, after photolysis of the disulfide bond. We find that the reencounter probability of residues that initially are in close contact decreases with time following an unusual power law that persists over the full time range and is independent of the primary sequence. Model simulations show that this power law arises from subdiffusional motion, indicating a wide distribution of trapping times in local minima of the energy landscape, and enable us to quantify the roughness of the energy landscape (4–5 kBT). Surprisingly, even under denaturing conditions, the energy landscape remains highly rugged with deep traps (>20 kBT) that result from multiple nonnative interactions and are sufficient for trapping on the millisecond timescale. Finally, we suggest that the subdiffusional motion of the protein backbone found here may promote rapid folding of proteins with low contact order by enhancing contact formation between nearby residues. PMID:23150572

Dynamics of partially folded and unfolded proteins investigated with quasielastic neutron spectroscopy

NASA Astrophysics Data System (ADS)

Stadler, Andreas M.

2018-05-01

Molecular dynamics in proteins animate and play a vital role for biologically relevant processes of these biomacromolecules. Quasielastic incoherent neutron scattering (QENS) is a well-suited experimental method to study protein dynamics from the picosecond to several nanoseconds and in the Ångström length-scale. In QENS experiments of protein solutions hydrogens act as reporters for the motions of methyl groups or amino acids to which they are bound. Neutron Spin-Echo spectroscopy (NSE) offers the highest energy resolution in the field of neutron spectroscopy and allows the study of slow collective motions in proteins up to several hundred nanoseconds and in the nanometer length-scale. In the following manuscript I will review recent studies that stress the relevance of molecular dynamics for protein folding and for conformational transitions of intrinsically disordered proteins (IDPs). During the folding collapse the protein is exploring its accessible conformational space via molecular motions. A large flexibility of partially folded and unfolded proteins, therefore, is mandatory for rapid protein folding. IDPs are a special case as they are largely unstructured under physiological conditions. A large flexibility is a characteristic property of IDPs as it allows, for example, the interaction with various binding partners or the rapid response to different conditions.

Characterizing RNA Dynamics at Atomic Resolution Using Solution-state NMR Spectroscopy

PubMed Central

Bothe, Jameson R.; Nikolova, Evgenia N.; Eichhorn, Catherine D.; Chugh, Jeetender; Hansen, Alexandar L.; Al-Hashimi, Hashim M.

2012-01-01

Many recently discovered non-coding RNAs do not fold into a single native conformation, but rather, sample many different conformations along their free energy landscape to carry out their biological function. Unprecedented insights into the RNA dynamic structure landscape are provided by solution-state NMR techniques that measure the structural, kinetic, and thermodynamic characteristics of motions spanning picosecond to second timescales at atomic resolution. From these studies a basic description of the RNA dynamic structure landscape is emerging, bringing new insights into how RNA structures change to carry out their function as well as applications in RNA-targeted drug discovery and RNA bioengineering. PMID:22036746

Single Molecule Measurement, a Tool for Exploring the Dynamic Mechanism of Biomolecules

NASA Astrophysics Data System (ADS)

Yanagida, Toshio

Biomolecules fluctuate in response to thermal agitation. These fluctuations are present at various biological levels ranging from single molecules to more complicated systems like perception. Despite thermal fluctuation often being considered noise, in some cases biomolecules actually utilize them to achieve function. How biomolecules do this is necessary to understand the mechanism underlying their function. Thermal noise causes fast, local motion in the time range of picosecond to nanosecond, which drives slower, collective motions [1]. These large, collective motions and conformational transitions are achieved in the time range of microsecond to millisecond, which is the time needed for a biomolecule to exceed its energy barrier in order to switch between two coordinates in its free-energy landscape. These slower conformational or state changes are likely rate limiting for biomolecule function.

Atomic-level characterization of the structural dynamics of proteins.

PubMed

Shaw, David E; Maragakis, Paul; Lindorff-Larsen, Kresten; Piana, Stefano; Dror, Ron O; Eastwood, Michael P; Bank, Joseph A; Jumper, John M; Salmon, John K; Shan, Yibing; Wriggers, Willy

2010-10-15

Molecular dynamics (MD) simulations are widely used to study protein motions at an atomic level of detail, but they have been limited to time scales shorter than those of many biologically critical conformational changes. We examined two fundamental processes in protein dynamics--protein folding and conformational change within the folded state--by means of extremely long all-atom MD simulations conducted on a special-purpose machine. Equilibrium simulations of a WW protein domain captured multiple folding and unfolding events that consistently follow a well-defined folding pathway; separate simulations of the protein's constituent substructures shed light on possible determinants of this pathway. A 1-millisecond simulation of the folded protein BPTI reveals a small number of structurally distinct conformational states whose reversible interconversion is slower than local relaxations within those states by a factor of more than 1000.

NMSim web server: integrated approach for normal mode-based geometric simulations of biologically relevant conformational transitions in proteins.

PubMed

Krüger, Dennis M; Ahmed, Aqeel; Gohlke, Holger

2012-07-01

The NMSim web server implements a three-step approach for multiscale modeling of protein conformational changes. First, the protein structure is coarse-grained using the FIRST software. Second, a rigid cluster normal-mode analysis provides low-frequency normal modes. Third, these modes are used to extend the recently introduced idea of constrained geometric simulations by biasing backbone motions of the protein, whereas side chain motions are biased toward favorable rotamer states (NMSim). The generated structures are iteratively corrected regarding steric clashes and stereochemical constraint violations. The approach allows performing three simulation types: unbiased exploration of conformational space; pathway generation by a targeted simulation; and radius of gyration-guided simulation. On a data set of proteins with experimentally observed conformational changes, the NMSim approach has been shown to be a computationally efficient alternative to molecular dynamics simulations for conformational sampling of proteins. The generated conformations and pathways of conformational transitions can serve as input to docking approaches or more sophisticated sampling techniques. The web server output is a trajectory of generated conformations, Jmol representations of the coarse-graining and a subset of the trajectory and data plots of structural analyses. The NMSim webserver, accessible at http://www.nmsim.de, is free and open to all users with no login requirement.

TALEs from a spring--superelasticity of Tal effector protein structures.

PubMed

Flechsig, Holger

2014-01-01

Transcription activator-like effectors (TALEs) are DNA-related proteins that recognise and bind specific target sequences to manipulate gene expression. Recently determined crystal structures show that their common architecture reveals a superhelical overall structure that may undergo drastic conformational changes. To establish a link between structure and dynamics in TALE proteins we have employed coarse-grained elastic-network modelling of currently available structural data and implemented a force-probe setup that allowed us to investigate their mechanical behaviour in computer experiments. Based on the measured force-extension curves we conclude that TALEs exhibit superelastic dynamical properties allowing for large-scale global conformational changes along their helical axis, which represents the soft direction in such proteins. For moderate external forcing the TALE models behave like linear springs, obeying Hooke's law, and the investigated structures can be characterised and compared by a corresponding spring constant. We show that conformational flexibility underlying the large-scale motions is not homogeneously distributed over the TALE structure, but instead soft spot residues around which strain is accumulated and which turn out to represent key agents in the transmission of conformational motions are identified. They correspond to the RVD loop residues that have been experimentally determined to play an eminent role in the binding process of target DNA.

TALEs from a Spring – Superelasticity of Tal Effector Protein Structures

PubMed Central

Flechsig, Holger

2014-01-01

Transcription activator-like effectors (TALEs) are DNA-related proteins that recognise and bind specific target sequences to manipulate gene expression. Recently determined crystal structures show that their common architecture reveals a superhelical overall structure that may undergo drastic conformational changes. To establish a link between structure and dynamics in TALE proteins we have employed coarse-grained elastic-network modelling of currently available structural data and implemented a force-probe setup that allowed us to investigate their mechanical behaviour in computer experiments. Based on the measured force-extension curves we conclude that TALEs exhibit superelastic dynamical properties allowing for large-scale global conformational changes along their helical axis, which represents the soft direction in such proteins. For moderate external forcing the TALE models behave like linear springs, obeying Hooke's law, and the investigated structures can be characterised and compared by a corresponding spring constant. We show that conformational flexibility underlying the large-scale motions is not homogeneously distributed over the TALE structure, but instead soft spot residues around which strain is accumulated and which turn out to represent key agents in the transmission of conformational motions are identified. They correspond to the RVD loop residues that have been experimentally determined to play an eminent role in the binding process of target DNA. PMID:25313859

Nucleotide-induced conformational dynamics in ABC transporters from structure-based coarse grained modelling.

NASA Astrophysics Data System (ADS)

Flechsig, Holger

2016-02-01

ATP-binding cassette (ABC) transporters are integral membrane proteins which mediate the exchange of diverse substrates across membranes powered by ATP molecules. Our understanding of their activity is still hampered since the conformational dynamics underlying the operation of such proteins cannot yet be resolved in detailed molecular dynamics studies. Here a coarse grained model which allows to mimic binding of nucleotides and follow subsequent conformational motions of full-length transporter structures in computer simulations is proposed and implemented. To justify its explanatory quality, the model is first applied to the maltose transporter system for which multiple conformations are known and we find that the model predictions agree remarkably well with the experimental data. For the MalK subunit the switching from open to the closed dimer configuration upon ATP binding is reproduced and, moreover, for the full-length maltose transporter, progression from inward-facing to the outward-facing state is correctly obtained. For the heme transporter HmuUV, for which only the free structure could yet be determined, the model was then applied to predict nucleotide-induced conformational motions. Upon binding of ATP-mimicking ligands the structure changed from a conformation in which the nucleotide-binding domains formed an open shape, to a conformation in which they were found in tight contact, while, at the same time, a pronounced rotation of the transmembrane domains was observed. This finding is supported by normal mode analysis, and, comparison with structural data of the homologous vitamin B12 transporter BtuCD suggests that the observed rotation mechanism may contribute a common functional aspect for this class of ABC transporters. Although in HmuuV noticeable rearrangement of essential transmembrane helices was detected, there are no indications from our simulations that ATP binding alone may facilitate propagation of substrate molecules in this transporter. Possible explanations are discussed in the light of currently debated transport scenarios of ABC transporters.

Coupling Protein Dynamics with Proton Transport in Human Carbonic Anhydrase II

PubMed Central

2016-01-01

The role of protein dynamics in enzyme catalysis is one of the most highly debated topics in enzymology. The main controversy centers around what may be defined as functionally significant conformational fluctuations and how, if at all, these fluctuations couple to enzyme catalyzed events. To shed light on this debate, the conformational dynamics along the transition path surmounting the highest free energy barrier have been herein investigated for the rate limiting proton transport event in human carbonic anhydrase (HCA) II. Special attention has been placed on whether the motion of an excess proton is correlated with fluctuations in the surrounding protein and solvent matrix, which may be rare on the picosecond and subpicosecond time scales of molecular motions. It is found that several active site residues, which do not directly participate in the proton transport event, have a significant impact on the dynamics of the excess proton. These secondary participants are shown to strongly influence the active site environment, resulting in the creation of water clusters that are conducive to fast, moderately slow, or slow proton transport events. The identification and characterization of these secondary participants illuminates the role of protein dynamics in the catalytic efficiency of HCA II. PMID:27063577

Dynamics, Conformational Entropy, and Frustration in Protein-Protein Interactions Involving an Intrinsically Disordered Protein Domain.

PubMed

Lindström, Ida; Dogan, Jakob

2018-05-18

Intrinsically disordered proteins (IDPs) are abundant in the eukaryotic proteome. However, little is known about the role of subnanosecond dynamics and the conformational entropy that it represents in protein-protein interactions involving IDPs. Using nuclear magnetic resonance side chain and backbone relaxation, stopped-flow kinetics, isothermal titration calorimetry, and computational studies, we have characterized the interaction between the globular TAZ1 domain of the CREB binding protein and the intrinsically disordered transactivation domain of STAT2 (TAD-STAT2). We show that the TAZ1/TAD-STAT2 complex retains considerable subnanosecond motions, with TAD-STAT2 undergoing only a partial disorder-to-order transition. We report here the first experimental determination of the conformational entropy change for both binding partners in an IDP binding interaction and find that the total change even exceeds in magnitude the binding enthalpy and is comparable to the contribution from the hydrophobic effect, demonstrating its importance in the binding energetics. Furthermore, we show that the conformational entropy change for TAZ1 is also instrumental in maintaining a biologically meaningful binding affinity. Strikingly, a spatial clustering of very high amplitude motions and a cluster of more rigid sites in the complex exist, which through computational studies we found to overlap with regions that experience energetic frustration and are less frustrated, respectively. Thus, the residual dynamics in the bound state could be necessary for faster dissociation, which is important for proteins that interact with multiple binding partners.

Relating protein conformational changes to packing efficiency and disorder

PubMed Central

Bhardwaj, Nitin; Gerstein, Mark

2009-01-01

Changes in protein conformation play key roles in facilitating various biochemical processes, ranging from signaling and phosphorylation to transport and catalysis. While various factors that drive these motions such as environmental changes and binding of small molecules are well understood, specific causative effects on the structural features of the protein due to these conformational changes have not been studied on a large scale. Here, we study protein conformational changes in relation to two key structural metrics: packing efficiency and disorder. Packing has been shown to be crucial for protein stability and function by many protein design and engineering studies. We study changes in packing efficiency during conformational changes, thus extending the analysis from a static context to a dynamic perspective and report some interesting observations. First, we study various proteins that adopt alternate conformations and find that tendencies to show motion and change in packing efficiency are correlated: residues that change their packing efficiency show larger motions. Second, our results suggest that residues that show higher changes in packing during motion are located on the changing interfaces which are formed during these conformational changes. These changing interfaces are slightly different from shear or static interfaces that have been analyzed in previous studies. Third, analysis of packing efficiency changes in the context of secondary structure shows that, as expected, residues buried in helices show the least change in packing efficiency, whereas those embedded in bends are most likely to change packing. Finally, by relating protein disorder to motions, we show that marginally disordered residues which are ordered enough to be crystallized but have sequence patterns indicative of disorder show higher dislocation and a higher change in packing than ordered ones and are located mostly on the changing interfaces. Overall, our results demonstrate that between the two conformations, the cores of the proteins remain mostly intact, whereas the interfaces display the most elasticity, both in terms of disorder and change in packing efficiency. By doing a variety of tests, we also show that our observations are robust to the solvation state of the proteins. PMID:19472340

Regulation of DNA conformations and dynamics in flows with hybrid field microfluidics.

PubMed

Ren, Fangfang; Zu, Yingbo; Kumar Rajagopalan, Kartik; Wang, Shengnian

2012-01-01

Visualizing single DNA dynamics in flow provides a wealth of physical insights in biophysics and complex flow study. However, large signal fluctuations, generated from diversified conformations, deformation history dependent dynamics and flow induced stochastic tumbling, often frustrate its wide adoption in single molecule and polymer flow study. We use a hybrid field microfluidic (HFM) approach, in which an electric field is imposed at desired locations and appropriate moments to balance the flow stress on charged molecules, to effectively regulate the initial conformations and the deformation dynamics of macromolecules in flow. With λ-DNA and a steady laminar shear flow as the model system, we herein studied the performance of HFM on regulating DNA trapping, relaxation, coil-stretch transition, and accumulation. DNA molecules were found to get captured in the focused planes when motions caused by flow, and the electric field were balanced. The trapped macromolecules relaxed in two different routes while eventually became more uniform in size and globule conformations. When removing the electric field, the sudden stretching dynamics of DNA molecules exhibited a more pronounced extension overshoot in their transient response under a true step function of flow stress while similar behaviors to what other pioneering work in steady shear flow. Such regulation strategies could be useful to control the conformations of other important macromolecules (e.g., proteins) and help better reveal their molecular dynamics.

A General, Adaptive, Roadmap-Based Algorithm for Protein Motion Computation.

PubMed

Molloy, Kevin; Shehu, Amarda

2016-03-01

Precious information on protein function can be extracted from a detailed characterization of protein equilibrium dynamics. This remains elusive in wet and dry laboratories, as function-modulating transitions of a protein between functionally-relevant, thermodynamically-stable and meta-stable structural states often span disparate time scales. In this paper we propose a novel, robotics-inspired algorithm that circumvents time-scale challenges by drawing analogies between protein motion and robot motion. The algorithm adapts the popular roadmap-based framework in robot motion computation to handle the more complex protein conformation space and its underlying rugged energy surface. Given known structures representing stable and meta-stable states of a protein, the algorithm yields a time- and energy-prioritized list of transition paths between the structures, with each path represented as a series of conformations. The algorithm balances computational resources between a global search aimed at obtaining a global view of the network of protein conformations and their connectivity and a detailed local search focused on realizing such connections with physically-realistic models. Promising results are presented on a variety of proteins that demonstrate the general utility of the algorithm and its capability to improve the state of the art without employing system-specific insight.

Conformational Plasticity of an Enzyme during Catalysis: Intricate Coupling between Cyclophilin A Dynamics and Substrate Turnover

PubMed Central

McGowan, Lauren C.; Hamelberg, Donald

2013-01-01

Enzyme catalysis is central to almost all biochemical processes, speeding up rates of reactions to biological relevant timescales. Enzymes make use of a large ensemble of conformations in recognizing their substrates and stabilizing the transition states, due to the inherent dynamical nature of biomolecules. The exact role of these diverse enzyme conformations and the interplay between enzyme conformational dynamics and catalysis is, according to the literature, not well understood. Here, we use molecular dynamics simulations to study human cyclophilin A (CypA), in order to understand the role of enzyme motions in the catalytic mechanism and recognition. Cyclophilin A is a tractable model system to study using classical simulation methods, because catalysis does not involve bond formation or breakage. We show that the conformational dynamics of active site residues of substrate-bound CypA is inherent in the substrate-free enzyme. CypA interacts with its substrate via conformational selection as the configurations of the substrate changes during catalysis. We also show that, in addition to tight intermolecular hydrophobic interactions between CypA and the substrate, an intricate enzyme-substrate intermolecular hydrogen-bonding network is extremely sensitive to the configuration of the substrate. These enzyme-substrate intermolecular interactions are loosely formed when the substrate is in the reactant and product states and become well formed and reluctant to break when the substrate is in the transition state. Our results clearly suggest coupling among enzyme-substrate intermolecular interactions, the dynamics of the enzyme, and the chemical step. This study provides further insights into the mechanism of peptidyl-prolyl cis/trans isomerases and the general interplay between enzyme conformational dynamics and catalysis. PMID:23332074

Conformational plasticity of an enzyme during catalysis: intricate coupling between cyclophilin A dynamics and substrate turnover.

PubMed

McGowan, Lauren C; Hamelberg, Donald

2013-01-08

Enzyme catalysis is central to almost all biochemical processes, speeding up rates of reactions to biological relevant timescales. Enzymes make use of a large ensemble of conformations in recognizing their substrates and stabilizing the transition states, due to the inherent dynamical nature of biomolecules. The exact role of these diverse enzyme conformations and the interplay between enzyme conformational dynamics and catalysis is, according to the literature, not well understood. Here, we use molecular dynamics simulations to study human cyclophilin A (CypA), in order to understand the role of enzyme motions in the catalytic mechanism and recognition. Cyclophilin A is a tractable model system to study using classical simulation methods, because catalysis does not involve bond formation or breakage. We show that the conformational dynamics of active site residues of substrate-bound CypA is inherent in the substrate-free enzyme. CypA interacts with its substrate via conformational selection as the configurations of the substrate changes during catalysis. We also show that, in addition to tight intermolecular hydrophobic interactions between CypA and the substrate, an intricate enzyme-substrate intermolecular hydrogen-bonding network is extremely sensitive to the configuration of the substrate. These enzyme-substrate intermolecular interactions are loosely formed when the substrate is in the reactant and product states and become well formed and reluctant to break when the substrate is in the transition state. Our results clearly suggest coupling among enzyme-substrate intermolecular interactions, the dynamics of the enzyme, and the chemical step. This study provides further insights into the mechanism of peptidyl-prolyl cis/trans isomerases and the general interplay between enzyme conformational dynamics and catalysis. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Direct Correlation of DNA Binding and Single Protein Domain Motion via Dual Illumination Fluorescence Microscopy

PubMed Central

2015-01-01

We report a dual illumination, single-molecule imaging strategy to dissect directly and in real-time the correlation between nanometer-scale domain motion of a DNA repair protein and its interaction with individual DNA substrates. The strategy was applied to XPD, an FeS cluster-containing DNA repair helicase. Conformational dynamics was assessed via FeS-mediated quenching of a fluorophore site-specifically incorporated into XPD. Simultaneously, binding of DNA molecules labeled with a spectrally distinct fluorophore was detected by colocalization of the DNA- and protein-derived signals. We show that XPD undergoes thermally driven conformational transitions that manifest in spatial separation of its two auxiliary domains. DNA binding does not strictly enforce a specific conformation. Interaction with a cognate DNA damage, however, stabilizes the compact conformation of XPD by increasing the weighted average lifetime of this state by 140% relative to an undamaged DNA. Our imaging strategy will be a valuable tool to study other FeS-containing nucleic acid processing enzymes. PMID:25204359

A principal component analysis of the dynamics of subdomains and binding sites in human serum albumin.

PubMed

Paris, Guillaume; Ramseyer, Christophe; Enescu, Mironel

2014-05-01

The conformational dynamics of human serum albumin (HSA) was investigated by principal component analysis (PCA) applied to three molecular dynamics trajectories of 200 ns each. The overlap of the essential subspaces spanned by the first 10 principal components (PC) of different trajectories was about 0.3 showing that the PCA based on a trajectory length of 200 ns is not completely convergent for this protein. The contributions of the relative motion of subdomains and of the subdomains (internal) distortion to the first 10 PCs were found to be comparable. Based on the distribution of the first 3 PC, 10 protein conformers are identified showing relative root mean square deviations (RMSD) between 2.3 and 4.6 Å. The main PCs are found to be delocalized over the whole protein structure indicating that the motions of different protein subdomains are coupled. This coupling is considered as being related to the allosteric effects observed upon ligand binding to HSA. On the other hand, the first PC of one of the three trajectories describes a conformational transition of the protein domain I that is close to that experimentally observed upon myristate binding. This is a theoretical support for the older hypothesis stating that changes of the protein onformation favorable to binding can precede the ligand complexation. A detailed all atoms PCA performed on the primary Sites 1 and 2 confirms the multiconformational character of the HSA binding sites as well as the significant coupling of their motions. Copyright © 2013 Wiley Periodicals, Inc.

QAARM: quasi-anharmonic autoregressive model reveals molecular recognition pathways in ubiquitin

PubMed Central

Savol, Andrej J.; Burger, Virginia M.; Agarwal, Pratul K.; Ramanathan, Arvind; Chennubhotla, Chakra S.

2011-01-01

Motivation: Molecular dynamics (MD) simulations have dramatically improved the atomistic understanding of protein motions, energetics and function. These growing datasets have necessitated a corresponding emphasis on trajectory analysis methods for characterizing simulation data, particularly since functional protein motions and transitions are often rare and/or intricate events. Observing that such events give rise to long-tailed spatial distributions, we recently developed a higher-order statistics based dimensionality reduction method, called quasi-anharmonic analysis (QAA), for identifying biophysically-relevant reaction coordinates and substates within MD simulations. Further characterization of conformation space should consider the temporal dynamics specific to each identified substate. Results: Our model uses hierarchical clustering to learn energetically coherent substates and dynamic modes of motion from a 0.5 μs ubiqutin simulation. Autoregressive (AR) modeling within and between states enables a compact and generative description of the conformational landscape as it relates to functional transitions between binding poses. Lacking a predictive component, QAA is extended here within a general AR model appreciative of the trajectory's temporal dependencies and the specific, local dynamics accessible to a protein within identified energy wells. These metastable states and their transition rates are extracted within a QAA-derived subspace using hierarchical Markov clustering to provide parameter sets for the second-order AR model. We show the learned model can be extrapolated to synthesize trajectories of arbitrary length. Contact: ramanathana@ornl.gov; chakracs@pitt.edu PMID:21685101

Apo adenylate kinase encodes its holo form: a principal component and varimax analysis.

PubMed

Cukier, Robert I

2009-02-12

Adenylate kinase undergoes large-scale motions of its LID and AMP-binding (AMPbd) domains when its apo, open form closes over its substrates, AMP and Mg2+-ATP. It may be an example of an enzyme that provides an ensemble of conformations in its apo state from which its substrates can select and bind to produce catalytically competent conformations. In this work, the fluctuations of the enzyme apo Escherichia coli adenylate kinase (AKE) are obtained with molecular dynamics. The resulting trajectory is analyzed with principal component analysis (PCA) that decomposes the atom motions into orthogonal modes ordered by their decreasing contributions to the total protein fluctuation. In apo AKE, a small set of the PCA modes describes the bulk of the fluctuations. Identification of the atom motions that are important contributors to these modes is improved with the use of a varimax rotation method that rotates the PCA modes to a new mode set that concentrates the atom contributions to a smaller set of atoms in these new modes. In this way, the nature of the important motions of the LID and AMPbd domains are clarified. The dominant PCA modes are used to investigate if apo AKE can fluctuate to conformations that are holo-like, even though the apo trajectory is mainly confined to a region around the initial apo structure. This is accomplished by expressing the difference between the protein coordinates, obtained from the holo and apo crystal structures, using as a basis the PCA modes from the apo AKE trajectory. The coherent motion described by a small set of the apo PCA modes is shown to be able to produce protein conformations that are quite similar to the holo conformation of the protein. In this sense, apo AKE does encode in its fluctuations information about holo-like conformations.

Hydrogen tunneling links protein dynamics to enzyme catalysis.

PubMed

Klinman, Judith P; Kohen, Amnon

2013-01-01

The relationship between protein dynamics and function is a subject of considerable contemporary interest. Although protein motions are frequently observed during ligand binding and release steps, the contribution of protein motions to the catalysis of bond making/breaking processes is more difficult to probe and verify. Here, we show how the quantum mechanical hydrogen tunneling associated with enzymatic C-H bond cleavage provides a unique window into the necessity of protein dynamics for achieving optimal catalysis. Experimental findings support a hierarchy of thermodynamically equilibrated motions that control the H-donor and -acceptor distance and active-site electrostatics, creating an ensemble of conformations suitable for H-tunneling. A possible extension of this view to methyl transfer and other catalyzed reactions is also presented. The impact of understanding these dynamics on the conceptual framework for enzyme activity, inhibitor/drug design, and biomimetic catalyst design is likely to be substantial.

Hydrogen Tunneling Links Protein Dynamics to Enzyme Catalysis

PubMed Central

Klinman, Judith P.; Kohen, Amnon

2014-01-01

The relationship between protein dynamics and function is a subject of considerable contemporary interest. Although protein motions are frequently observed during ligand binding and release steps, the contribution of protein motions to the catalysis of bond making/breaking processes is more difficult to probe and verify. Here, we show how the quantum mechanical hydrogen tunneling associated with enzymatic C–H bond cleavage provides a unique window into the necessity of protein dynamics for achieving optimal catalysis. Experimental findings support a hierarchy of thermodynamically equilibrated motions that control the H-donor and -acceptor distance and active-site electrostatics, creating an ensemble of conformations suitable for H-tunneling. A possible extension of this view to methyl transfer and other catalyzed reactions is also presented. The impact of understanding these dynamics on the conceptual framework for enzyme activity, inhibitor/drug design, and biomimetic catalyst design is likely to be substantial. PMID:23746260

Global and local molecular dynamics of a bacterial carboxylesterase provide insight into its catalytic mechanism

PubMed Central

Yu, Xiaozhen; Sigler, Sara C.; Hossain, Delwar; Wierdl, Monika; Gwaltney, Steven R.; Potter, Philip M.; Wadkins, Randy M.

2013-01-01

Carboxylesterases (CEs) are ubiquitous enzymes responsible for the detoxification of xenobiotics. In humans, substrates for these enzymes are far-ranging, and include the street drug heroin and the anticancer agent irinotecan. Hence, their ability to bind and metabolize substrates is of broad interest to biomedical science. In this study, we focused our attention on dynamic motions of a CE from B. subtilis (pnbCE), with emphasis on the question of what individual domains of the enzyme might contribute to its catalytic activity. We used a 10 ns all-atom molecular dynamics simulation, normal mode calculations, and enzyme kinetics to understand catalytic consequences of structural changes within this enzyme. Our results shed light on how molecular motions are coupled with catalysis. During molecular dynamics, we observed a distinct C-C bond rotation between two conformations of Glu310. Such a bond rotation would alternately facilitate and impede protonation of the active site His399 and act as a mechanism by which the enzyme alternates between its active and inactive conformation. Our normal mode results demonstrate that the distinct low-frequency motions of two loops in pnbCE, coil_5 and coil_21, are important in substrate conversion and seal the active site. Mutant CEs lacking these external loops show significantly reduced rates of substrate conversion, suggesting this sealing motion prevents escape of substrate. Overall, the results of our studies give new insight into the structure-function relationship of CEs and have implications for the entire family of α/β fold family of hydrolases, of which this CE is a member. PMID:22127613

Conformational dynamics of a crystalline protein from microsecond-scale molecular dynamics simulations and diffuse X-ray scattering.

PubMed

Wall, Michael E; Van Benschoten, Andrew H; Sauter, Nicholas K; Adams, Paul D; Fraser, James S; Terwilliger, Thomas C

2014-12-16

X-ray diffraction from protein crystals includes both sharply peaked Bragg reflections and diffuse intensity between the peaks. The information in Bragg scattering is limited to what is available in the mean electron density. The diffuse scattering arises from correlations in the electron density variations and therefore contains information about collective motions in proteins. Previous studies using molecular-dynamics (MD) simulations to model diffuse scattering have been hindered by insufficient sampling of the conformational ensemble. To overcome this issue, we have performed a 1.1-μs MD simulation of crystalline staphylococcal nuclease, providing 100-fold more sampling than previous studies. This simulation enables reproducible calculations of the diffuse intensity and predicts functionally important motions, including transitions among at least eight metastable states with different active-site geometries. The total diffuse intensity calculated using the MD model is highly correlated with the experimental data. In particular, there is excellent agreement for the isotropic component of the diffuse intensity, and substantial but weaker agreement for the anisotropic component. Decomposition of the MD model into protein and solvent components indicates that protein-solvent interactions contribute substantially to the overall diffuse intensity. We conclude that diffuse scattering can be used to validate predictions from MD simulations and can provide information to improve MD models of protein motions.

Sucrose in Aqueous Solution Revisited: 2. Adaptively Biased Molecular Dynamics Simulations and Computational Analysis of NMR Relaxation

PubMed Central

Xia, Junchao; Case, David A.

2012-01-01

We report 100 ns molecular dynamics simulations, at various temperatures, of sucrose in water (with concentrations of sucrose ranging from 0.02 to 4 M), and in a 7:3 water-DMSO mixture. Convergence of the resulting conformational ensembles was checked using adaptive-biased simulations along the glycosidic φ and ψ torsion angles. NMR relaxation parameters, including longitudinal (R1) and transverse (R2) relaxation rates, nuclear Overhauser enhancements (NOE), and generalized order parameter (S2) were computed from the resulting time-correlation functions. The amplitude and time scales of molecular motions change with temperature and concentration in ways that track closely with experimental results, and are consistent with a model in which sucrose conformational fluctuations are limited (with 80–90% of the conformations having φ – ψ values within 20° of an average conformation), but with some important differences in conformation between pure water and DMSO-water mixtures. PMID:22058066

Exploring protein kinase conformation using swarm-enhanced sampling molecular dynamics.

PubMed

Atzori, Alessio; Bruce, Neil J; Burusco, Kepa K; Wroblowski, Berthold; Bonnet, Pascal; Bryce, Richard A

2014-10-27

Protein plasticity, while often linked to biological function, also provides opportunities for rational design of selective and potent inhibitors of their function. The application of computational methods to the prediction of concealed protein concavities is challenging, as the motions involved can be significant and occur over long time scales. Here we introduce the swarm-enhanced sampling molecular dynamics (sesMD) method as a tool to improve sampling of conformational landscapes. In this approach, a swarm of replica simulations interact cooperatively via a set of pairwise potentials incorporating attractive and repulsive components. We apply the sesMD approach to explore the conformations of the DFG motif in the protein p38α mitogen-activated protein kinase. In contrast to multiple MD simulations, sesMD trajectories sample a range of DFG conformations, some of which map onto existing crystal structures. Simulated structures intermediate between the DFG-in and DFG-out conformations are predicted to have druggable pockets of interest for structure-based ligand design.

Structure Calculation and Reconstruction of Discrete-State Dynamics from Residual Dipolar Couplings.

PubMed

Cole, Casey A; Mukhopadhyay, Rishi; Omar, Hanin; Hennig, Mirko; Valafar, Homayoun

2016-04-12

Residual dipolar couplings (RDCs) acquired by nuclear magnetic resonance (NMR) spectroscopy are an indispensable source of information in investigation of molecular structures and dynamics. Here, we present a comprehensive strategy for structure calculation and reconstruction of discrete-state dynamics from RDC data that is based on the singular value decomposition (SVD) method of order tensor estimation. In addition to structure determination, we provide a mechanism of producing an ensemble of conformations for the dynamical regions of a protein from RDC data. The developed methodology has been tested on simulated RDC data with ±1 Hz of error from an 83 residue α protein (PDB ID 1A1Z ) and a 213 residue α/β protein DGCR8 (PDB ID 2YT4 ). In nearly all instances, our method reproduced the structure of the protein including the conformational ensemble to within less than 2 Å. On the basis of our investigations, arc motions with more than 30° of rotation are identified as internal dynamics and are reconstructed with sufficient accuracy. Furthermore, states with relative occupancies above 20% are consistently recognized and reconstructed successfully. Arc motions with a magnitude of 15° or relative occupancy of less than 10% are consistently unrecognizable as dynamical regions within the context of ±1 Hz of error.

Structural Plasticity and Conformational Transitions of HIV Envelope Glycoprotein gp120

PubMed Central

Korkut, Anil; Hendrickson, Wayne A.

2012-01-01

HIV envelope glycoproteins undergo large-scale conformational changes as they interact with cellular receptors to cause the fusion of viral and cellular membranes that permits viral entry to infect targeted cells. Conformational dynamics in HIV gp120 are also important in masking conserved receptor epitopes from being detected for effective neutralization by the human immune system. Crystal structures of HIV gp120 and its complexes with receptors and antibody fragments provide high-resolution pictures of selected conformational states accessible to gp120. Here we describe systematic computational analyses of HIV gp120 plasticity in such complexes with CD4 binding fragments, CD4 mimetic proteins, and various antibody fragments. We used three computational approaches: an isotropic elastic network analysis of conformational plasticity, a full atomic normal mode analysis, and simulation of conformational transitions with our coarse-grained virtual atom molecular mechanics (VAMM) potential function. We observe collective sub-domain motions about hinge points that coordinate those motions, correlated local fluctuations at the interfacial cavity formed when gp120 binds to CD4, and concerted changes in structural elements that form at the CD4 interface during large-scale conformational transitions to the CD4-bound state from the deformed states of gp120 in certain antibody complexes. PMID:23300605

Distributions of experimental protein structures on coarse-grained free energy landscapes

PubMed Central

Liu, Jie; Jernigan, Robert L.

2015-01-01

Predicting conformational changes of proteins is needed in order to fully comprehend functional mechanisms. With the large number of available structures in sets of related proteins, it is now possible to directly visualize the clusters of conformations and their conformational transitions through the use of principal component analysis. The most striking observation about the distributions of the structures along the principal components is their highly non-uniform distributions. In this work, we use principal component analysis of experimental structures of 50 diverse proteins to extract the most important directions of their motions, sample structures along these directions, and estimate their free energy landscapes by combining knowledge-based potentials and entropy computed from elastic network models. When these resulting motions are visualized upon their coarse-grained free energy landscapes, the basis for conformational pathways becomes readily apparent. Using three well-studied proteins, T4 lysozyme, serum albumin, and sarco-endoplasmic reticular Ca2+ adenosine triphosphatase (SERCA), as examples, we show that such free energy landscapes of conformational changes provide meaningful insights into the functional dynamics and suggest transition pathways between different conformational states. As a further example, we also show that Monte Carlo simulations on the coarse-grained landscape of HIV-1 protease can directly yield pathways for force-driven conformational changes. PMID:26723638

Cosolvent-Based Molecular Dynamics for Ensemble Docking: Practical Method for Generating Druggable Protein Conformations.

PubMed

Uehara, Shota; Tanaka, Shigenori

2017-04-24

Protein flexibility is a major hurdle in current structure-based virtual screening (VS). In spite of the recent advances in high-performance computing, protein-ligand docking methods still demand tremendous computational cost to take into account the full degree of protein flexibility. In this context, ensemble docking has proven its utility and efficiency for VS studies, but it still needs a rational and efficient method to select and/or generate multiple protein conformations. Molecular dynamics (MD) simulations are useful to produce distinct protein conformations without abundant experimental structures. In this study, we present a novel strategy that makes use of cosolvent-based molecular dynamics (CMD) simulations for ensemble docking. By mixing small organic molecules into a solvent, CMD can stimulate dynamic protein motions and induce partial conformational changes of binding pocket residues appropriate for the binding of diverse ligands. The present method has been applied to six diverse target proteins and assessed by VS experiments using many actives and decoys of DEKOIS 2.0. The simulation results have revealed that the CMD is beneficial for ensemble docking. Utilizing cosolvent simulation allows the generation of druggable protein conformations, improving the VS performance compared with the use of a single experimental structure or ensemble docking by standard MD with pure water as the solvent.

Insight into microtubule destabilization mechanism of 3,4,5-trimethoxyphenyl indanone derivatives using molecular dynamics simulation and conformational modes analysis

NASA Astrophysics Data System (ADS)

Tripathi, Shubhandra; Srivastava, Gaurava; Singh, Aastha; Prakasham, A. P.; Negi, Arvind S.; Sharma, Ashok

2018-03-01

Colchicine site inhibitors are microtubule destabilizers having promising role in cancer therapeutics. In the current study, four such indanone derivatives (t1, t9, t14 and t17) with 3,4,5-trimethoxyphenyl fragment (ring A) and showing significant microtubule destabilization property have been explored. The interaction mechanism and conformational modes triggered by binding of these indanone derivatives and combretastatin at colchicine binding site (CBS) of αβ-tubulin dimer were studied using molecular dynamics (MD) simulation, principle component analysis and free energy landscape analysis. In the MD results, t1 showed binding similar to colchicine interacting in the deep hydrophobic core at the CBS. While t9, t14 and t17 showed binding conformation similar to combretastatin, with ring A superficially binding at the CBS. Results demonstrated that ring A played a vital role in binding via hydrophobic interactions and got anchored between the S8 and S9 sheets, H8 helix and T7 loop at the CBS. Conformational modes study revealed that twisting and bending conformational motions (as found in the apo system) were nearly absent in the ligand bound systems. Absence of twisting motion might causes loss of lateral contacts in microtubule, thus promoting microtubule destabilization. This study provides detailed account of microtubule destabilization mechanism by indanone ligands and combretastatin, and would be helpful for designing microtubule destabilizers with higher activity.

New open conformation of SMYD3 implicates conformational selection and allostery

PubMed Central

Spellmon, Nicholas; Sun, Xiaonan; Xue, Wen; Holcomb, Joshua; Chakravarthy, Srinivas; Shang, Weifeng; Edwards, Brian; Sirinupong, Nualpun; Li, Chunying; Yang, Zhe

2016-01-01

SMYD3 plays a key role in cancer cell viability, adhesion, migration and invasion. SMYD3 promotes formation of inducible regulatory T cells and is involved in reducing autoimmunity. However, the nearly “closed” substrate-binding site and poor in vitro H3K4 methyltransferase activity have obscured further understanding of this oncogenically related protein. Here we reveal that SMYD3 can adopt an “open” conformation using molecular dynamics simulation and small-angle X-ray scattering. This ligand-binding-capable open state is related to the crystal structure-like closed state by a striking clamshell-like inter-lobe dynamics. The two states are characterized by many distinct structural and dynamical differences and the conformational transition pathway is mediated by a reversible twisting motion of the C-terminal domain (CTD). The spontaneous transition from the closed to open states suggests two possible, mutually non-exclusive models for SMYD3 functional regulation and the conformational selection mechanism and allostery may regulate the catalytic or ligand binding competence of SMYD3. This study provides an immediate clue to the puzzling role of SMYD3 in epigenetic gene regulation. PMID:28050603

Conformational Dynamics of Mechanically Compliant DNA Nanostructures from Coarse-Grained Molecular Dynamics Simulations.

PubMed

Shi, Ze; Castro, Carlos E; Arya, Gaurav

2017-05-23

Structural DNA nanotechnology, the assembly of rigid 3D structures of complex yet precise geometries, has recently been used to design dynamic, mechanically compliant nanostructures with tunable equilibrium conformations and conformational distributions. Here we use coarse-grained molecular dynamics simulations to provide insights into the conformational dynamics of a set of mechanically compliant DNA nanostructures-DNA hinges that use single-stranded DNA "springs" to tune the equilibrium conformation of a layered double-stranded DNA "joint" connecting two stiff "arms" constructed from DNA helix bundles. The simulations reproduce the experimentally measured equilibrium angles between hinge arms for a range of hinge designs. The hinges are found to be structurally stable, except for some fraying of the open ends of the DNA helices comprising the hinge arms and some loss of base-pairing interactions in the joint regions coinciding with the crossover junctions, especially in hinges designed to exhibit a small bending angle that exhibit large local stresses resulting in strong kinks in their joints. Principal component analysis reveals that while the hinge dynamics are dominated by bending motion, some twisting and sliding of hinge arms relative to each other also exists. Forced deformation of the hinges reveals distinct bending mechanisms for hinges with short, inextensible springs versus those with longer, more extensible springs. Lastly, we introduce an approach for rapidly predicting equilibrium hinge angles from individual force-deformation behaviors of its single- and double-stranded DNA components. Taken together, these results demonstrate that coarse-grained modeling is a promising approach for designing, predicting, and studying the dynamics of compliant DNA nanostructures, where conformational fluctuations become important, multiple deformation mechanisms exist, and continuum approaches may not yield accurate properties.

Markov State Models Provide Insights into Dynamic Modulation of Protein Function

PubMed Central

2015-01-01

Conspectus Protein function is inextricably linked to protein dynamics. As we move from a static structural picture to a dynamic ensemble view of protein structure and function, novel computational paradigms are required for observing and understanding conformational dynamics of proteins and its functional implications. In principle, molecular dynamics simulations can provide the time evolution of atomistic models of proteins, but the long time scales associated with functional dynamics make it difficult to observe rare dynamical transitions. The issue of extracting essential functional components of protein dynamics from noisy simulation data presents another set of challenges in obtaining an unbiased understanding of protein motions. Therefore, a methodology that provides a statistical framework for efficient sampling and a human-readable view of the key aspects of functional dynamics from data analysis is required. The Markov state model (MSM), which has recently become popular worldwide for studying protein dynamics, is an example of such a framework. In this Account, we review the use of Markov state models for efficient sampling of the hierarchy of time scales associated with protein dynamics, automatic identification of key conformational states, and the degrees of freedom associated with slow dynamical processes. Applications of MSMs for studying long time scale phenomena such as activation mechanisms of cellular signaling proteins has yielded novel insights into protein function. In particular, from MSMs built using large-scale simulations of GPCRs and kinases, we have shown that complex conformational changes in proteins can be described in terms of structural changes in key structural motifs or “molecular switches” within the protein, the transitions between functionally active and inactive states of proteins proceed via multiple pathways, and ligand or substrate binding modulates the flux through these pathways. Finally, MSMs also provide a theoretical toolbox for studying the effect of nonequilibrium perturbations on conformational dynamics. Considering that protein dynamics in vivo occur under nonequilibrium conditions, MSMs coupled with nonequilibrium statistical mechanics provide a way to connect cellular components to their functional environments. Nonequilibrium perturbations of protein folding MSMs reveal the presence of dynamically frozen glass-like states in their conformational landscape. These frozen states are also observed to be rich in β-sheets, which indicates their possible role in the nucleation of β-sheet rich aggregates such as those observed in amyloid-fibril formation. Finally, we describe how MSMs have been used to understand the dynamical behavior of intrinsically disordered proteins such as amyloid-β, human islet amyloid polypeptide, and p53. While certainly not a panacea for studying functional dynamics, MSMs provide a rigorous theoretical foundation for understanding complex entropically dominated processes and a convenient lens for viewing protein motions. PMID:25625937

Studying the role of protein dynamics in an SN2 enzyme reaction using free-energy surfaces and solvent coordinates

NASA Astrophysics Data System (ADS)

García-Meseguer, Rafael; Martí, Sergio; Ruiz-Pernía, J. Javier; Moliner, Vicent; Tuñón, Iñaki

2013-07-01

Conformational changes are known to be able to drive an enzyme through its catalytic cycle, allowing, for example, substrate binding or product release. However, the influence of protein motions on the chemical step is a controversial issue. One proposal is that the simple equilibrium fluctuations incorporated into transition-state theory are insufficient to account for the catalytic effect of enzymes and that protein motions should be treated dynamically. Here, we propose the use of free-energy surfaces, obtained as a function of both a chemical coordinate and an environmental coordinate, as an efficient way to elucidate the role of protein structure and motions during the reaction. We show that the structure of the protein provides an adequate environment for the progress of the reaction, although a certain degree of flexibility is needed to attain the full catalytic effect. However, these motions do not introduce significant dynamical corrections to the rate constant and can be described as equilibrium fluctuations.

The principal motions involved in the coupling mechanism of the recovery stroke of the myosin motor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mesentean, Sidonia; Koppole, Sampath; Smith, Jeremy C

2006-12-01

Muscle contraction is driven by a cycle of conformational changes in the myosin II head. After myosin binds ATP and releases from the actin fibril, myosin prepares for the next power stroke by rotating back the converter domain that carries the lever arm by {approx}60 degrees. This recovery stroke is coupled to the activation of myosin's ATPase by a mechanism that is essential for an efficient motor cycle. The mechanics of this coupling have been proposed to occur via two distinct and successive motions of the two helices that hold the converter domain: in a first phase a see-saw motionmore » of the relay helix, followed by a piston/seesaw motion of the SH1 helix in a second phase. To test this model, we have determined the principal motions of these structural elements during equilibrium molecular dynamics simulations of the crystallographic end states of the recovery stroke by using Principal Component Analysis. This reveals that the only principal motions of these two helices that make a large amplitude contribution towards the conformational change of the recovery stroke are indeed the predicted seesaw and piston motions.« less

Lid opening and conformational stability of T1 Lipase is mediated by increasing chain length polar solvents

PubMed Central

Mohamad Ali, Mohd Shukuri; Salleh, Abu Bakar; Rahman, Raja Noor Zaliha Raja Abd; Normi, Yahaya M.; Mohd Shariff, Fairolniza

2017-01-01

The dynamics and conformational landscape of proteins in organic solvents are events of potential interest in nonaqueous process catalysis. Conformational changes, folding transitions, and stability often correspond to structural rearrangements that alter contacts between solvent molecules and amino acid residues. However, in nonaqueous enzymology, organic solvents limit stability and further application of proteins. In the present study, molecular dynamics (MD) of a thermostable Geobacillus zalihae T1 lipase was performed in different chain length polar organic solvents (methanol, ethanol, propanol, butanol, and pentanol) and water mixture systems to a concentration of 50%. On the basis of the MD results, the structural deviations of the backbone atoms elucidated the dynamic effects of water/organic solvent mixtures on the equilibrium state of the protein simulations in decreasing solvent polarity. The results show that the solvent mixture gives rise to deviations in enzyme structure from the native one simulated in water. The drop in the flexibility in H2O, MtOH, EtOH and PrOH simulation mixtures shows that greater motions of residues were influenced in BtOH and PtOH simulation mixtures. Comparing the root mean square fluctuations value with the accessible solvent area (SASA) for every residue showed an almost correspondingly high SASA value of residues to high flexibility and low SASA value to low flexibility. The study further revealed that the organic solvents influenced the formation of more hydrogen bonds in MtOH, EtOH and PrOH and thus, it is assumed that increased intraprotein hydrogen bonding is ultimately correlated to the stability of the protein. However, the solvent accessibility analysis showed that in all solvent systems, hydrophobic residues were exposed and polar residues tended to be buried away from the solvent. Distance variation of the tetrahedral intermediate packing of the active pocket was not conserved in organic solvent systems, which could lead to weaknesses in the catalytic H-bond network and most likely a drop in catalytic activity. The conformational variation of the lid domain caused by the solvent molecules influenced its gradual opening. Formation of additional hydrogen bonds and hydrophobic interactions indicates that the contribution of the cooperative network of interactions could retain the stability of the protein in some solvent systems. Time-correlated atomic motions were used to characterize the correlations between the motions of the atoms from atomic coordinates. The resulting cross-correlation map revealed that the organic solvent mixtures performed functional, concerted, correlated motions in regions of residues of the lid domain to other residues. These observations suggest that varying lengths of polar organic solvents play a significant role in introducing dynamic conformational diversity in proteins in a decreasing order of polarity. PMID:28533982

Investigation of structural dynamics of Thrombocytopenia Cargeeg mutants of human apoptotic cytochrome c: A molecular dynamics simulation approach.

PubMed

Muneeswaran, Gurusamy; Kartheeswaran, Subramanian; Pandiaraj, Manickam; Muthukumar, Kaliappan; Sankaralingam, Muniyandi; Arunachalam, Saravanavadivu

2017-11-01

Naturally occurring mutations to cytochrome c (cyt-c) have been identified recently in patients with mild autosomal dominant thrombocytopenia (low platelet levels), which yield cyt-c mutants with enhanced apoptotic activity. However, the molecular mechanism underlying this low platelet production and enhanced apoptosis remain unclear. Therefore, an attempt is made herein for the first time to investigate the effects of mutations of glycine 41 by serine (G41S) and tyrosine 48 by histidine (Y48H) on the conformational and dynamic changes of apoptotic (Fe 3+ ) cyt-c using all atom molecular dynamics (MD) simulations in explicit water solvent. Our 30ns MD simulations demonstrate considerable structural differences in G41S and Y48H compared to wild type (WT) cyt-c, such as increasing distances between the critical electron transfer residues results in open conformation at the heme active site, large fluctuations in β-turns and α-helices. Additionally, although the β-sheets remain mostly unaffected in all the three cyt-c simulations, the α-helices undergo conformational switch to β-turns in both the mutant simulations. Importantly, this conformational switch of α-helix to β-turn around heme active site should attributes to the loss of intraprotein H-bonds in the mutant simulations especially between NE2 (His26) and O (Pro44) in agreement with the experimental report. Further, essential dynamics analysis reveals that overall motions of WT cyt-c is mainly involved only in the first eigenvector, but in G41S and Y48H the overall motions are mainly in three and two eigenvectors respectively. Overall, the detailed atomistic level information provide a unifying description for the molecular mechanism of structural destabilization, disregulation of platelet formation and enhanced peroxidase activity of the mutant cyt-c's in the pathology of intrinsic apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Myosin isoform determines the conformational dynamics and cooperativity of actin filaments in the strongly bound actomyosin complex

PubMed Central

Prochniewicz, Ewa; Chin, Harvey F.; Henn, Arnon; Hannemann, Diane E.; Olivares, Adrian O.; Thomas, David D.; De La Cruz, Enrique M.

2010-01-01

SUMMARY We have used transient phosphorescence anisotropy (TPA) to detect the microsecond rotational dynamics of erythrosin iodoacetamide (ErIA)-labeled actin strongly bound to single-headed fragments of muscle myosin (muscle S1) and non-muscle myosin V (MV). The conformational dynamics of actin filaments in solution are markedly influenced by the isoform of bound myosin. Both myosins increase the final anisotropy of actin at sub-stoichiometric binding densities, indicating long-range, non-nearest neighbor cooperative restriction of filament rotational dynamics amplitude, but the cooperative unit is larger with MV than muscle S1. Both myosin isoforms also cooperatively affect the actin filament rotational correlation time, but with opposite effects; muscle S1 decreases rates of intrafilament torsional motion, while binding of MV increases the rates of motion. The cooperative effects on the rates of intrafilament motions correlate with the kinetics of myosin binding to actin filaments such that MV binds more rapidly, and muscle myosin more slowly, to partially decorated filaments than to bare filaments. The two isoforms also differ in their effects on the phosphorescence lifetime of the actin-bound ErIA; while muscle S1 increases the lifetime, suggesting decreased aqueous exposure of the probe, MV does not induce a significant change. We conclude that the dynamics and structure of actin in the strongly bound actomyosin complex is determined by the isoform of the bound myosin, in a manner likely to accommodate the diverse functional roles of actomyosin in muscle and non-muscle cells. PMID:19962990

Glutamate 338 is an electrostatic facilitator of C–Co bond breakage in a dynamic/electrostatic model of catalysis by ornithine aminomutase

PubMed Central

Menon, Binuraj R K; Menon, Navya; Fisher, Karl; Rigby, Stephen E J; Leys, David; Scrutton, Nigel S

2015-01-01

How cobalamin-dependent enzymes promote C–Co homolysis to initiate radical catalysis has been debated extensively. For the pyridoxal 5′-phosphate and cobalamin-dependent enzymes lysine 5,6-aminomutase and ornithine 4,5-aminomutase (OAM), large-scale re-orientation of the cobalamin-binding domain linked to C–Co bond breakage has been proposed. In these models, substrate binding triggers dynamic sampling of the B12-binding Rossmann domain to achieve a catalytically competent ‘closed’ conformational state. In ‘closed’ conformations of OAM, Glu338 is thought to facilitate C–Co bond breakage by close association with the cobalamin adenosyl group. We investigated this using stopped-flow continuous-wave photolysis, viscosity dependence kinetic measurements, and electron paramagnetic resonance spectroscopy of a series of Glu338 variants. We found that substrate-induced C–Co bond homolysis is compromised in Glu388 variant forms of OAM, although photolysis of the C–Co bond is not affected by the identity of residue 338. Electrostatic interactions of Glu338 with the 5′-deoxyadenosyl group of B12 potentiate C–Co bond homolysis in ‘closed’ conformations only; these conformations are unlocked by substrate binding. Our studies extend earlier models that identified a requirement for large-scale motion of the cobalamin domain. Our findings indicate that large-scale motion is required to pre-organize the active site by enabling transient formation of ‘closed’ conformations of OAM. In ‘closed’ conformations, Glu338 interacts with the 5′-deoxyadenosyl group of cobalamin. This interaction is required to potentiate C–Co homolysis, and is a crucial component of the approximately 1012 rate enhancement achieved by cobalamin-dependent enzymes for C–Co bond homolysis. PMID:25627283

Molecular Dynamics, Monte Carlo Simulations, and Langevin Dynamics: A Computational Review

PubMed Central

Paquet, Eric; Viktor, Herna L.

2015-01-01

Macromolecular structures, such as neuraminidases, hemagglutinins, and monoclonal antibodies, are not rigid entities. Rather, they are characterised by their flexibility, which is the result of the interaction and collective motion of their constituent atoms. This conformational diversity has a significant impact on their physicochemical and biological properties. Among these are their structural stability, the transport of ions through the M2 channel, drug resistance, macromolecular docking, binding energy, and rational epitope design. To assess these properties and to calculate the associated thermodynamical observables, the conformational space must be efficiently sampled and the dynamic of the constituent atoms must be simulated. This paper presents algorithms and techniques that address the abovementioned issues. To this end, a computational review of molecular dynamics, Monte Carlo simulations, Langevin dynamics, and free energy calculation is presented. The exposition is made from first principles to promote a better understanding of the potentialities, limitations, applications, and interrelations of these computational methods. PMID:25785262

Integrative, Dynamic Structural Biology at Atomic Resolution—It’s About Time

PubMed Central

van den Bedem, Henry; Fraser, James S.

2015-01-01

Biomolecules adopt a dynamic ensemble of conformations, each with the potential to interact with binding partners or perform the chemical reactions required for a multitude of cellular functions. Recent advances in X-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy, and other techniques are helping us realize the dream of seeing—in atomic detail—how different parts of biomolecules exchange between functional sub-states using concerted motions. Integrative structural biology has advanced our understanding of the formation of large macromolecular complexes and how their components interact in assemblies by leveraging data from many low-resolution methods. Here, we review the growing opportunities for integrative, dynamic structural biology at the atomic scale, contending there is increasing synergistic potential between X-ray crystallography, NMR, and computer simulations to reveal a structural basis for protein conformational dynamics at high resolution. PMID:25825836

Information flow and protein dynamics: the interplay between nuclear magnetic resonance spectroscopy and molecular dynamics simulations

PubMed Central

Pastor, Nina; Amero, Carlos

2015-01-01

Proteins participate in information pathways in cells, both as links in the chain of signals, and as the ultimate effectors. Upon ligand binding, proteins undergo conformation and motion changes, which can be sensed by the following link in the chain of information. Nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations represent powerful tools for examining the time-dependent function of biological molecules. The recent advances in NMR and the availability of faster computers have opened the door to more detailed analyses of structure, dynamics, and interactions. Here we briefly describe the recent applications that allow NMR spectroscopy and MD simulations to offer unique insight into the basic motions that underlie information transfer within and between cells. PMID:25999971

Computer simulations reveal changes in the conformational space of the transcriptional regulator MosR upon the formation of a disulphide bond and in the collective motions that regulate its DNA-binding affinity

PubMed Central

Câmara, Amanda Souza

2018-01-01

M. tuberculosis oxidation sense Regulator (MosR) is a transcriptional regulator from Mycobacterium tuberculosis. It senses the environment oxidation and regulates the expression of a secreted oxidoreductase, thus defending the bacilli against oxidative stress from the phagosome. While most of the members of the Multiple antibiotics resistance Regulator (MarR) family are ligand-responsive, MosR may dissociate from its DNA site upon formation of an intrachain disulphide bond. However, the structure of MosR in its oxidized state is not known, and it is not clear how the formation of this disulphide bond would lead to the conformational changes required for dissociation of the DNA. Nonetheless, MosR presents two crystallographically resolved conformations in its reduced state: bound and unbound to DNA. We managed to simulate MosR unbound to the DNA, both in the presence and in the absence of the disulphide bond. Our results indicate that this disulphide bond precludes the N-terminal residues from adopting a conformation that stands in-between the helix α1 and the DNA binding domain (DBD) from the other chain. Once this conformation is achieved in the reduced state, this DBD detaches from the dimerization domain and becomes more flexible, being able to perform motions with higher amplitude and higher degree of collectivity. Only then, MosR may achieve a conformation where its recognition helices fit into the major grooves of its DNA site. The analysis of the collective motions performed by MosR, during the different situations sampled by the molecular dynamics (MDs), was only possible by the method of filtering harmonic modes with specific frequencies. The frequency of the collective motions performed by the DBD of MosR in the reduced state to achieve a DNA-binding conformation is in the range of 20 to 50 MHz, but it may be associated to more sporadic events since it requires the combination of a suitable conformation of the N-terminal residues. PMID:29470546

Computer simulations reveal changes in the conformational space of the transcriptional regulator MosR upon the formation of a disulphide bond and in the collective motions that regulate its DNA-binding affinity.

PubMed

Câmara, Amanda Souza; Horjales, Eduardo

2018-01-01

M. tuberculosis oxidation sense Regulator (MosR) is a transcriptional regulator from Mycobacterium tuberculosis. It senses the environment oxidation and regulates the expression of a secreted oxidoreductase, thus defending the bacilli against oxidative stress from the phagosome. While most of the members of the Multiple antibiotics resistance Regulator (MarR) family are ligand-responsive, MosR may dissociate from its DNA site upon formation of an intrachain disulphide bond. However, the structure of MosR in its oxidized state is not known, and it is not clear how the formation of this disulphide bond would lead to the conformational changes required for dissociation of the DNA. Nonetheless, MosR presents two crystallographically resolved conformations in its reduced state: bound and unbound to DNA. We managed to simulate MosR unbound to the DNA, both in the presence and in the absence of the disulphide bond. Our results indicate that this disulphide bond precludes the N-terminal residues from adopting a conformation that stands in-between the helix α1 and the DNA binding domain (DBD) from the other chain. Once this conformation is achieved in the reduced state, this DBD detaches from the dimerization domain and becomes more flexible, being able to perform motions with higher amplitude and higher degree of collectivity. Only then, MosR may achieve a conformation where its recognition helices fit into the major grooves of its DNA site. The analysis of the collective motions performed by MosR, during the different situations sampled by the molecular dynamics (MDs), was only possible by the method of filtering harmonic modes with specific frequencies. The frequency of the collective motions performed by the DBD of MosR in the reduced state to achieve a DNA-binding conformation is in the range of 20 to 50 MHz, but it may be associated to more sporadic events since it requires the combination of a suitable conformation of the N-terminal residues.

Conformational dynamics of a crystalline protein from microsecond-scale molecular dynamics simulations and diffuse X-ray scattering

DOE PAGES

Wall, Michael E.; Van Benschoten, Andrew H.; Sauter, Nicholas K.; ...

2014-12-01

X-ray diffraction from protein crystals includes both sharply peaked Bragg reflections and diffuse intensity between the peaks. The information in Bragg scattering is limited to what is available in the mean electron density. The diffuse scattering arises from correlations in the electron density variations and therefore contains information about collective motions in proteins. Previous studies using molecular-dynamics (MD) simulations to model diffuse scattering have been hindered by insufficient sampling of the conformational ensemble. To overcome this issue, we have performed a 1.1-μs MD simulation of crystalline staphylococcal nuclease, providing 100-fold more sampling than previous studies. This simulation enables reproducible calculationsmore » of the diffuse intensity and predicts functionally important motions, including transitions among at least eight metastable states with different active-site geometries. The total diffuse intensity calculated using the MD model is highly correlated with the experimental data. In particular, there is excellent agreement for the isotropic component of the diffuse intensity, and substantial but weaker agreement for the anisotropic component. The decomposition of the MD model into protein and solvent components indicates that protein–solvent interactions contribute substantially to the overall diffuse intensity. In conclusion, diffuse scattering can be used to validate predictions from MD simulations and can provide information to improve MD models of protein motions.« less

Activity loss by H46A mutation in Mycobacterium tuberculosis isocitrate lyase is due to decrease in structural plasticity and collective motions of the active site.

PubMed

Shukla, Rohit; Shukla, Harish; Tripathi, Timir

2018-01-01

Mycobacterium tuberculosis isocitrate lyase (MtbICL) is a crucial enzyme of the glyoxylate cycle and is a validated anti-tuberculosis drug target. Structurally distant, non-active site mutation (H46A) in MtbICL has been found to cause loss of enzyme activity. The aim of the present work was to explore the structural alterations induced by H46A mutation that caused the loss of enzyme activity. The structural and dynamic consequences of H46A mutation were studied using multiple computational methods such as docking, molecular dynamics simulation and residue interaction network analysis (RIN). Principal component analysis and cross correlation analysis revealed the difference in conformational flexibility and collective modes of motions between the wild-type and mutant enzyme, particularly in the active site region. RIN analysis revealed that the active site geometry was disturbed in the mutant enzyme. Thus, the dynamic perturbation of the active site led to enzyme transition from its active form to inactive form upon mutation. The computational analyses elucidated the mutant-specific conformational alterations, differential dominant motions, and anomalous residue level interactions that contributed to the abrogated function of mutant MtbICL. An understanding of interactions of mutant enzymes may help in modifying the existing drugs and designing improved drugs for successful control of tuberculosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Conformational dynamics of a crystalline protein from microsecond-scale molecular dynamics simulations and diffuse X-ray scattering

PubMed Central

Wall, Michael E.; Van Benschoten, Andrew H.; Sauter, Nicholas K.; Adams, Paul D.; Fraser, James S.; Terwilliger, Thomas C.

2014-01-01

X-ray diffraction from protein crystals includes both sharply peaked Bragg reflections and diffuse intensity between the peaks. The information in Bragg scattering is limited to what is available in the mean electron density. The diffuse scattering arises from correlations in the electron density variations and therefore contains information about collective motions in proteins. Previous studies using molecular-dynamics (MD) simulations to model diffuse scattering have been hindered by insufficient sampling of the conformational ensemble. To overcome this issue, we have performed a 1.1-μs MD simulation of crystalline staphylococcal nuclease, providing 100-fold more sampling than previous studies. This simulation enables reproducible calculations of the diffuse intensity and predicts functionally important motions, including transitions among at least eight metastable states with different active-site geometries. The total diffuse intensity calculated using the MD model is highly correlated with the experimental data. In particular, there is excellent agreement for the isotropic component of the diffuse intensity, and substantial but weaker agreement for the anisotropic component. Decomposition of the MD model into protein and solvent components indicates that protein–solvent interactions contribute substantially to the overall diffuse intensity. We conclude that diffuse scattering can be used to validate predictions from MD simulations and can provide information to improve MD models of protein motions. PMID:25453071

Internal protein motions in molecular-dynamics simulations of Bragg and diffuse X-ray scattering.

PubMed

Wall, Michael E

2018-03-01

Molecular-dynamics (MD) simulations of Bragg and diffuse X-ray scattering provide a means of obtaining experimentally validated models of protein conformational ensembles. This paper shows that compared with a single periodic unit-cell model, the accuracy of simulating diffuse scattering is increased when the crystal is modeled as a periodic supercell consisting of a 2 × 2 × 2 layout of eight unit cells. The MD simulations capture the general dependence of correlations on the separation of atoms. There is substantial agreement between the simulated Bragg reflections and the crystal structure; there are local deviations, however, indicating both the limitation of using a single structure to model disordered regions of the protein and local deviations of the average structure away from the crystal structure. Although it was anticipated that a simulation of longer duration might be required to achieve maximal agreement of the diffuse scattering calculation with the data using the supercell model, only a microsecond is required, the same as for the unit cell. Rigid protein motions only account for a minority fraction of the variation in atom positions from the simulation. The results indicate that protein crystal dynamics may be dominated by internal motions rather than packing interactions, and that MD simulations can be combined with Bragg and diffuse X-ray scattering to model the protein conformational ensemble.

Internal protein motions in molecular-dynamics simulations of Bragg and diffuse X-ray scattering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wall, Michael E.

Molecular-dynamics (MD) simulations of Bragg and diffuse X-ray scattering provide a means of obtaining experimentally validated models of protein conformational ensembles. This paper shows that compared with a single periodic unit-cell model, the accuracy of simulating diffuse scattering is increased when the crystal is modeled as a periodic supercell consisting of a 2 × 2 × 2 layout of eight unit cells. The MD simulations capture the general dependence of correlations on the separation of atoms. There is substantial agreement between the simulated Bragg reflections and the crystal structure; there are local deviations, however, indicating both the limitation of using a single structuremore » to model disordered regions of the protein and local deviations of the average structure away from the crystal structure. Although it was anticipated that a simulation of longer duration might be required to achieve maximal agreement of the diffuse scattering calculation with the data using the supercell model, only a microsecond is required, the same as for the unit cell. Rigid protein motions only account for a minority fraction of the variation in atom positions from the simulation. The results indicate that protein crystal dynamics may be dominated by internal motions rather than packing interactions, and that MD simulations can be combined with Bragg and diffuse X-ray scattering to model the protein conformational ensemble.« less

Internal protein motions in molecular-dynamics simulations of Bragg and diffuse X-ray scattering

DOE PAGES

Wall, Michael E.

2018-01-25

Molecular-dynamics (MD) simulations of Bragg and diffuse X-ray scattering provide a means of obtaining experimentally validated models of protein conformational ensembles. This paper shows that compared with a single periodic unit-cell model, the accuracy of simulating diffuse scattering is increased when the crystal is modeled as a periodic supercell consisting of a 2 × 2 × 2 layout of eight unit cells. The MD simulations capture the general dependence of correlations on the separation of atoms. There is substantial agreement between the simulated Bragg reflections and the crystal structure; there are local deviations, however, indicating both the limitation of using a single structuremore » to model disordered regions of the protein and local deviations of the average structure away from the crystal structure. Although it was anticipated that a simulation of longer duration might be required to achieve maximal agreement of the diffuse scattering calculation with the data using the supercell model, only a microsecond is required, the same as for the unit cell. Rigid protein motions only account for a minority fraction of the variation in atom positions from the simulation. The results indicate that protein crystal dynamics may be dominated by internal motions rather than packing interactions, and that MD simulations can be combined with Bragg and diffuse X-ray scattering to model the protein conformational ensemble.« less

Comparing the Ability of Enhanced Sampling Molecular Dynamics Methods To Reproduce the Behavior of Fluorescent Labels on Proteins.

PubMed

Walczewska-Szewc, Katarzyna; Deplazes, Evelyne; Corry, Ben

2015-07-14

Adequately sampling the large number of conformations accessible to proteins and other macromolecules is one of the central challenges in molecular dynamics (MD) simulations; this activity can be difficult, even for relatively simple systems. An example where this problem arises is in the simulation of dye-labeled proteins, which are now being widely used in the design and interpretation of Förster resonance energy transfer (FRET) experiments. In this study, MD simulations are used to characterize the motion of two commonly used FRET dyes attached to an immobilized chain of polyproline. Even in this simple system, the dyes exhibit complex behavior that is a mixture of fast and slow motions. Consequently, very long MD simulations are required to sufficiently sample the entire range of dye motion. Here, we compare the ability of enhanced sampling methods to reproduce the behavior of fluorescent labels on proteins. In particular, we compared Accelerated Molecular Dynamics (AMD), metadynamics, Replica Exchange Molecular Dynamics (REMD), and High Temperature Molecular Dynamics (HTMD) to equilibrium MD simulations. We find that, in our system, all of these methods improve the sampling of the dye motion, but the most significant improvement is achieved using REMD.

Solvent-dependent gating motions of an extremophilic lipase from Pseudomonas aeruginosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Quentin R.; Nellas, Ricky B.; Shen, Tongye

2012-07-25

Understanding how organic solvent-stable proteins can function in anhydrous and often complex solutions is essential for the study of the interaction of protein and molecular immiscible interfaces and the design of efficient industrial enzymes in nonaqueous solvents. Using an extremophilic lipase from Pseudomonas aeruginosa as an example, we investigated the conformational dynamics of an organic solvent-tolerant enzyme in complex solvent milieux. Four 100-ns molecular dynamics simulations of the lipase were performed in solvent systems: water, hexane, and two mixtures of hexane and water, 5% and 95% (w/w) hexane. Our results show a solvent-dependent structural change of the protein, especially inmore » the region that regulates the admission of the substrate. We observed that the lipase is much less flexible in hexane than in aqueous solution or at the immiscible interface. Quantified by the size of the accessible channel, the lipase in water has a closed-gate conformation and no access to the active site, while in the hexane-containing systems, the lipase is at various degrees of open-gate state, with the immiscible interface setup being in the widely open conformation ensembles. Furthermore, the composition of explicit solvents in the access channel showed a significant influence on the conformational dynamics of the protein. Interestingly, the slowest step (bottleneck) of the hexane-induced conformational switch seems to be correlated with the slow dehydration dynamics of the channel.« less

Artificial neural networks for efficient clustering of conformational ensembles and their potential for medicinal chemistry.

PubMed

Pandini, Alessandro; Fraccalvieri, Domenico; Bonati, Laura

2013-01-01

The biological function of proteins is strictly related to their molecular flexibility and dynamics: enzymatic activity, protein-protein interactions, ligand binding and allosteric regulation are important mechanisms involving protein motions. Computational approaches, such as Molecular Dynamics (MD) simulations, are now routinely used to study the intrinsic dynamics of target proteins as well as to complement molecular docking approaches. These methods have also successfully supported the process of rational design and discovery of new drugs. Identification of functionally relevant conformations is a key step in these studies. This is generally done by cluster analysis of the ensemble of structures in the MD trajectory. Recently Artificial Neural Network (ANN) approaches, in particular methods based on Self-Organising Maps (SOMs), have been reported performing more accurately and providing more consistent results than traditional clustering algorithms in various data-mining problems. In the specific case of conformational analysis, SOMs have been successfully used to compare multiple ensembles of protein conformations demonstrating a potential in efficiently detecting the dynamic signatures central to biological function. Moreover, examples of the use of SOMs to address problems relevant to other stages of the drug-design process, including clustering of docking poses, have been reported. In this contribution we review recent applications of ANN algorithms in analysing conformational and structural ensembles and we discuss their potential in computer-based approaches for medicinal chemistry.

Estimation of conformational entropy in protein-ligand interactions: a computational perspective.

PubMed

Polyansky, Anton A; Zubac, Ruben; Zagrovic, Bojan

2012-01-01

Conformational entropy is an important component of the change in free energy upon binding of a ligand to its target protein. As a consequence, development of computational techniques for reliable estimation of conformational entropies is currently receiving an increased level of attention in the context of computational drug design. Here, we review the most commonly used techniques for conformational entropy estimation from classical molecular dynamics simulations. Although by-and-large still not directly used in practical drug design, these techniques provide a golden standard for developing other, computationally less-demanding methods for such applications, in addition to furthering our understanding of protein-ligand interactions in general. In particular, we focus on the quasi-harmonic approximation and discuss different approaches that can be used to go beyond it, most notably, when it comes to treating anharmonic and/or correlated motions. In addition to reviewing basic theoretical formalisms, we provide a concrete set of steps required to successfully calculate conformational entropy from molecular dynamics simulations, as well as discuss a number of practical issues that may arise in such calculations.

Equilibrium stochastic dynamics of a Brownian particle in inhomogeneous space: Derivation of an alternative model

NASA Astrophysics Data System (ADS)

Bhattacharyay, A.

2018-03-01

An alternative equilibrium stochastic dynamics for a Brownian particle in inhomogeneous space is derived. Such a dynamics can model the motion of a complex molecule in its conformation space when in equilibrium with a uniform heat bath. The derivation is done by a simple generalization of the formulation due to Zwanzig for a Brownian particle in homogeneous heat bath. We show that, if the system couples to different number of bath degrees of freedom at different conformations then the alternative model gets derived. We discuss results of an experiment by Faucheux and Libchaber which probably has indicated possible limitation of the Boltzmann distribution as equilibrium distribution of a Brownian particle in inhomogeneous space and propose experimental verification of the present theory using similar methods.

Structural intermediates and directionality of the swiveling motion of Pyruvate Phosphate Dikinase

NASA Astrophysics Data System (ADS)

Minges, Alexander; Ciupka, Daniel; Winkler, Christian; Höppner, Astrid; Gohlke, Holger; Groth, Georg

2017-03-01

Pyruvate phosphate dikinase (PPDK) is a vital enzyme in cellular energy metabolism catalyzing the ATP- and Pi-dependent formation of phosphoenolpyruvate from pyruvate in C4 -plants, but the reverse reaction forming ATP in bacteria and protozoa. The multi-domain enzyme is considered an efficient molecular machine that performs one of the largest single domain movements in proteins. However, a comprehensive understanding of the proposed swiveling domain motion has been limited by not knowing structural intermediates or molecular dynamics of the catalytic process. Here, we present crystal structures of PPDKs from Flaveria, a model genus for studying the evolution of C4 -enzymes from phylogenetic ancestors. These structures resolve yet unknown conformational intermediates and provide the first detailed view on the large conformational transitions of the protein in the catalytic cycle. Independently performed unrestrained MD simulations and configurational free energy calculations also identified these intermediates. In all, our experimental and computational data reveal strict coupling of the CD swiveling motion to the conformational state of the NBD. Moreover, structural asymmetries and nucleotide binding states in the PPDK dimer support an alternate binding change mechanism for this intriguing bioenergetic enzyme.

Direct observation of fast protein conformational switching.

PubMed

Ishikawa, Haruto; Kwak, Kyungwon; Chung, Jean K; Kim, Seongheun; Fayer, Michael D

2008-06-24

Folded proteins can exist in multiple conformational substates. Each substate reflects a local minimum on the free-energy landscape with a distinct structure. By using ultrafast 2D-IR vibrational echo chemical-exchange spectroscopy, conformational switching between two well defined substates of a myoglobin mutant is observed on the approximately 50-ps time scale. The conformational dynamics are directly measured through the growth of cross peaks in the 2D-IR spectra of CO bound to the heme active site. The conformational switching involves motion of the distal histidine/E helix that changes the location of the imidazole side group of the histidine. The exchange between substates changes the frequency of the CO, which is detected by the time dependence of the 2D-IR vibrational echo spectrum. These results demonstrate that interconversion between protein conformational substates can occur on very fast time scales. The implications for larger structural changes that occur on much longer time scales are discussed.

Mechanism of the αβ Conformational Change in F1-ATPase after ATP Hydrolysis: Free-Energy Simulations

PubMed Central

Ito, Yuko; Ikeguchi, Mitsunori

2015-01-01

One of the motive forces for F1-ATPase rotation is the conformational change of the catalytically active β subunit due to closing and opening motions caused by ATP binding and hydrolysis, respectively. The closing motion is accomplished in two steps: the hydrogen-bond network around ATP changes and then the entire structure changes via B-helix sliding, as shown in our previous study. Here, we investigated the opening motion induced by ATP hydrolysis using all-atom free-energy simulations, combining the nudged elastic band method and umbrella sampling molecular-dynamics simulations. Because hydrolysis requires residues in the α subunit, the simulations were performed with the αβ dimer. The results indicate that the large-scale opening motion is also achieved by the B-helix sliding (in the reverse direction). However, the sliding mechanism is different from that of ATP binding because sliding is triggered by separation of the hydrolysis products ADP and Pi. We also addressed several important issues: 1), the timing of the product Pi release; 2), the unresolved half-closed β structure; and 3), the ADP release mechanism. These issues are fundamental for motor function; thus, the rotational mechanism of the entire F1-ATPase is also elucidated through this αβ study. During the conformational change, conserved residues among the ATPase proteins play important roles, suggesting that the obtained mechanism may be shared with other ATPase proteins. When combined with our previous studies, these results provide a comprehensive view of the β-subunit conformational change that drives the ATPase. PMID:25564855

Toward structural dynamics: protein motions viewed by chemical shift modulations and direct detection of C'N multiple-quantum relaxation.

PubMed

Mori, Mirko; Kateb, Fatiha; Bodenhausen, Geoffrey; Piccioli, Mario; Abergel, Daniel

2010-03-17

Multiple quantum relaxation in proteins reveals unexpected relationships between correlated or anti-correlated conformational backbone dynamics in alpha-helices or beta-sheets. The contributions of conformational exchange to the relaxation rates of C'N coherences (i.e., double- and zero-quantum coherences involving backbone carbonyl (13)C' and neighboring amide (15)N nuclei) depend on the kinetics of slow exchange processes, as well as on the populations of the conformations and chemical shift differences of (13)C' and (15)N nuclei. The relaxation rates of C'N coherences, which reflect concerted fluctuations due to slow chemical shift modulations (CSMs), were determined by direct (13)C detection in diamagnetic and paramagnetic proteins. In well-folded proteins such as lanthanide-substituted calbindin (CaLnCb), copper,zinc superoxide dismutase (Cu,Zn SOD), and matrix metalloproteinase (MMP12), slow conformational exchange occurs along the entire backbone. Our observations demonstrate that relaxation rates of C'N coherences arising from slow backbone dynamics have positive signs (characteristic of correlated fluctuations) in beta-sheets and negative signs (characteristic of anti-correlated fluctuations) in alpha-helices. This extends the prospects of structure-dynamics relationships to slow time scales that are relevant for protein function and enzymatic activity.

Principal component analysis of molecular dynamics: On the use of Cartesian vs. internal coordinates

NASA Astrophysics Data System (ADS)

Sittel, Florian; Jain, Abhinav; Stock, Gerhard

2014-07-01

Principal component analysis of molecular dynamics simulations is a popular method to account for the essential dynamics of the system on a low-dimensional free energy landscape. Using Cartesian coordinates, first the translation and overall rotation need to be removed from the trajectory. Since the rotation depends via the moment of inertia on the molecule's structure, this separation is only straightforward for relatively rigid systems. Adopting millisecond molecular dynamics simulations of the folding of villin headpiece and the functional dynamics of BPTI provided by D. E. Shaw Research, it is demonstrated via a comparison of local and global rotational fitting that the structural dynamics of flexible molecules necessarily results in a mixing of overall and internal motion. Even for the small-amplitude functional motion of BPTI, the conformational distribution obtained from a Cartesian principal component analysis therefore reflects to some extend the dominant overall motion rather than the much smaller internal motion of the protein. Internal coordinates such as backbone dihedral angles, on the other hand, are found to yield correct and well-resolved energy landscapes for both examples. The virtues and shortcomings of the choice of various fitting schemes and coordinate sets as well as the generality of these results are discussed in some detail.

Principal component analysis of molecular dynamics: on the use of Cartesian vs. internal coordinates.

PubMed

Sittel, Florian; Jain, Abhinav; Stock, Gerhard

2014-07-07

Principal component analysis of molecular dynamics simulations is a popular method to account for the essential dynamics of the system on a low-dimensional free energy landscape. Using Cartesian coordinates, first the translation and overall rotation need to be removed from the trajectory. Since the rotation depends via the moment of inertia on the molecule's structure, this separation is only straightforward for relatively rigid systems. Adopting millisecond molecular dynamics simulations of the folding of villin headpiece and the functional dynamics of BPTI provided by D. E. Shaw Research, it is demonstrated via a comparison of local and global rotational fitting that the structural dynamics of flexible molecules necessarily results in a mixing of overall and internal motion. Even for the small-amplitude functional motion of BPTI, the conformational distribution obtained from a Cartesian principal component analysis therefore reflects to some extend the dominant overall motion rather than the much smaller internal motion of the protein. Internal coordinates such as backbone dihedral angles, on the other hand, are found to yield correct and well-resolved energy landscapes for both examples. The virtues and shortcomings of the choice of various fitting schemes and coordinate sets as well as the generality of these results are discussed in some detail.

Conformational Transitions and Stop-and-Go Nanopore Transport of Single Stranded DNA on Charged Graphene

PubMed Central

Shankla, Manish; Aksimentiev, Aleksei

2014-01-01

Control over interactions with biomolecules holds the key to applications of graphene in biotechnology. One such application is nanopore sequencing, where a DNA molecule is electrophoretically driven through a graphene nanopore. Here, we investigate how interactions of single-stranded DNA and a graphene membrane can be controlled by electrically biasing the membrane. The results of our molecular dynamics simulations suggest that electric charge on graphene can force a DNA homopolymer to adopt a range of strikingly different conformations. The conformational response is sensitive to even very subtle nucleotide modifications, such as DNA methylation. The speed of DNA motion through a graphene nanopore is strongly affected by the graphene charge: a positive charge accelerates the motion whereas a negative charge arrests it. As a possible application of the effect, we demonstrate stop-and-go transport of DNA controlled by the charge of graphene. Such on-demand transport of DNA is essential for realizing nanopore sequencing. PMID:25296960

Conformational transitions and stop-and-go nanopore transport of single-stranded DNA on charged graphene

NASA Astrophysics Data System (ADS)

Shankla, Manish; Aksimentiev, Aleksei

2014-10-01

Control over interactions with biomolecules holds the key to applications of graphene in biotechnology. One such application is nanopore sequencing, where a DNA molecule is electrophoretically driven through a graphene nanopore. Here we investigate how interactions of single-stranded DNA and a graphene membrane can be controlled by electrically biasing the membrane. The results of our molecular dynamics simulations suggest that electric charge on graphene can force a DNA homopolymer to adopt a range of strikingly different conformations. The conformational response is sensitive to even very subtle nucleotide modifications, such as DNA methylation. The speed of DNA motion through a graphene nanopore is strongly affected by the graphene charge: a positive charge accelerates the motion, whereas a negative charge arrests it. As a possible application of the effect, we demonstrate stop-and-go transport of DNA controlled by the charge of graphene. Such on-demand transport of DNA is essential for realizing nanopore sequencing.

Visualizing Protein Interactions and Dynamics: Evolving a Visual Language for Molecular Animation

ERIC Educational Resources Information Center

Jenkinson, Jodie; McGill, Gael

2012-01-01

Undergraduate biology education provides students with a number of learning challenges. Subject areas that are particularly difficult to understand include protein conformational change and stability, diffusion and random molecular motion, and molecular crowding. In this study, we examined the relative effectiveness of three-dimensional…

Visualizing protein interactions and dynamics: evolving a visual language for molecular animation.

PubMed

Jenkinson, Jodie; McGill, Gaël

2012-01-01

Undergraduate biology education provides students with a number of learning challenges. Subject areas that are particularly difficult to understand include protein conformational change and stability, diffusion and random molecular motion, and molecular crowding. In this study, we examined the relative effectiveness of three-dimensional visualization techniques for learning about protein conformation and molecular motion in association with a ligand-receptor binding event. Increasingly complex versions of the same binding event were depicted in each of four animated treatments. Students (n = 131) were recruited from the undergraduate biology program at University of Toronto, Mississauga. Visualization media were developed in the Center for Molecular and Cellular Dynamics at Harvard Medical School. Stem cell factor ligand and cKit receptor tyrosine kinase were used as a classical example of a ligand-induced receptor dimerization and activation event. Each group completed a pretest, viewed one of four variants of the animation, and completed a posttest and, at 2 wk following the assessment, a delayed posttest. Overall, the most complex animation was the most effective at fostering students' understanding of the events depicted. These results suggest that, in select learning contexts, increasingly complex representations may be more desirable for conveying the dynamic nature of cell binding events.

Dynamics of living matter: can we ``see'' collective motions in proteins?

NASA Astrophysics Data System (ADS)

Hekstra, Doeke

2015-03-01

Proteins are ideal model systems for quantitative study of the interplay of physical and evolutionary forces. Collective, anharmonic motions of amino acid residues within proteins are thought to be central to their function, and to explain, in large part, the complex dependence of protein function on its constituent parts. Currently, the experimental characterization of such motions poses a major stumbling block on the way to a physical understanding of protein function and evolution. We are addressing this problem in two ways. First, alternate conformations of protein residues can often be distinguished in the electron density estimated from room-temperature X-ray crystallography. The dense packing of residues in the folded protein requires that such conformational variations must propagate through networks of amino acids to preclude local steric clashes. Fraser and colleagues showed that such steric conflicts can be used to extract contact networks of residues collectively switching conformation. We ask if these networks are conserved over homologous sequences and connected to the functional reaction coordinate, both of which would demonstrate their fundamental importance. I will describe initial results for the family of PDZ domains: small ligand-binding proteins for which a network of energetically and conformationally coupled residues controlling ligand affinity has been demonstrated previously by a range of methods. Second, the analysis of collective motions in proteins, by nearly any means, is indirect: nothing is seen moving. To directly induce and ``see'' motions on a range of time scales, we developed a new approach based on (a) electric field pulses to induce motions within a protein crystal and (b) time-resolved crystallography to observe these motions. Since proteins generically have a heterogeneous, modifiable charge distribution, this method could provide a powerful, general way of probing the collective motions, and excited states, of proteins in kinetic and atomic detail. I will present initial experiments showing the method is feasible. Taken together, these experiments begin to provide a basis for the development of a physical theory of proteins consistent with their function and adaptation - the source of their survival throughout the evolutionary process.

Investigation of sliding DNA clamp dynamics by single-molecule fluorescence, mass spectrometry and structure-based modeling

PubMed Central

Gadkari, Varun V; Harvey, Sophie R; Raper, Austin T; Chu, Wen-Ting; Wang, Jin; Wysocki, Vicki H; Suo, Zucai

2018-01-01

Abstract Proliferating cell nuclear antigen (PCNA) is a trimeric ring-shaped clamp protein that encircles DNA and interacts with many proteins involved in DNA replication and repair. Despite extensive structural work to characterize the monomeric, dimeric, and trimeric forms of PCNA alone and in complex with interacting proteins, no structure of PCNA in a ring-open conformation has been published. Here, we use a multidisciplinary approach, including single-molecule Förster resonance energy transfer (smFRET), native ion mobility-mass spectrometry (IM-MS), and structure-based computational modeling, to explore the conformational dynamics of a model PCNA from Sulfolobus solfataricus (Sso), an archaeon. We found that Sso PCNA samples ring-open and ring-closed conformations even in the absence of its clamp loader complex, replication factor C, and transition to the ring-open conformation is modulated by the ionic strength of the solution. The IM-MS results corroborate the smFRET findings suggesting that PCNA dynamics are maintained in the gas phase and further establishing IM-MS as a reliable strategy to investigate macromolecular motions. Our molecular dynamic simulations agree with the experimental data and reveal that ring-open PCNA often adopts an out-of-plane left-hand geometry. Collectively, these results implore future studies to define the roles of PCNA dynamics in DNA loading and other PCNA-mediated interactions. PMID:29529283

Thermal fluctuations of haemoglobin from different species: adaptation to temperature via conformational dynamics

PubMed Central

Stadler, A. M.; Garvey, C. J.; Bocahut, A.; Sacquin-Mora, S.; Digel, I.; Schneider, G. J.; Natali, F.; Artmann, G. M.; Zaccai, G.

2012-01-01

Thermodynamic stability, configurational motions and internal forces of haemoglobin (Hb) of three endotherms (platypus, Ornithorhynchus anatinus; domestic chicken, Gallus gallus domesticus and human, Homo sapiens) and an ectotherm (salt water crocodile, Crocodylus porosus) were investigated using circular dichroism, incoherent elastic neutron scattering and coarse-grained Brownian dynamics simulations. The experimental results from Hb solutions revealed a direct correlation between protein resilience, melting temperature and average body temperature of the different species on the 0.1 ns time scale. Molecular forces appeared to be adapted to permit conformational fluctuations with a root mean square displacement close to 1.2 Å at the corresponding average body temperature of the endotherms. Strong forces within crocodile Hb maintain the amplitudes of motion within a narrow limit over the entire temperature range in which the animal lives. In fully hydrated powder samples of human and chicken, Hb mean square displacements and effective force constants on the 1 ns time scale showed no differences over the whole temperature range from 10 to 300 K, in contrast to the solution case. A complementary result of the study, therefore, is that one hydration layer is not sufficient to activate all conformational fluctuations of Hb in the pico- to nanosecond time scale which might be relevant for biological function. Coarse-grained Brownian dynamics simulations permitted to explore residue-specific effects. They indicated that temperature sensing of human and chicken Hb occurs mainly at residues lining internal cavities in the β-subunits. PMID:22696485

Thermal fluctuations of haemoglobin from different species: adaptation to temperature via conformational dynamics.

PubMed

Stadler, A M; Garvey, C J; Bocahut, A; Sacquin-Mora, S; Digel, I; Schneider, G J; Natali, F; Artmann, G M; Zaccai, G

2012-11-07

Thermodynamic stability, configurational motions and internal forces of haemoglobin (Hb) of three endotherms (platypus, Ornithorhynchus anatinus; domestic chicken, Gallus gallus domesticus and human, Homo sapiens) and an ectotherm (salt water crocodile, Crocodylus porosus) were investigated using circular dichroism, incoherent elastic neutron scattering and coarse-grained Brownian dynamics simulations. The experimental results from Hb solutions revealed a direct correlation between protein resilience, melting temperature and average body temperature of the different species on the 0.1 ns time scale. Molecular forces appeared to be adapted to permit conformational fluctuations with a root mean square displacement close to 1.2 Å at the corresponding average body temperature of the endotherms. Strong forces within crocodile Hb maintain the amplitudes of motion within a narrow limit over the entire temperature range in which the animal lives. In fully hydrated powder samples of human and chicken, Hb mean square displacements and effective force constants on the 1 ns time scale showed no differences over the whole temperature range from 10 to 300 K, in contrast to the solution case. A complementary result of the study, therefore, is that one hydration layer is not sufficient to activate all conformational fluctuations of Hb in the pico- to nanosecond time scale which might be relevant for biological function. Coarse-grained Brownian dynamics simulations permitted to explore residue-specific effects. They indicated that temperature sensing of human and chicken Hb occurs mainly at residues lining internal cavities in the β-subunits.

Molecular Dynamics Simulations of KirBac1.1 Mutants Reveal Global Gating Changes of Kir Channels.

PubMed

Linder, Tobias; Wang, Shizhen; Zangerl-Plessl, Eva-Maria; Nichols, Colin G; Stary-Weinzinger, Anna

2015-04-27

Prokaryotic inwardly rectifying (KirBac) potassium channels are homologous to mammalian Kir channels. Their activity is controlled by dynamical conformational changes that regulate ion flow through a central pore. Understanding the dynamical rearrangements of Kir channels during gating requires high-resolution structure information from channels crystallized in different conformations and insight into the transition steps, which are difficult to access experimentally. In this study, we use MD simulations on wild type KirBac1.1 and an activatory mutant to investigate activation gating of KirBac channels. Full atomistic MD simulations revealed that introducing glutamate in position 143 causes significant widening at the helix bundle crossing gate, enabling water flux into the cavity. Further, global rearrangements including a twisting motion as well as local rearrangements at the subunit interface in the cytoplasmic domain were observed. These structural rearrangements are similar to recently reported KirBac3.1 crystal structures in closed and open conformation, suggesting that our simulations capture major conformational changes during KirBac1.1 opening. In addition, an important role of protein-lipid interactions during gating was observed. Slide-helix and C-linker interactions with lipids were strengthened during activation gating.

Chemistry in motion: tiny synthetic motors.

PubMed

Colberg, Peter H; Reigh, Shang Yik; Robertson, Bryan; Kapral, Raymond

2014-12-16

CONSPECTUS: Diffusion is the principal transport mechanism that controls the motion of solute molecules and other species in solution; however, the random walk process that underlies diffusion is slow and often nonspecific. Although diffusion is an essential mechanism for transport in the biological realm, biological systems have devised more efficient transport mechanisms using molecular motors. Most biological motors utilize some form of chemical energy derived from their surroundings to induce conformational changes in order to carry out specific functions. These small molecular motors operate in the presence of strong thermal fluctuations and in the regime of low Reynolds numbers, where viscous forces dominate inertial forces. Thus, their dynamical behavior is fundamentally different from that of macroscopic motors, and different mechanisms are responsible for the production of useful mechanical motion. There is no reason why our interest should be confined to the small motors that occur naturally in biological systems. Recently, micron and nanoscale motors that use chemical energy to produce directed motion by a number of different mechanisms have been made in the laboratory. These small synthetic motors also experience strong thermal fluctuations and operate in regimes where viscous forces dominate. Potentially, these motors could be directed to perform different transport tasks, analogous to those of biological motors, for both in vivo and in vitro applications. Although some synthetic motors execute conformational changes to effect motion, the majority do not, and, instead, they use other mechanisms to convert chemical energy into directed motion. In this Account, we describe how synthetic motors that operate by self-diffusiophoresis make use of a self-generated concentration gradient to drive motor motion. A description of propulsion by self-diffusiophoresis is presented for Janus particle motors comprising catalytic and noncatalytic faces. The properties of the dynamics of chemically powered motors are illustrated by presenting the results of particle-based simulations of sphere-dimer motors constructed from linked catalytic and noncatalytic spheres. The geometries of both Janus and sphere-dimer motors with asymmetric catalytic activity support the formation of concentration gradients around the motors. Because directed motion can occur only when the system is not in equilibrium, the nature of the environment and the role it plays in motor dynamics are described. Rotational Brownian motion also acts to limit directed motion, and it has especially strong effects for very small motors. We address the following question: how small can motors be and still exhibit effects due to propulsion, even if only to enhance diffusion? Synthetic motors have the potential to transform the manner in which chemical dynamical processes are carried out for a wide range of applications.

Direct design of an energy landscape with bistable DNA origami mechanisms.

PubMed

Zhou, Lifeng; Marras, Alexander E; Su, Hai-Jun; Castro, Carlos E

2015-03-11

Structural DNA nanotechnology provides a feasible technique for the design and fabrication of complex geometries even exhibiting controllable dynamic behavior. Recently we have demonstrated the possibility of implementing macroscopic engineering design approaches to construct DNA origami mechanisms (DOM) with programmable motion and tunable flexibility. Here, we implement the design of compliant DNA origami mechanisms to extend from prescribing motion to prescribing an energy landscape. Compliant mechanisms facilitate motion via deformation of components with tunable stiffness resulting in well-defined mechanical energy stored in the structure. We design, fabricate, and characterize a DNA origami nanostructure with an energy landscape defined by two stable states (local energy minima) separated by a designed energy barrier. This nanostructure is a four-bar bistable mechanism with two undeformed states. Traversing between those states requires deformation, and hence mechanical energy storage, in a compliant arm of the linkage. The energy barrier for switching between two states was obtained from the conformational distribution based on a Boltzmann probability function and closely follows a predictive mechanical model. Furthermore, we demonstrated the ability to actuate the mechanism into one stable state via additional DNA inputs and then release the actuation via DNA strand displacement. This controllable multistate system establishes a foundation for direct design of energy landscapes that regulate conformational dynamics similar to biomolecular complexes.

Fast, clash-free RNA conformational morphing using molecular junctions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heliou, Amelie; Budday, Dominik; Fonseca, Rasmus

Non-coding ribonucleic acids (ncRNA) are functional RNA molecules that are not translated into protein. They are extremely dynamic, adopting diverse conformational substates, which enables them to modulate their interaction with a large number of other molecules. The flexibility of ncRNA provides a challenge for probing their complex 3D conformational landscape, both experimentally and computationally. As a result, despite their conformational diversity, ncRNAs mostly preserve their secondary structure throughout the dynamic ensemble. Here we present a kinematics-based procedure to morph an RNA molecule between conformational substates, while avoiding inter-atomic clashes. We represent an RNA as a kinematic linkage, with fixed groupsmore » of atoms as rigid bodies and rotatable bonds as degrees of freedom. Our procedure maintains RNA secondary structure by treating hydrogen bonds between base pairs as constraints. The constraints define a lower-dimensional, secondary-structure constraint manifold in conformation space, where motions are largely governed by molecular junctions of unpaired nucleotides. On a large benchmark set, we show that our morphing procedure compares favorably to peer algorithms, and can approach goal conformations to within a low all-atom RMSD by directing fewer than 1% of its atoms. Furthermore, our results suggest that molecular junctions can modulate 3D structural rearrangements, while secondary structure elements guide large parts of the molecule along the transition to the correct final conformation.« less

Fast, clash-free RNA conformational morphing using molecular junctions

DOE PAGES

Heliou, Amelie; Budday, Dominik; Fonseca, Rasmus; ...

2017-03-13

Non-coding ribonucleic acids (ncRNA) are functional RNA molecules that are not translated into protein. They are extremely dynamic, adopting diverse conformational substates, which enables them to modulate their interaction with a large number of other molecules. The flexibility of ncRNA provides a challenge for probing their complex 3D conformational landscape, both experimentally and computationally. As a result, despite their conformational diversity, ncRNAs mostly preserve their secondary structure throughout the dynamic ensemble. Here we present a kinematics-based procedure to morph an RNA molecule between conformational substates, while avoiding inter-atomic clashes. We represent an RNA as a kinematic linkage, with fixed groupsmore » of atoms as rigid bodies and rotatable bonds as degrees of freedom. Our procedure maintains RNA secondary structure by treating hydrogen bonds between base pairs as constraints. The constraints define a lower-dimensional, secondary-structure constraint manifold in conformation space, where motions are largely governed by molecular junctions of unpaired nucleotides. On a large benchmark set, we show that our morphing procedure compares favorably to peer algorithms, and can approach goal conformations to within a low all-atom RMSD by directing fewer than 1% of its atoms. Furthermore, our results suggest that molecular junctions can modulate 3D structural rearrangements, while secondary structure elements guide large parts of the molecule along the transition to the correct final conformation.« less

Interpreting medium ring canonical conformers by a triangular plane tessellation of the macrocycle

NASA Astrophysics Data System (ADS)

Khalili, Pegah; Barnett, Christopher B.; Naidoo, Kevin J.

2013-05-01

Cyclic conformational coordinates are essential for the distinction of molecular ring conformers as the use of Cremer-Pople coordinates have illustrated for five- and six-membered rings. Here, by tessellating medium rings into triangular planes and using the relative angles made between triangular planes we are able to assign macrocyclic pucker conformations into canonical pucker conformers such as chairs, boats, etc. We show that the definition is straightforward compared with other methods popularly used for small rings and that it is computationally simple to implement for complex macrocyclic rings. These cyclic conformational coordinates directly couple to the motion of individual nodes of a ring. Therefore, they are useful for correlating the physical properties of macrocycles with their ring pucker and measuring the dynamic ring conformational behavior. We illustrate the triangular tessellation, assignment, and pucker analysis on 7- and 8-membered rings. Sets of canonical states are given for cycloheptane and cyclooctane that have been previously experimentally analysed.

Slowdown of Interhelical Motions Induces a Glass Transition in RNA

PubMed Central

Frank, Aaron T.; Zhang, Qi; Al-Hashimi, Hashim M.; Andricioaei, Ioan

2015-01-01

RNA function depends crucially on the details of its dynamics. The simplest RNA dynamical unit is a two-way interhelical junction. Here, for such a unit—the transactivation response RNA element—we present evidence from molecular dynamics simulations, supported by nuclear magnetic resonance relaxation experiments, for a dynamical transition near 230 K. This glass transition arises from the freezing out of collective interhelical motional modes. The motions, resolved with site-specificity, are dynamically heterogeneous and exhibit non-Arrhenius relaxation. The microscopic origin of the glass transition is a low-dimensional, slow manifold consisting largely of the Euler angles describing interhelical reorientation. Principal component analysis over a range of temperatures covering the glass transition shows that the abrupt slowdown of motion finds its explanation in a localization transition that traps probability density into several disconnected conformational pools over the low-dimensional energy landscape. Upon temperature increase, the probability density pools then flood a larger basin, akin to a lakes-to-sea transition. Simulations on transactivation response RNA are also used to backcalculate inelastic neutron scattering data that match previous inelastic neutron scattering measurements on larger and more complex RNA structures and which, upon normalization, give temperature-dependent fluctuation profiles that overlap onto a glass transition curve that is quasi-universal over a range of systems and techniques. PMID:26083927

Conformational dynamics of cancer-associated MyD88-TIR domain mutant L252P (L265P) allosterically tilts the landscape toward homo-dimerization

PubMed Central

Zhan, Chendi; Qi, Ruxi; Wei, Guanghong; Guven-Maiorov, Emine; Nussinov, Ruth; Ma, Buyong

2016-01-01

MyD88 is an essential adaptor protein, which mediates the signaling of the toll-like and interleukin-1 receptors’ superfamily. The MyD88 L252P (L265P) mutation has been identified in diffuse large B-cell lymphoma. The identification of this mutation has been a major advance in the diagnosis of patients with aldenstrom macroglobulinemia and related lymphoid neoplasms. Here we used computational methods to characterize the conformational effects of the mutation. Our molecular dynamics simulations revealed that the mutation allosterically quenched the global conformational dynamics of the toll/IL-1R (TIR) domain, and readjusted its salt bridges and dynamic community network. Specifically, the mutation changed the orientation and reduced the fluctuation of α-helix 3, possibly through eliminating/weakening ~8 salt bridges and enhancing the salt bridge D225-K258. Using the energy landscape of the TIR domains of MyD88, we identified two dynamic conformational basins, which correspond to the binding sites used in homo- and hetero-oligomerization, respectively. Our results indicate that the mutation stabilizes the core of the homo-dimer interface of the MyD88-TIR domain, and increases the population of homo-dimer-compatible conformational states in MyD88 family proteins. However, the dampened motion restricts its ability to heterodimerize with other TIR domains, thereby curtailing physiological signaling. In conclusion, the L252P both shifts the landscape toward homo-dimerization and restrains the dynamics of the MyD88-TIR domain, which disfavors its hetero-dimerization with other TIR domains. We further put these observations within the framework of MyD88-mediated cell signaling. PMID:27503954

Common functionally important motions of the nucleotide-binding domain of Hsp70.

PubMed

Gołaś, Ewa I; Czaplewski, Cezary; Scheraga, Harold A; Liwo, Adam

2015-02-01

The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones involved in protein folding, aggregate prevention, and protein disaggregation. They consist of the substrate-binding domain (SBD) that binds client substrates, and the nucleotide-binding domain (NBD), whose cycles of nucleotide hydrolysis and exchange underpin the activity of the chaperone. To characterize the structure-function relationships that link the binding state of the NBD to its conformational behavior, we analyzed the dynamics of the NBD of the Hsp70 chaperone from Bos taurus (PDB 3C7N:B) by all-atom canonical molecular dynamics simulations. It was found that essential motions within the NBD fall into three major classes: the mutual class, reflecting tendencies common to all binding states, and the ADP- and ATP-unique classes, which reflect conformational trends that are unique to either the ADP- or ATP-bound states, respectively. "Mutual" class motions generally describe "in-plane" and/or "out-of-plane" (scissor-like) rotation of the subdomains within the NBD. This result is consistent with experimental nuclear magnetic resonance data on the NBD. The "unique" class motions target specific regions on the NBD, usually surface loops or sites involved in nucleotide binding and are, therefore, expected to be involved in allostery and signal transmission. For all classes, and especially for those of the "unique" type, regions of enhanced mobility can be identified; these are termed "hot spots," and their locations generally parallel those found by NMR spectroscopy. The presence of magnesium and potassium cations in the nucleotide-binding pocket was also found to influence the dynamics of the NBD significantly. © 2014 Wiley Periodicals, Inc.

Internal Coordinate Molecular Dynamics: A Foundation for Multiscale Dynamics

PubMed Central

2015-01-01

Internal coordinates such as bond lengths, bond angles, and torsion angles (BAT) are natural coordinates for describing a bonded molecular system. However, the molecular dynamics (MD) simulation methods that are widely used for proteins, DNA, and polymers are based on Cartesian coordinates owing to the mathematical simplicity of the equations of motion. However, constraints are often needed with Cartesian MD simulations to enhance the conformational sampling. This makes the equations of motion in the Cartesian coordinates differential-algebraic, which adversely impacts the complexity and the robustness of the simulations. On the other hand, constraints can be easily placed in BAT coordinates by removing the degrees of freedom that need to be constrained. Thus, the internal coordinate MD (ICMD) offers an attractive alternative to Cartesian coordinate MD for developing multiscale MD method. The torsional MD method is a special adaptation of the ICMD method, where all the bond lengths and bond angles are kept rigid. The advantages of ICMD simulation methods are the longer time step size afforded by freezing high frequency degrees of freedom and performing a conformational search in the more important low frequency torsional degrees of freedom. However, the advancements in the ICMD simulations have been slow and stifled by long-standing mathematical bottlenecks. In this review, we summarize the recent mathematical advancements we have made based on spatial operator algebra, in developing a robust long time scale ICMD simulation toolkit useful for various applications. We also present the applications of ICMD simulations to study conformational changes in proteins and protein structure refinement. We review the advantages of the ICMD simulations over the Cartesian simulations when used with enhanced sampling methods and project the future use of ICMD simulations in protein dynamics. PMID:25517406

Conformational changes in intact dengue virus reveal serotype-specific expansion

PubMed Central

Lim, Xin-Xiang; Chandramohan, Arun; Lim, Xin Ying Elisa; Bag, Nirmalya; Sharma, Kamal Kant; Wirawan, Melissa; Wohland, Thorsten; Lok, Shee-Mei; Anand, Ganesh S.

2017-01-01

Dengue virus serotype 2 (DENV2) alone undergoes structural expansion at 37 °C (associated with host entry), despite high sequence and structural homology among the four known serotypes. The basis for this differential expansion across strains and serotypes is unknown and necessitates mapping of the dynamics of dengue whole viral particles to describe their coordinated motions and conformational changes when exposed to host-like environments. Here we capture the dynamics of intact viral particles of two serotypes, DENV1 and DENV2, by amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and time resolved Förster Resonance Energy Transfer. Our results show temperature-dependent dynamics hotspots on DENV2 and DENV1 particles with DENV1 showing expansion at 40 °C but not at 37 °C. HDXMS measurement of virion dynamics in solution offers a powerful approach to identify potential epitopes, map virus-antibody complex structure and dynamics, and test effects of multiple host-specific perturbations on viruses and virus-antibody complexes. PMID:28186093

Force-momentum-based self-guided Langevin dynamics: A rapid sampling method that approaches the canonical ensemble

NASA Astrophysics Data System (ADS)

Wu, Xiongwu; Brooks, Bernard R.

2011-11-01

The self-guided Langevin dynamics (SGLD) is a method to accelerate conformational searching. This method is unique in the way that it selectively enhances and suppresses molecular motions based on their frequency to accelerate conformational searching without modifying energy surfaces or raising temperatures. It has been applied to studies of many long time scale events, such as protein folding. Recent progress in the understanding of the conformational distribution in SGLD simulations makes SGLD also an accurate method for quantitative studies. The SGLD partition function provides a way to convert the SGLD conformational distribution to the canonical ensemble distribution and to calculate ensemble average properties through reweighting. Based on the SGLD partition function, this work presents a force-momentum-based self-guided Langevin dynamics (SGLDfp) simulation method to directly sample the canonical ensemble. This method includes interaction forces in its guiding force to compensate the perturbation caused by the momentum-based guiding force so that it can approximately sample the canonical ensemble. Using several example systems, we demonstrate that SGLDfp simulations can approximately maintain the canonical ensemble distribution and significantly accelerate conformational searching. With optimal parameters, SGLDfp and SGLD simulations can cross energy barriers of more than 15 kT and 20 kT, respectively, at similar rates for LD simulations to cross energy barriers of 10 kT. The SGLDfp method is size extensive and works well for large systems. For studies where preserving accessible conformational space is critical, such as free energy calculations and protein folding studies, SGLDfp is an efficient approach to search and sample the conformational space.

Three-dimensional structural dynamics of DNA origami Bennett linkages using individual-particle electron tomography

DOE PAGES

Lei, Dongsheng; Marras, Alexander E.; Liu, Jianfang; ...

2018-02-09

Scaffolded DNA origami has proven to be a powerful and efficient technique to fabricate functional nanomachines by programming the folding of a single-stranded DNA template strand into three-dimensional (3D) nanostructures, designed to be precisely motion-controlled. Although two-dimensional (2D) imaging of DNA nanomachines using transmission electron microscopy and atomic force microscopy suggested these nanomachines are dynamic in 3D, geometric analysis based on 2D imaging was insufficient to uncover the exact motion in 3D. In this paper, we use the individual-particle electron tomography method and reconstruct 129 density maps from 129 individual DNA origami Bennett linkage mechanisms at ~6-14 nm resolution. The statisticalmore » analyses of these conformations lead to understanding the 3D structural dynamics of Bennett linkage mechanisms. Moreover, our effort provides experimental verification of a theoretical kinematics model of DNA origami, which can be used as feedback to improve the design and control of motion via optimized DNA sequences and routing.« less

Three-dimensional structural dynamics of DNA origami Bennett linkages using individual-particle electron tomography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lei, Dongsheng; Marras, Alexander E.; Liu, Jianfang

Scaffolded DNA origami has proven to be a powerful and efficient technique to fabricate functional nanomachines by programming the folding of a single-stranded DNA template strand into three-dimensional (3D) nanostructures, designed to be precisely motion-controlled. Although two-dimensional (2D) imaging of DNA nanomachines using transmission electron microscopy and atomic force microscopy suggested these nanomachines are dynamic in 3D, geometric analysis based on 2D imaging was insufficient to uncover the exact motion in 3D. In this paper, we use the individual-particle electron tomography method and reconstruct 129 density maps from 129 individual DNA origami Bennett linkage mechanisms at ~6-14 nm resolution. The statisticalmore » analyses of these conformations lead to understanding the 3D structural dynamics of Bennett linkage mechanisms. Moreover, our effort provides experimental verification of a theoretical kinematics model of DNA origami, which can be used as feedback to improve the design and control of motion via optimized DNA sequences and routing.« less

Disordering Chain Motions in Fluoropolymers

NASA Astrophysics Data System (ADS)

Holt, David B.; Farmer, Barry L.

1998-03-01

Rotational and conformational disorder play important roles in the solid state phases of fluoropolymers such as polytetrafluoro- ethylene (PTFE). Modeling disordering processes and transitions which occur in fluoropolymers has been hampered due to a lack of force field parameters that adequately describe both the intra- and intermolecular characteristics (conformations and distances) of these polymers in the solid state. A force field has been developed which overcomes these inadequacies and has been utilized in molecular dynamics simulations on a system of PTFE oligomers to investigate two of the primary disordering processes that occur in the solid phases: rotations of chains about their helical axes and the formation and subsequent behavior of helix reversals. The simulation results confirm helix reversal activity at low temperatures and demonstrate correlations between chain segment rotations or librations and helix reversal motion. A mechanism for large scale chain segment rotations is proposed.

Hydride transfer catalysed by Escherichia coli and Bacillus subtilis dihydrofolate reductase: coupled motions and distal mutations.

PubMed

Hammes-Schiffer, Sharon; Watney, James B

2006-08-29

This paper reviews the results from hybrid quantum/classical molecular dynamics simulations of the hydride transfer reaction catalysed by wild-type (WT) and mutant Escherichia coli and WT Bacillus subtilis dihydrofolate reductase (DHFR). Nuclear quantum effects such as zero point energy and hydrogen tunnelling are significant in these reactions and substantially decrease the free energy barrier. The donor-acceptor distance decreases to ca 2.7 A at transition-state configurations to enable the hydride transfer. A network of coupled motions representing conformational changes along the collective reaction coordinate facilitates the hydride transfer reaction by decreasing the donor-acceptor distance and providing a favourable geometric and electrostatic environment. Recent single-molecule experiments confirm that at least some of these thermally averaged equilibrium conformational changes occur on the millisecond time-scale of the hydride transfer. Distal mutations can lead to non-local structural changes and significantly impact the probability of sampling configurations conducive to the hydride transfer, thereby altering the free-energy barrier and the rate of hydride transfer. E. coli and B. subtilis DHFR enzymes, which have similar tertiary structures and hydride transfer rates with 44% sequence identity, exhibit both similarities and differences in the equilibrium motions and conformational changes correlated to hydride transfer, suggesting a balance of conservation and flexibility across species.

Simulations of a Membrane-Anchored Peptide: Structure, Dynamics, and Influence on Bilayer Properties

PubMed Central

Jensen, Morten Ø.; Mouritsen, Ole G.; Peters, Günther H.

2004-01-01

A three-dimensional structure of a model decapeptide is obtained by performing molecular dynamics simulations of the peptide in explicit water. Interactions between an N-myristoylated form of the folded peptide anchored to dipalmitoylphosphatidylcholine fluid phase lipid membranes are studied at different applied surface tensions by molecular dynamics simulations. The lipid membrane environment influences the conformational space explored by the peptide. The overall secondary structure of the anchored peptide is found to deviate at times from its structure in aqueous solution through reversible conformational transitions. The peptide is, despite the anchor, highly mobile at the membrane surface with the peptide motion along the bilayer normal being integrated into the collective modes of the membrane. Peptide anchoring moderately alters the lateral compressibility of the bilayer by changing the equilibrium area of the membrane. Although membrane anchoring moderately affects the elastic properties of the bilayer, the model peptide studied here exhibits conformational flexibility and our results therefore suggest that peptide acylation is a feasible way to reinforce peptide-membrane interactions whereby, e.g., the lifetime of receptor-ligand interactions can be prolonged. PMID:15189854

Fractional Brownian motion and the critical dynamics of zipping polymers.

PubMed

Walter, J-C; Ferrantini, A; Carlon, E; Vanderzande, C

2012-03-01

We consider two complementary polymer strands of length L attached by a common-end monomer. The two strands bind through complementary monomers and at low temperatures form a double-stranded conformation (zipping), while at high temperature they dissociate (unzipping). This is a simple model of DNA (or RNA) hairpin formation. Here we investigate the dynamics of the strands at the equilibrium critical temperature T=T(c) using Monte Carlo Rouse dynamics. We find that the dynamics is anomalous, with a characteristic time scaling as τ∼L(2.26(2)), exceeding the Rouse time ∼L(2.18). We investigate the probability distribution function, velocity autocorrelation function, survival probability, and boundary behavior of the underlying stochastic process. These quantities scale as expected from a fractional Brownian motion with a Hurst exponent H=0.44(1). We discuss similarities to and differences from unbiased polymer translocation.

Mitochondrial dynamics and optical conformation changes in DsRed as studied by Fourier imaging correlation spectroscopy

NASA Astrophysics Data System (ADS)

Senning, Eric Nicolas

Novel experiments that probe the dynamics of intracellular species, including the center-of-mass displacements and internal conformational transitions of biological macromolecules, have the potential to reveal the complex biochemical mechanisms operating within the cell. This work presents the implementation and development of Fourier imaging correlation spectroscopy (FICS), a phase-selective approach to fluorescence spectroscopy that measures the collective coordinate fluctuations of fluorescently labeled microscopic particles. In FICS experiments, a spatially modulated optical grating excites a fluorescently labeled sample. Phase-synchronous detection of the fluorescence, with respect to the phase of the exciting optical grating, can be used to monitor the fluctuations of partially averaged spatial coordinates. These data are then analyzed by two-point and four-point time correlation functions to provide a statistically meaningful understanding of the dynamics under observation. FICS represents a unique route to elevate signal levels, while acquiring detailed information about molecular coordinate trajectories. Mitochondria of mammalian cells are known to associate with cytoskeletal proteins, and their motions are affected by the stability of microtubules and microfilaments. Within the cell it is possible to fluorescently label the mitochondria and study its dynamic behavior with FICS. The dynamics of S. cerevisiae yeast mitochondria are characterized at four discrete length scales (ranging from 0.6--1.19 mum) and provide detailed information about the influence of specific cytoskeletal elements. Using the microtubule and microfilament destabilizing agents, Nocodazole and Latrunculin A, it is determined that microfilaments are required for normal yeast mitochondrial motion while microtubules have no effect. Experiments with specific actin mutants revealed that actin is responsible for enhanced mobility on length scales greater than 0.6 mum. The versatility of FICS expands when individual molecules are labeled with fluorescent chromophores. In recent experiments on the tetrameric fluorescent protein DsRed, polarization-modulated FICS (PM-FICS) is demonstrated to separate conformational dynamics from molecular translational dynamics. The optical switching pathways of DsRed, a tetrameric complex of fluorescent protein subunits, are examined. An analysis of PM-FICS coordinate trajectories, in terms of 2D spectra and joint probability distributions, provides detailed information about the transition pathways between distinct dipole-coupled DsRed conformations. This dissertation includes co-authored and previously published material.

Optical observation of correlated motions in dihydrofolate reductase

NASA Astrophysics Data System (ADS)

Xu, Mengyang; Niessen, Katherine; Pace, James; Cody, Vivian; Markelz, Andrea

2015-03-01

Enzyme function relies on its structural flexibility to make conformational changes for substrate binding and product release. An example of a metabolic enzyme where such structural changes are vital is dihydrofolate reductase (DHFR). DHFR is essential in both prokaryotes and eukaryotes for the nucleotide biosynthesis by catalyzing the reduction of dihydrofolate to tetrahydrofolate. NMR dynamical measurements found large amplitude fast dynamics that could indicate rigid-body, twisting-hinge motion for ecDHFR that may mediate flux. The role of such long-range correlated motions in function was suggested by the observed sharp decrease in enzyme activity for the single point mutation G121V, which is remote from active sites. This decrease in activity may be caused by the mutation interfering with the long-range intramolecular vibrations necessary for rapid access to functional configurations. We use our new technique of crystal anisotropy terahertz microscopy (CATM), to observe correlated motions in ecDHFR crystals with the bonding of NADPH and methotrexate. We compare the measured intramolecular vibrational spectrum with calculations using normal mode analysis.

Coarse-Grained Models Reveal Functional Dynamics – II. Molecular Dynamics Simulation at the Coarse-Grained Level – Theories and Biological Applications

PubMed Central

Chng, Choon-Peng; Yang, Lee-Wei

2008-01-01

Molecular dynamics (MD) simulation has remained the most indispensable tool in studying equilibrium/non-equilibrium conformational dynamics since its advent 30 years ago. With advances in spectroscopy accompanying solved biocomplexes in growing sizes, sampling their dynamics that occur at biologically interesting spatial/temporal scales becomes computationally intractable; this motivated the use of coarse-grained (CG) approaches. CG-MD models are used to study folding and conformational transitions in reduced resolution and can employ enlarged time steps due to the absence of some of the fastest motions in the system. The Boltzmann-Inversion technique, heavily used in parameterizing these models, provides a smoothed-out effective potential on which molecular conformation evolves at a faster pace thus stretching simulations into tens of microseconds. As a result, a complete catalytic cycle of HIV-1 protease or the assembly of lipid-protein mixtures could be investigated by CG-MD to gain biological insights. In this review, we survey the theories developed in recent years, which are categorized into Folding-based and Molecular-Mechanics-based. In addition, physical bases in the selection of CG beads/time-step, the choice of effective potentials, representation of solvent, and restoration of molecular representations back to their atomic details are systematically discussed. PMID:19812774

Overcoming potential energy distortions in constrained internal coordinate molecular dynamics simulations.

PubMed

Kandel, Saugat; Salomon-Ferrer, Romelia; Larsen, Adrien B; Jain, Abhinandan; Vaidehi, Nagarajan

2016-01-28

The Internal Coordinate Molecular Dynamics (ICMD) method is an attractive molecular dynamics (MD) method for studying the dynamics of bonded systems such as proteins and polymers. It offers a simple venue for coarsening the dynamics model of a system at multiple hierarchical levels. For example, large scale protein dynamics can be studied using torsional dynamics, where large domains or helical structures can be treated as rigid bodies and the loops connecting them as flexible torsions. ICMD with such a dynamic model of the protein, combined with enhanced conformational sampling method such as temperature replica exchange, allows the sampling of large scale domain motion involving high energy barrier transitions. Once these large scale conformational transitions are sampled, all-torsion, or even all-atom, MD simulations can be carried out for the low energy conformations sampled via coarse grained ICMD to calculate the energetics of distinct conformations. Such hierarchical MD simulations can be carried out with standard all-atom forcefields without the need for compromising on the accuracy of the forces. Using constraints to treat bond lengths and bond angles as rigid can, however, distort the potential energy landscape of the system and reduce the number of dihedral transitions as well as conformational sampling. We present here a two-part solution to overcome such distortions of the potential energy landscape with ICMD models. To alleviate the intrinsic distortion that stems from the reduced phase space in torsional MD, we use the Fixman compensating potential. To additionally alleviate the extrinsic distortion that arises from the coupling between the dihedral angles and bond angles within a force field, we propose a hybrid ICMD method that allows the selective relaxing of bond angles. This hybrid ICMD method bridges the gap between all-atom MD and torsional MD. We demonstrate with examples that these methods together offer a solution to eliminate the potential energy distortions encountered in constrained ICMD simulations of peptide molecules.

Overcoming potential energy distortions in constrained internal coordinate molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Kandel, Saugat; Salomon-Ferrer, Romelia; Larsen, Adrien B.; Jain, Abhinandan; Vaidehi, Nagarajan

2016-01-01

The Internal Coordinate Molecular Dynamics (ICMD) method is an attractive molecular dynamics (MD) method for studying the dynamics of bonded systems such as proteins and polymers. It offers a simple venue for coarsening the dynamics model of a system at multiple hierarchical levels. For example, large scale protein dynamics can be studied using torsional dynamics, where large domains or helical structures can be treated as rigid bodies and the loops connecting them as flexible torsions. ICMD with such a dynamic model of the protein, combined with enhanced conformational sampling method such as temperature replica exchange, allows the sampling of large scale domain motion involving high energy barrier transitions. Once these large scale conformational transitions are sampled, all-torsion, or even all-atom, MD simulations can be carried out for the low energy conformations sampled via coarse grained ICMD to calculate the energetics of distinct conformations. Such hierarchical MD simulations can be carried out with standard all-atom forcefields without the need for compromising on the accuracy of the forces. Using constraints to treat bond lengths and bond angles as rigid can, however, distort the potential energy landscape of the system and reduce the number of dihedral transitions as well as conformational sampling. We present here a two-part solution to overcome such distortions of the potential energy landscape with ICMD models. To alleviate the intrinsic distortion that stems from the reduced phase space in torsional MD, we use the Fixman compensating potential. To additionally alleviate the extrinsic distortion that arises from the coupling between the dihedral angles and bond angles within a force field, we propose a hybrid ICMD method that allows the selective relaxing of bond angles. This hybrid ICMD method bridges the gap between all-atom MD and torsional MD. We demonstrate with examples that these methods together offer a solution to eliminate the potential energy distortions encountered in constrained ICMD simulations of peptide molecules.

Insights from molecular dynamics simulations for computational protein design.

PubMed

Childers, Matthew Carter; Daggett, Valerie

2017-02-01

A grand challenge in the field of structural biology is to design and engineer proteins that exhibit targeted functions. Although much success on this front has been achieved, design success rates remain low, an ever-present reminder of our limited understanding of the relationship between amino acid sequences and the structures they adopt. In addition to experimental techniques and rational design strategies, computational methods have been employed to aid in the design and engineering of proteins. Molecular dynamics (MD) is one such method that simulates the motions of proteins according to classical dynamics. Here, we review how insights into protein dynamics derived from MD simulations have influenced the design of proteins. One of the greatest strengths of MD is its capacity to reveal information beyond what is available in the static structures deposited in the Protein Data Bank. In this regard simulations can be used to directly guide protein design by providing atomistic details of the dynamic molecular interactions contributing to protein stability and function. MD simulations can also be used as a virtual screening tool to rank, select, identify, and assess potential designs. MD is uniquely poised to inform protein design efforts where the application requires realistic models of protein dynamics and atomic level descriptions of the relationship between dynamics and function. Here, we review cases where MD simulations was used to modulate protein stability and protein function by providing information regarding the conformation(s), conformational transitions, interactions, and dynamics that govern stability and function. In addition, we discuss cases where conformations from protein folding/unfolding simulations have been exploited for protein design, yielding novel outcomes that could not be obtained from static structures.

Insights from molecular dynamics simulations for computational protein design

PubMed Central

Childers, Matthew Carter; Daggett, Valerie

2017-01-01

A grand challenge in the field of structural biology is to design and engineer proteins that exhibit targeted functions. Although much success on this front has been achieved, design success rates remain low, an ever-present reminder of our limited understanding of the relationship between amino acid sequences and the structures they adopt. In addition to experimental techniques and rational design strategies, computational methods have been employed to aid in the design and engineering of proteins. Molecular dynamics (MD) is one such method that simulates the motions of proteins according to classical dynamics. Here, we review how insights into protein dynamics derived from MD simulations have influenced the design of proteins. One of the greatest strengths of MD is its capacity to reveal information beyond what is available in the static structures deposited in the Protein Data Bank. In this regard simulations can be used to directly guide protein design by providing atomistic details of the dynamic molecular interactions contributing to protein stability and function. MD simulations can also be used as a virtual screening tool to rank, select, identify, and assess potential designs. MD is uniquely poised to inform protein design efforts where the application requires realistic models of protein dynamics and atomic level descriptions of the relationship between dynamics and function. Here, we review cases where MD simulations was used to modulate protein stability and protein function by providing information regarding the conformation(s), conformational transitions, interactions, and dynamics that govern stability and function. In addition, we discuss cases where conformations from protein folding/unfolding simulations have been exploited for protein design, yielding novel outcomes that could not be obtained from static structures. PMID:28239489

Visualizing Protein Interactions and Dynamics: Evolving a Visual Language for Molecular Animation

PubMed Central

Jenkinson, Jodie; McGill, Gaël

2012-01-01

Undergraduate biology education provides students with a number of learning challenges. Subject areas that are particularly difficult to understand include protein conformational change and stability, diffusion and random molecular motion, and molecular crowding. In this study, we examined the relative effectiveness of three-dimensional visualization techniques for learning about protein conformation and molecular motion in association with a ligand–receptor binding event. Increasingly complex versions of the same binding event were depicted in each of four animated treatments. Students (n = 131) were recruited from the undergraduate biology program at University of Toronto, Mississauga. Visualization media were developed in the Center for Molecular and Cellular Dynamics at Harvard Medical School. Stem cell factor ligand and cKit receptor tyrosine kinase were used as a classical example of a ligand-induced receptor dimerization and activation event. Each group completed a pretest, viewed one of four variants of the animation, and completed a posttest and, at 2 wk following the assessment, a delayed posttest. Overall, the most complex animation was the most effective at fostering students' understanding of the events depicted. These results suggest that, in select learning contexts, increasingly complex representations may be more desirable for conveying the dynamic nature of cell binding events. PMID:22383622

NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformers.

PubMed

Sekhar, Ashok; Kay, Lewis E

2013-08-06

The importance of dynamics to biomolecular function is becoming increasingly clear. A description of the structure-function relationship must, therefore, include the role of motion, requiring a shift in paradigm from focus on a single static 3D picture to one where a given biomolecule is considered in terms of an ensemble of interconverting conformers, each with potentially diverse activities. In this Perspective, we describe how recent developments in solution NMR spectroscopy facilitate atomic resolution studies of sparsely populated, transiently formed biomolecular conformations that exchange with the native state. Examples of how this methodology is applied to protein folding and misfolding, ligand binding, and molecular recognition are provided as a means of illustrating both the power of the new techniques and the significant roles that conformationally excited protein states play in biology.

Effects of ligand binding on the conformation and internal dynamics in specific regions of porcine pancreatic phospholipase A2 with tryptophan as a probe: a study combining time-resolved fluorescence spectroscopy and site-directed mutagenesis (same as p. 100)

NASA Astrophysics Data System (ADS)

Kuipers, Oscar; Vincent, Michel; Brochon, Jean-Claude; Verheij, Bert; de Haas, Gerard; Gallay, Jacques

1990-05-01

Exploration of the effect of ligand-protein interactions on conformational substates and internal dynamics in different regions of phospholipase A2 from porcine pancreas (PLA2), was performed by combining site-directed mutagenesis and time-resolved fluorescence measurements. The single tryptophan residue (Trp-3) in the wild type protein was replaced by a phenylalanine residue, whereafter Trp was substituted either for leucine-31 ,located in the calcium binding loop, or for phenylalanine-94, located at the "back side" of the enzyme, in a-helix E (Dijkstra et al., J. Mol. Biol., 147, 97-123, 1981). Analyses by the Maximum Entropy Method (MIEM) of the total fluorescence intensity decays, provide in each case a distribution of separate lifetime classes, which can be interpreted as reflecting the existence of discrete conformational substates in slow exchange with respect to the time-scale of the decay kinetics. The fluorescence decay of the W94 mutant is dominated by an extremely short excited state lifetime of ~60 ps, probably arising from the presence of two proximate disulfide bridges. Time-resolved fluorescence anisotropy studies show that the Trp residue near the NH2 terminus (Trp-3) undergoes a more limited rotational motion than the Trp-3 1 located in the calcium binding loop. The widest angular rotation is observed at position 94, in a-helix E. Calcium binding displays the strongest influence on the lifetime distribution of Trp-31: a major local conformation corresponding to a lifetime class with a barycenter value of ~5.5 ns and contributing to ~50% of the decay is selected. The conformations giving rise to the short lifetimes ((tau)1 and (tau)2 lifetime classes) become less important. The contribution of the third lifetime class (c3) stays at a constant value of 30%. In the presence of calcium, the amplitude of motion is wider than without the ion. There is virtually no effect of calcium binding on the lifetime distribution of the Trp residue at the 3 or the 94 position. Binding of the monomeric substrate analog n-dodecylphosphocholine (C12PN) in the presence of calcium hardly affects neither the Trp-3 excited state population distribution, nor its rotational dynamics. The binding of C12PN monomers to the W31 mutant further increases the contribution of the t4lifetime class at the expense of c2. A more restricted rotation of the Trp-31 residue is also induced. The binding of the micellar substrate analog n-hexadecylphosphocholine (C16PN) in the presence of calcium is very efficient in modifying the lifetime distribution of Trp-3. Essentially, one major broad lifetime population (centered at ~2.6 ns) is revealed by MEM analysis of the total intensity decay. The internal motion is slowed down and the angle of rotation is much smaller in this conformation. Neither the excited state lifetime distribution of Trp-31 nor its dynamics are affected by micelle binding relative to monomer binding. In conclusion, by placing a single Tip-residue at strategic positions along the peptide chain of PLA2, relevant to the binding of biological ligands, an excellent model system for the study of selective perturbations of conformational substates and internal dynamics is provided.

Effects of ligand binding on the conformation and internal dynamics in specific regions of porcine pancreatic phospholipase A2 with tryptophan as a probe: a study combinging time-resolved fluorescence spectroscopy and site-directed mutagenesis (same as p. 628)

NASA Astrophysics Data System (ADS)

Kuipers, Oscar; Vincent, Michel; Brochon, Jean-Claude; Verheij, Bert; de Haas, Gerard; Gallay, Jacques

1990-05-01

Exploration of the effect of ligand-protein interactions on conformational substates and internal dynamics in different regions of phospholipase A2 from porcine pancreas (PLA2), was performed by combining site-directed mutagenesis and time-resolved fluorescence measurements. The single tryptophan residue (Trp-3) in the wild type protein was replaced by a phenylalanine residue, whereafter Tip was substituted either for leucine-3 1 ,located in the calcium binding ioop, or for phenylalanine-94, located at the "back side" of the enzyme, in a-helix E (Dijkstra et al., J. Mol. Biol., 147, 97-123, 1981). Analyses by the Maximum Entropy Method (MEM) of the total fluorescence intensity decays, provide in each case a distribution of separate lifetime classes, which can be interpreted as reflecting the existence of discrete conformational substates in slow exchange with respect to the time-scale of the decay kinetics. The fluorescence decay of the W94 mutant is. dominated by an extremely short excited state lifetime of ~60 ps, probably arising from the presence of two proximate disulfide bridges. Time-resolved fluorescence anisotropy studies show that the Trp residue near the NH2 terminus (Trp-3) undergoes a more limited rotational motion than the Trp-3 1 located in the calcium binding loop. The widest angular rotation is observed at position 94, in a-helix E. Calcium binding displays the strongest influence on the lifetime distribution of Trp-3 1: a major local conformation corresponding to a lifetime class with a barycenter value of -5.5 ns and contributing to ~50% of the decay is selected. The conformations giving rise to the short lifetimes (τ1 and τ2 lifetime classes) become less important. The contribution of the third lifetime class (c3) stays at a constant value of 30%. In the presence of calcium, the amplitude of motion is wider than without the ion. There is virtually no effect of calcium binding on the lifetime distribution of the Trp residue at the 3 or the 94 position. Binding of the monomeric substrate analog n-dodecylphosphocholine (C12PN) in the presence of calcium hardly affects neither the Trp-3 excited state population distribution, nor its rotational dynamics. The binding of C12PN monomers to the W31 mutant further increases the contribution of the τ4 lifetime class at the expense of c2. A more restricted rotation of the Trp-3 1 residue is also induced. The binding of the micellar substrate analog n-hexadecylphosphocholine (C16PN) in the presence of calcium is very efficient in modifying the lifetime distribution of Trp-3. Essentially, one major broad lifetime population (centered at ~2.6 ns) is revealed by MEM analysis of the total intensity decay. The internal motion is slowed down and the angle of rotation is much smaller in this conformation. Neither the excited state lifetime distribution of Trp-31 nor its dynamics are affected by micelle binding relative to monomer binding. In conclusion, by placing a single Tip-residue at strategic positions along the peptide chain of PLA2, relevant to the binding of biological ligands, an excellent model system for the study of selective perturbations of conformational substates and internal dynamics is provided.

Effects of protein conformational flexibilities and electrostatic interactions on the low-frequency vibrational spectrum of hydration water.

PubMed

Pal, Somedatta; Bandyopadhyay, Sanjoy

2013-05-16

The conformational flexibility of a protein and its ability to form hydrogen bonds with water are expected to influence the microscopic properties of water layer hydrating the protein. Detailed molecular dynamics simulations with an aqueous solution of the globular protein barstar have been carried out to explore such influence on the low-frequency vibrational spectrum of the hydration water molecules. The calculations reveal that enhanced degree of confinement at the protein surface on freezing its local motions leads to increasingly restricted oscillatory motions of the hydration water molecules as evident from larger blue shifts of the corresponding band. Interestingly, conformational fluctuations of the protein and electrostatic component of its interaction with the solvent have been found to affect the transverse and longitudinal oscillations of hydration water molecules in a nonuniform manner. It is further noticed that the distributions of the low-frequency modes for the water molecules hydrogen bonded to the residues of different segments of the protein are heterogeneously altered. The effect is more around the frozen protein matrix and agrees well with slower protein-water hydrogen bond relaxations.

Probing Molecular Mechanisms of the Hsp90 Chaperone: Biophysical Modeling Identifies Key Regulators of Functional Dynamics

PubMed Central

Dixit, Anshuman; Verkhivker, Gennady M.

2012-01-01

Deciphering functional mechanisms of the Hsp90 chaperone machinery is an important objective in cancer biology aiming to facilitate discovery of targeted anti-cancer therapies. Despite significant advances in understanding structure and function of molecular chaperones, organizing molecular principles that control the relationship between conformational diversity and functional mechanisms of the Hsp90 activity lack a sufficient quantitative characterization. We combined molecular dynamics simulations, principal component analysis, the energy landscape model and structure-functional analysis of Hsp90 regulatory interactions to systematically investigate functional dynamics of the molecular chaperone. This approach has identified a network of conserved regions common to the Hsp90 chaperones that could play a universal role in coordinating functional dynamics, principal collective motions and allosteric signaling of Hsp90. We have found that these functional motifs may be utilized by the molecular chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, and protein client binding. These findings have provided support to a long-standing assertion that allosteric regulation and catalysis may have emerged via common evolutionary routes. The interaction networks regulating functional motions of Hsp90 may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based “conformational selection” of functional states is likely to be activated based on the nature of the binding partner. This mechanistic model of Hsp90 dynamics and function is consistent with the notion that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected residue clusters. Hence, allosteric signaling through a small network of distantly connected residue clusters may be a rather general functional requirement encoded across molecular chaperones. The obtained insights may be useful in guiding discovery of allosteric Hsp90 inhibitors targeting protein interfaces with co-chaperones and protein binding clients. PMID:22624053

Zipping and Unzipping of Adenylate Kinase: Atomistic Insights into the Ensemble of Open ↔ Closed Transitions

PubMed Central

Beckstein, Oliver; Denning, Elizabeth J.; Perilla, Juan R.; Woolf, Thomas B.

2009-01-01

Adenylate kinase (AdK), a phosphotransferase enzyme, plays an important role in cellular energy homeostasis. It undergoes a large conformational change between an open and a closed state, even in the absence of substrate. We investigate the apo-AdK transition at the atomic level both with free energy calculations and our new dynamic importance sampling (DIMS) molecular dynamics (MD) method. DIMS is shown to sample biologically relevant conformations as verified by comparing an ensemble of hundreds of DIMS transitions to AdK crystal structure intermediates. The simulations reveal in atomic detail how hinge regions partially and intermittently unfold during the transition. Conserved salt bridges are seen to have important structural and dynamic roles; in particular four ionic bonds are identified that open in a sequential, zipper-like fashion and thus dominate the free energy landscape of the transition. Transitions between the closed and open conformations only have to overcome moderate free energy barriers. Unexpectedly, the closed and open state encompass broad free energy basins that contain conformations differing in domain hinge motions by up to 40°. The significance of these extended states is discussed in relation to recent experimental FRET measurements. Taken together, these results demonstrate how a small number of cooperative key interactions can shape the overall dynamics of an enzyme and suggest an “all-or-nothing” mechanism for the opening and closing of AdK. Our efficient DIMS-MD computer simulation approach can provide a detailed picture of a functionally important macromolecular transition and thus help to interpret and suggest experiments to probe the conformational landscape of dynamic proteins such as AdK. PMID:19751742

A Method for Extracting the Free Energy Surface and Conformational Dynamics of Fast-Folding Proteins from Single Molecule Photon Trajectories

PubMed Central

2015-01-01

Single molecule fluorescence spectroscopy holds the promise of providing direct measurements of protein folding free energy landscapes and conformational motions. However, fulfilling this promise has been prevented by technical limitations, most notably, the difficulty in analyzing the small packets of photons per millisecond that are typically recorded from individual biomolecules. Such limitation impairs the ability to accurately determine conformational distributions and resolve sub-millisecond processes. Here we develop an analytical procedure for extracting the conformational distribution and dynamics of fast-folding proteins directly from time-stamped photon arrival trajectories produced by single molecule FRET experiments. Our procedure combines the maximum likelihood analysis originally developed by Gopich and Szabo with a statistical mechanical model that describes protein folding as diffusion on a one-dimensional free energy surface. Using stochastic kinetic simulations, we thoroughly tested the performance of the method in identifying diverse fast-folding scenarios, ranging from two-state to one-state downhill folding, as a function of relevant experimental variables such as photon count rate, amount of input data, and background noise. The tests demonstrate that the analysis can accurately retrieve the original one-dimensional free energy surface and microsecond folding dynamics in spite of the sub-megahertz photon count rates and significant background noise levels of current single molecule fluorescence experiments. Therefore, our approach provides a powerful tool for the quantitative analysis of single molecule FRET experiments of fast protein folding that is also potentially extensible to the analysis of any other biomolecular process governed by sub-millisecond conformational dynamics. PMID:25988351

A Method for Extracting the Free Energy Surface and Conformational Dynamics of Fast-Folding Proteins from Single Molecule Photon Trajectories.

PubMed

Ramanathan, Ravishankar; Muñoz, Victor

2015-06-25

Single molecule fluorescence spectroscopy holds the promise of providing direct measurements of protein folding free energy landscapes and conformational motions. However, fulfilling this promise has been prevented by technical limitations, most notably, the difficulty in analyzing the small packets of photons per millisecond that are typically recorded from individual biomolecules. Such limitation impairs the ability to accurately determine conformational distributions and resolve sub-millisecond processes. Here we develop an analytical procedure for extracting the conformational distribution and dynamics of fast-folding proteins directly from time-stamped photon arrival trajectories produced by single molecule FRET experiments. Our procedure combines the maximum likelihood analysis originally developed by Gopich and Szabo with a statistical mechanical model that describes protein folding as diffusion on a one-dimensional free energy surface. Using stochastic kinetic simulations, we thoroughly tested the performance of the method in identifying diverse fast-folding scenarios, ranging from two-state to one-state downhill folding, as a function of relevant experimental variables such as photon count rate, amount of input data, and background noise. The tests demonstrate that the analysis can accurately retrieve the original one-dimensional free energy surface and microsecond folding dynamics in spite of the sub-megahertz photon count rates and significant background noise levels of current single molecule fluorescence experiments. Therefore, our approach provides a powerful tool for the quantitative analysis of single molecule FRET experiments of fast protein folding that is also potentially extensible to the analysis of any other biomolecular process governed by sub-millisecond conformational dynamics.

A mass weighted chemical elastic network model elucidates closed form domain motions in proteins

PubMed Central

Kim, Min Hyeok; Seo, Sangjae; Jeong, Jay Il; Kim, Bum Joon; Liu, Wing Kam; Lim, Byeong Soo; Choi, Jae Boong; Kim, Moon Ki

2013-01-01

An elastic network model (ENM), usually Cα coarse-grained one, has been widely used to study protein dynamics as an alternative to classical molecular dynamics simulation. This simple approach dramatically saves the computational cost, but sometimes fails to describe a feasible conformational change due to unrealistically excessive spring connections. To overcome this limitation, we propose a mass-weighted chemical elastic network model (MWCENM) in which the total mass of each residue is assumed to be concentrated on the representative alpha carbon atom and various stiffness values are precisely assigned according to the types of chemical interactions. We test MWCENM on several well-known proteins of which both closed and open conformations are available as well as three α-helix rich proteins. Their normal mode analysis reveals that MWCENM not only generates more plausible conformational changes, especially for closed forms of proteins, but also preserves protein secondary structures thus distinguishing MWCENM from traditional ENMs. In addition, MWCENM also reduces computational burden by using a more sparse stiffness matrix. PMID:23456820

SIMS: A Hybrid Method for Rapid Conformational Analysis

PubMed Central

Gipson, Bryant; Moll, Mark; Kavraki, Lydia E.

2013-01-01

Proteins are at the root of many biological functions, often performing complex tasks as the result of large changes in their structure. Describing the exact details of these conformational changes, however, remains a central challenge for computational biology due the enormous computational requirements of the problem. This has engendered the development of a rich variety of useful methods designed to answer specific questions at different levels of spatial, temporal, and energetic resolution. These methods fall largely into two classes: physically accurate, but computationally demanding methods and fast, approximate methods. We introduce here a new hybrid modeling tool, the Structured Intuitive Move Selector (sims), designed to bridge the divide between these two classes, while allowing the benefits of both to be seamlessly integrated into a single framework. This is achieved by applying a modern motion planning algorithm, borrowed from the field of robotics, in tandem with a well-established protein modeling library. sims can combine precise energy calculations with approximate or specialized conformational sampling routines to produce rapid, yet accurate, analysis of the large-scale conformational variability of protein systems. Several key advancements are shown, including the abstract use of generically defined moves (conformational sampling methods) and an expansive probabilistic conformational exploration. We present three example problems that sims is applied to and demonstrate a rapid solution for each. These include the automatic determination of “active” residues for the hinge-based system Cyanovirin-N, exploring conformational changes involving long-range coordinated motion between non-sequential residues in Ribose-Binding Protein, and the rapid discovery of a transient conformational state of Maltose-Binding Protein, previously only determined by Molecular Dynamics. For all cases we provide energetic validations using well-established energy fields, demonstrating this framework as a fast and accurate tool for the analysis of a wide range of protein flexibility problems. PMID:23935893

Prediction and validation of protein intermediate states from structurally rich ensembles and coarse-grained simulations

NASA Astrophysics Data System (ADS)

Orellana, Laura; Yoluk, Ozge; Carrillo, Oliver; Orozco, Modesto; Lindahl, Erik

2016-08-01

Protein conformational changes are at the heart of cell functions, from signalling to ion transport. However, the transient nature of the intermediates along transition pathways hampers their experimental detection, making the underlying mechanisms elusive. Here we retrieve dynamic information on the actual transition routes from principal component analysis (PCA) of structurally-rich ensembles and, in combination with coarse-grained simulations, explore the conformational landscapes of five well-studied proteins. Modelling them as elastic networks in a hybrid elastic-network Brownian dynamics simulation (eBDIMS), we generate trajectories connecting stable end-states that spontaneously sample the crystallographic motions, predicting the structures of known intermediates along the paths. We also show that the explored non-linear routes can delimit the lowest energy passages between end-states sampled by atomistic molecular dynamics. The integrative methodology presented here provides a powerful framework to extract and expand dynamic pathway information from the Protein Data Bank, as well as to validate sampling methods in general.

Molecular dynamics coupled with a virtual system for effective conformational sampling.

PubMed

Hayami, Tomonori; Kasahara, Kota; Nakamura, Haruki; Higo, Junichi

2018-07-15

An enhanced conformational sampling method is proposed: virtual-system coupled canonical molecular dynamics (VcMD). Although VcMD enhances sampling along a reaction coordinate, this method is free from estimation of a canonical distribution function along the reaction coordinate. This method introduces a virtual system that does not necessarily obey a physical law. To enhance sampling the virtual system couples with a molecular system to be studied. Resultant snapshots produce a canonical ensemble. This method was applied to a system consisting of two short peptides in an explicit solvent. Conventional molecular dynamics simulation, which is ten times longer than VcMD, was performed along with adaptive umbrella sampling. Free-energy landscapes computed from the three simulations mutually converged well. The VcMD provided quicker association/dissociation motions of peptides than the conventional molecular dynamics did. The VcMD method is applicable to various complicated systems because of its methodological simplicity. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

Coupling between Catalytic Loop Motions and Enzyme Global Dynamics

PubMed Central

Kurkcuoglu, Zeynep; Bakan, Ahmet; Kocaman, Duygu; Bahar, Ivet; Doruker, Pemra

2012-01-01

Catalytic loop motions facilitate substrate recognition and binding in many enzymes. While these motions appear to be highly flexible, their functional significance suggests that structure-encoded preferences may play a role in selecting particular mechanisms of motions. We performed an extensive study on a set of enzymes to assess whether the collective/global dynamics, as predicted by elastic network models (ENMs), facilitates or even defines the local motions undergone by functional loops. Our dataset includes a total of 117 crystal structures for ten enzymes of different sizes and oligomerization states. Each enzyme contains a specific functional/catalytic loop (10–21 residues long) that closes over the active site during catalysis. Principal component analysis (PCA) of the available crystal structures (including apo and ligand-bound forms) for each enzyme revealed the dominant conformational changes taking place in these loops upon substrate binding. These experimentally observed loop reconfigurations are shown to be predominantly driven by energetically favored modes of motion intrinsically accessible to the enzyme in the absence of its substrate. The analysis suggests that robust global modes cooperatively defined by the overall enzyme architecture also entail local components that assist in suitable opening/closure of the catalytic loop over the active site. PMID:23028297

Structure and dynamics of GeoCyp: a thermophilic cyclophilin with a novel substrate binding mechanism that functions efficiently at low temperatures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holliday, Michael; Camilloni, Carlo; Armstrong, Geoffrey S.

2015-05-26

Thermophilic proteins have found extensive use in research and industrial applications due to their high stability and functionality at elevated temperatures while simultaneously providing valuable insight into our understanding of protein folding, stability, dynamics, and function. Cyclophilins, a ubiquitously expressed family of peptidyl-prolyl isomerases with a range of biological functions and disease associations, have been utilized both for conferring stress tolerances and in exploring the link between conformational dynamics and enzymatic function. To date, however, no active thermophilic cyclophilin has been fully biophysically characterized. Here, we determine the structure of a thermophilic cyclophilin (GeoCyp) from Geobacillus kaustophilus, characterize its dynamicmore » motions over several timescales using an array of methodologies that include chemical shift-based methods and relaxation experiments over a range of temperatures, and measure catalytic activity over a range of temperatures in order to compare structure, dynamics, and function to a mesophilic counterpart, human Cyclophilin A (CypA). Unlike most thermophile/mesophile pairs, GeoCyp catalysis is not substantially impaired at low temperatures as compared to CypA, retaining ~70% of the activity of its mesophilic counterpart. Examination of substrate-bound ensembles reveals a mechanism by which the two cyclophilins may have adapted to their environments through altering dynamic loop motions and a critical residue that acts as a clamp to regulate substrate binding differentially in CypA and GeoCyp. Despite subtle differences in conformational movements, dynamics over fast (ps-ns) and slow (μs) timescales are largely conserved between the two proteins.« less

Conformal killing tensors and covariant Hamiltonian dynamics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cariglia, M., E-mail: marco@iceb.ufop.br; Gibbons, G. W., E-mail: G.W.Gibbons@damtp.cam.ac.uk; LE STUDIUM, Loire Valley Institute for Advanced Studies, Tours and Orleans

2014-12-15

A covariant algorithm for deriving the conserved quantities for natural Hamiltonian systems is combined with the non-relativistic framework of Eisenhart, and of Duval, in which the classical trajectories arise as geodesics in a higher dimensional space-time, realized by Brinkmann manifolds. Conserved quantities which are polynomial in the momenta can be built using time-dependent conformal Killing tensors with flux. The latter are associated with terms proportional to the Hamiltonian in the lower dimensional theory and with spectrum generating algebras for higher dimensional quantities of order 1 and 2 in the momenta. Illustrations of the general theory include the Runge-Lenz vector formore » planetary motion with a time-dependent gravitational constant G(t), motion in a time-dependent electromagnetic field of a certain form, quantum dots, the Hénon-Heiles and Holt systems, respectively, providing us with Killing tensors of rank that ranges from one to six.« less

Role of dynamics in enzyme catalysis: substantial versus semantic controversies.

PubMed

Kohen, Amnon

2015-02-17

CONSPECTUS: The role of the enzyme's dynamic motions in catalysis is at the center of heated contemporary debates among both theoreticians and experimentalists. Resolving these apparent disputes is of both intellectual and practical importance: incorporation of enzyme dynamics could be critical for any calculation of enzymatic function and may have profound implications for structure-based drug design and the design of biomimetic catalysts. Analysis of the literature suggests that while part of the dispute may reflect substantial differences between theoretical approaches, much of the debate is semantic. For example, the term "protein dynamics" is often used by some researchers when addressing motions that are in thermal equilibrium with their environment, while other researchers only use this term for nonequilibrium events. The last cases are those in which thermal energy is "stored" in a specific protein mode and "used" for catalysis before it can dissipate to its environment (i.e., "nonstatistical dynamics"). This terminology issue aside, a debate has arisen among theoreticians around the roles of nonstatistical vs statistical dynamics in catalysis. However, the author knows of no experimental findings available today that examined this question in enzyme catalyzed reactions. Another source of perhaps nonsubstantial argument might stem from the varying time scales of enzymatic motions, which range from seconds to femtoseconds. Motions at different time scales play different roles in the many events along the catalytic cascade (reactant binding, reprotonation of reactants, structural rearrangement toward the transition state, product release, etc.). In several cases, when various experimental tools have been used to probe catalytic events at differing time scales, illusory contradictions seem to have emerged. In this Account, recent attempts to sort the merits of those questions are discussed along with possible future directions. A possible summary of current studies could be that enzyme, substrate, and solvent dynamics contribute to enzyme catalyzed reactions in several ways: first via mutual "induced-fit" shifting of their conformational ensemble upon binding; then via thermal search of the conformational space toward the reaction's transition-state (TS) and the rare event of the barrier crossing toward products, which is likely to be on faster time scales then the first and following events; and finally via the dynamics associated with products release, which are rate-limiting for many enzymatic reactions. From a chemical perspective, close to the TS, enzymatic systems seem to stiffen, restricting motions orthogonal to the chemical coordinate and enabling dynamics along the reaction coordinate to occur selectively. Studies of how enzymes evolved to support those efficient dynamics at various time scales are still in their infancy, and further experiments and calculations are needed to reveal these phenomena in both enzymes and uncatalyzed reactions.

Exploring transition pathway and free-energy profile of large-scale protein conformational change by combining normal mode analysis and umbrella sampling molecular dynamics.

PubMed

Wang, Jinan; Shao, Qiang; Xu, Zhijian; Liu, Yingtao; Yang, Zhuo; Cossins, Benjamin P; Jiang, Hualiang; Chen, Kaixian; Shi, Jiye; Zhu, Weiliang

2014-01-09

Large-scale conformational changes of proteins are usually associated with the binding of ligands. Because the conformational changes are often related to the biological functions of proteins, understanding the molecular mechanisms of these motions and the effects of ligand binding becomes very necessary. In the present study, we use the combination of normal-mode analysis and umbrella sampling molecular dynamics simulation to delineate the atomically detailed conformational transition pathways and the associated free-energy landscapes for three well-known protein systems, viz., adenylate kinase (AdK), calmodulin (CaM), and p38α kinase in the absence and presence of respective ligands. For each protein under study, the transient conformations along the conformational transition pathway and thermodynamic observables are in agreement with experimentally and computationally determined ones. The calculated free-energy profiles reveal that AdK and CaM are intrinsically flexible in structures without obvious energy barrier, and their ligand binding shifts the equilibrium from the ligand-free to ligand-bound conformation (population shift mechanism). In contrast, the ligand binding to p38α leads to a large change in free-energy barrier (ΔΔG ≈ 7 kcal/mol), promoting the transition from DFG-in to DFG-out conformation (induced fit mechanism). Moreover, the effect of the protonation of D168 on the conformational change of p38α is also studied, which reduces the free-energy difference between the two functional states of p38α and thus further facilitates the conformational interconversion. Therefore, the present study suggests that the detailed mechanism of ligand binding and the associated conformational transition is not uniform for all kinds of proteins but correlated to their respective biological functions.

Molecular Dynamics Simulations of the Human Glucose Transporter GLUT1

PubMed Central

Park, Min-Sun

2015-01-01

Glucose transporters (GLUTs) provide a pathway for glucose transport across membranes. Human GLUTs are implicated in devastating diseases such as heart disease, hyper- and hypo-glycemia, type 2 diabetes and caner. The human GLUT1 has been recently crystalized in the inward-facing open conformation. However, there is no other structural information for other conformations. The X-ray structures of E. coli Xylose permease (XylE), a glucose transporter homolog, are available in multiple conformations with and without the substrates D-xylose and D-glucose. XylE has high sequence homology to human GLUT1 and key residues in the sugar-binding pocket are conserved. Here we construct a homology model for human GLUT1 based on the available XylE crystal structure in the partially occluded outward-facing conformation. A long unbiased all atom molecular dynamics simulation starting from the model can capture a new fully opened outward-facing conformation. Our investigation of molecular interactions at the interface between the transmembrane (TM) domains and the intracellular helices (ICH) domain in the outward- and inward-facing conformation supports that the ICH domain likely stabilizes the outward-facing conformation in GLUT1. Furthermore, inducing a conformational transition, our simulations manifest a global asymmetric rocker switch motion and detailed molecular interactions between the substrate and residues through the water-filled selective pore along a pathway from the extracellular to the intracellular side. The results presented here are consistent with previously published biochemical, mutagenesis and functional studies. Together, this study shed light on the structure and functional relationships of GLUT1 in multiple conformational states. PMID:25919356

Excited state conformational dynamics in carotenoids: dark intermediates and excitation energy transfer.

PubMed

Beck, Warren F; Bishop, Michael M; Roscioli, Jerome D; Ghosh, Soumen; Frank, Harry A

2015-04-15

A consideration of the excited state potential energy surfaces of carotenoids develops a new hypothesis for the nature of the conformational motions that follow optical preparation of the S2 (1(1)Bu(+)) state. After an initial displacement from the Franck-Condon geometry along bond length alternation coordinates, it is suggested that carotenoids pass over a transition-state barrier leading to twisted conformations. This hypothesis leads to assignments for several dark intermediate states encountered in femtosecond spectroscopic studies. The Sx state is assigned to the structure reached upon the onset of torsional motions near the transition state barrier that divides planar and twisted structures on the S2 state potential energy surface. The X state, detected recently in two-dimensional electronic spectra, corresponds to a twisted structure well past the barrier and approaching the S2 state torsional minimum. Lastly, the S(∗) state is assigned to a low lying S1 state structure with intramolecular charge transfer character (ICT) and a pyramidal conformation. It follows that the bent and twisted structures of carotenoids that are found in photosynthetic light-harvesting proteins yield excited-state structures that favor the development of an ICT character and optimized energy transfer yields to (bacterio)chlorophyll acceptors. Copyright © 2015 Elsevier Inc. All rights reserved.

iMODS: internal coordinates normal mode analysis server.

PubMed

López-Blanco, José Ramón; Aliaga, José I; Quintana-Ortí, Enrique S; Chacón, Pablo

2014-07-01

Normal mode analysis (NMA) in internal (dihedral) coordinates naturally reproduces the collective functional motions of biological macromolecules. iMODS facilitates the exploration of such modes and generates feasible transition pathways between two homologous structures, even with large macromolecules. The distinctive internal coordinate formulation improves the efficiency of NMA and extends its applicability while implicitly maintaining stereochemistry. Vibrational analysis, motion animations and morphing trajectories can be easily carried out at different resolution scales almost interactively. The server is versatile; non-specialists can rapidly characterize potential conformational changes, whereas advanced users can customize the model resolution with multiple coarse-grained atomic representations and elastic network potentials. iMODS supports advanced visualization capabilities for illustrating collective motions, including an improved affine-model-based arrow representation of domain dynamics. The generated all-heavy-atoms conformations can be used to introduce flexibility for more advanced modeling or sampling strategies. The server is free and open to all users with no login requirement at http://imods.chaconlab.org. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Binding Leverage as a Molecular Basis for Allosteric Regulation

PubMed Central

Mitternacht, Simon; Berezovsky, Igor N.

2011-01-01

Allosteric regulation involves conformational transitions or fluctuations between a few closely related states, caused by the binding of effector molecules. We introduce a quantity called binding leverage that measures the ability of a binding site to couple to the intrinsic motions of a protein. We use Monte Carlo simulations to generate potential binding sites and either normal modes or pairs of crystal structures to describe relevant motions. We analyze single catalytic domains and multimeric allosteric enzymes with complex regulation. For the majority of the analyzed proteins, we find that both catalytic and allosteric sites have high binding leverage. Furthermore, our analysis of the catabolite activator protein, which is allosteric without conformational change, shows that its regulation involves other types of motion than those modulated at sites with high binding leverage. Our results point to the importance of incorporating dynamic information when predicting functional sites. Because it is possible to calculate binding leverage from a single crystal structure it can be used for characterizing proteins of unknown function and predicting latent allosteric sites in any protein, with implications for drug design. PMID:21935347

Celestial dynamics and astrometry in expanding universe

NASA Astrophysics Data System (ADS)

Kopeikin, Sergei

2012-08-01

Post - Newtonian theory of motion of celestial bodies and propagation of light was instrumental in conducting the critical experimental tests of general relativity and in building the astronomical ephemerides of celestial bodies in the solar system with an unparalleled precision. The cornerstone of the theory is the postulate that the solar system is gravitationally isolated from the rest of the universe and the background spacetime is asymptotically flat. The present talk abolishes this postulate and lays down the principles of celestial dynamics of particles and light moving in gravitational field of a localized astronomical system embedded to the expanding universe. We formulate the precise mathematical concept of the Newtonian limit of Einstein ’s field equations in the conformally - flat spacetime and analyse the geodesic equations of motion o f particles and light in this limit. We demonstrate that the equations of motion of particles and light can be reduced to their Newtonian counterparts by doing conformal transformations of time and space coordinates. However, the Newtonian equations for particles and light differ by terms of the first order in the Hubble constant. This leads to the important conclusion that the equations of motion used currently by Space Navigation Centres and Astronomical Observatories for calculating orbits of celestial bodies, are incomplete and missing some terms of cosmological origin. We explicitly identify the missing terms and demonstrate that they bring about a noticeable discrepancy between the observed and calculated astronomical ephemerides. We argue that a number of observed celestial anomalies in the solar system can be explained as caused by the Hubble expansion of the universe.

Simulations of Biased Agonists in the β2 Adrenergic Receptor with Accelerated Molecular Dynamics

PubMed Central

2013-01-01

The biased agonism of the G protein-coupled receptors (GPCRs), where in addition to a traditional G protein-signaling pathway a GPCR promotes intracellular signals though β-arrestin, is a novel paradigm in pharmacology. Biochemical and biophysical studies have suggested that a GPCR forms a distinct ensemble of conformations signaling through the G protein and β-arrestin. Here we report on the dynamics of the β2 adrenergic receptor bound to the β-arrestin and G protein-biased agonists and the empty receptor to further characterize the receptor conformational changes caused by biased agonists. We use conventional and accelerated molecular dynamics (aMD) simulations to explore the conformational transitions of the GPCR from the active state to the inactive state. We found that aMD simulations enable monitoring of the transition within the nanosecond time scale while capturing the known microscopic characteristics of the inactive states, such as the ionic lock, the inward position of F6.44, and water clusters. Distinct conformational states are shown to be stabilized by each biased agonist. In particular, in simulations of the receptor with the β-arrestin-biased agonist N-cyclopentylbutanepherine, we observe a different pattern of motions in helix 7 when compared to simulations with the G protein-biased agonist salbutamol that involves perturbations of the network of interactions within the NPxxY motif. Understanding the network of interactions induced by biased ligands and the subsequent receptor conformational shifts will lead to development of more efficient drugs. PMID:23879802

Conformational Dynamics of Thermus aquaticus DNA Polymerase I during Catalysis

PubMed Central

Suo, Zucai

2014-01-01

Despite the fact that DNA polymerases have been investigated for many years and are commonly used as tools in a number of molecular biology assays, many details of the kinetic mechanism they use to catalyze DNA synthesis remain unclear. Structural and kinetic studies have characterized a rapid, pre-catalytic open-to-close conformational change of the Finger domain during nucleotide binding for many DNA polymerases including Thermus aquaticus DNA polymerase I (Taq Pol), a thermostable enzyme commonly used for DNA amplification in PCR. However, little has been done to characterize the motions of other structural domains of Taq Pol or any other DNA polymerase during catalysis. Here, we used stopped-flow Förster resonance energy transfer (FRET) to investigate the conformational dynamics of all five structural domains of the full-length Taq Pol relative to the DNA substrate during nucleotide binding and incorporation. Our study provides evidence for a rapid conformational change step induced by dNTP binding and a subsequent global conformational transition involving all domains of Taq Pol during catalysis. Additionally, our study shows that the rate of the global transition was greatly increased with the truncated form of Taq Pol lacking the N-terminal domain. Finally, we utilized a mutant of Taq Pol containing a de novo disulfide bond to demonstrate that limiting protein conformational flexibility greatly reduced the polymerization activity of Taq Pol. PMID:24931550

NMR Studies of Dynamic Biomolecular Conformational Ensembles

PubMed Central

Torchia, Dennis A.

2015-01-01

Multidimensional heteronuclear NMR approaches can provide nearly complete sequential signal assignments of isotopically enriched biomolecules. The availability of assignments together with measurements of spin relaxation rates, residual spin interactions, J-couplings and chemical shifts provides information at atomic resolution about internal dynamics on timescales ranging from ps to ms, both in solution and in the solid state. However, due to the complexity of biomolecules, it is not possible to extract a unique atomic-resolution description of biomolecular motions even from extensive NMR data when many conformations are sampled on multiple timescales. For this reason, powerful computational approaches are increasingly applied to large NMR data sets to elucidate conformational ensembles sampled by biomolecules. In the past decade, considerable attention has been directed at an important class of biomolecules that function by binding to a wide variety of target molecules. Questions of current interest are: “Does the free biomolecule sample a conformational ensemble that encompasses the conformations found when it binds to various targets; and if so, on what time scale is the ensemble sampled?” This article reviews recent efforts to answer these questions, with a focus on comparing ensembles obtained for the same biomolecules by different investigators. A detailed comparison of results obtained is provided for three biomolecules: ubiquitin, calmodulin and the HIV-1 trans-activation response RNA. PMID:25669739

Entropy in molecular recognition by proteins

PubMed Central

Caro, José A.; Harpole, Kyle W.; Kasinath, Vignesh; Lim, Jackwee; Granja, Jeffrey; Valentine, Kathleen G.; Sharp, Kim A.

2017-01-01

Molecular recognition by proteins is fundamental to molecular biology. Dissection of the thermodynamic energy terms governing protein–ligand interactions has proven difficult, with determination of entropic contributions being particularly elusive. NMR relaxation measurements have suggested that changes in protein conformational entropy can be quantitatively obtained through a dynamical proxy, but the generality of this relationship has not been shown. Twenty-eight protein–ligand complexes are used to show a quantitative relationship between measures of fast side-chain motion and the underlying conformational entropy. We find that the contribution of conformational entropy can range from favorable to unfavorable, which demonstrates the potential of this thermodynamic variable to modulate protein–ligand interactions. For about one-quarter of these complexes, the absence of conformational entropy would render the resulting affinity biologically meaningless. The dynamical proxy for conformational entropy or “entropy meter” also allows for refinement of the contributions of solvent entropy and the loss in rotational-translational entropy accompanying formation of high-affinity complexes. Furthermore, structure-based application of the approach can also provide insight into long-lived specific water–protein interactions that escape the generic treatments of solvent entropy based simply on changes in accessible surface area. These results provide a comprehensive and unified view of the general role of entropy in high-affinity molecular recognition by proteins. PMID:28584100

BIM (BCL-2 interacting mediator of cell death) SAHB (stabilized α helix of BCL2) not always convinces BAX (BCL-2-associated X protein) for apoptosis.

PubMed

Verma, Sharad; Goyal, Sukriti; Tyagi, Chetna; Jamal, Salma; Singh, Aditi; Grover, Abhinav

2016-06-01

The interaction of BAX (BCL-2-associated X protein) with BIM (BCL-2 interacting mediator of cell death) SAHB (stabilized α helix of BCL2) directly initiates BAX-mediated mitochondrial apoptosis. This molecular dynamics study reveals that BIM SAHB forms a stable complex with BAX but it remains in a non-functional conformation. N terminal of BAX folds towards the core which has been reported exposed in the functional monomer. The α1-α2 loop, which has been reported in open conformation in functional BAX, acquires a closed conformation during the simulation. BH3/α2 remains less exposed as compared to initial structure. The hydrophobic residues of BIM accommodates in the rear pocket of BAX during the simulation. A steep decrease in radius of gyration and solvent accessible surface area (SASA) indicates the complex folding to acquire a more stable but inactive conformation. Further the covariance matrix reveals that the backbone atoms' motions favour the inactive conformation of the complex. This is the first report on the non-functional BAX-BIM SAHB complex by molecular dynamics simulation in the best of our knowledge. Copyright © 2016 Elsevier Inc. All rights reserved.

Entropy in molecular recognition by proteins.

PubMed

Caro, José A; Harpole, Kyle W; Kasinath, Vignesh; Lim, Jackwee; Granja, Jeffrey; Valentine, Kathleen G; Sharp, Kim A; Wand, A Joshua

2017-06-20

Molecular recognition by proteins is fundamental to molecular biology. Dissection of the thermodynamic energy terms governing protein-ligand interactions has proven difficult, with determination of entropic contributions being particularly elusive. NMR relaxation measurements have suggested that changes in protein conformational entropy can be quantitatively obtained through a dynamical proxy, but the generality of this relationship has not been shown. Twenty-eight protein-ligand complexes are used to show a quantitative relationship between measures of fast side-chain motion and the underlying conformational entropy. We find that the contribution of conformational entropy can range from favorable to unfavorable, which demonstrates the potential of this thermodynamic variable to modulate protein-ligand interactions. For about one-quarter of these complexes, the absence of conformational entropy would render the resulting affinity biologically meaningless. The dynamical proxy for conformational entropy or "entropy meter" also allows for refinement of the contributions of solvent entropy and the loss in rotational-translational entropy accompanying formation of high-affinity complexes. Furthermore, structure-based application of the approach can also provide insight into long-lived specific water-protein interactions that escape the generic treatments of solvent entropy based simply on changes in accessible surface area. These results provide a comprehensive and unified view of the general role of entropy in high-affinity molecular recognition by proteins.

Conformational Dynamics, Ligand Binding and Effects of Mutations in NirE an S-Adenosyl-L-Methionine Dependent Methyltransferase

NASA Astrophysics Data System (ADS)

Singh, Warispreet; Karabencheva-Christova, Tatyana G.; Black, Gary W.; Ainsley, Jon; Dover, Lynn; Christov, Christo Z.

2016-01-01

Heme d1, a vital tetrapyrrol involved in the denitrification processes is synthesized from its precursor molecule precorrin-2 in a chemical reaction catalysed by an S-adenosyl-L-methionine (SAM) dependent Methyltransferase (NirE). The NirE enzyme catalyses the transfer of a methyl group from the SAM to uroporphyrinogen III and serves as a novel potential drug target for the pharmaceutical industry. An important insight into the structure-activity relationships of NirE has been revealed by elucidating its crystal structure, but there is still no understanding about how conformational flexibility influences structure, cofactor and substrate binding by the enzyme as well as the structural effects of mutations of residues involved in binding and catalysis. In order to provide this missing but very important information we performed a comprehensive atomistic molecular dynamics study which revealed that i) the binding of the substrate contributes to the stabilization of the structure of the full complex; ii) conformational changes influence the orientation of the pyrrole rings in the substrate, iii) more open conformation of enzyme active site to accommodate the substrate as an outcome of conformational motions; and iv) the mutations of binding and active site residues lead to sensitive structural changes which influence binding and catalysis.

Application of time series analysis on molecular dynamics simulations of proteins: a study of different conformational spaces by principal component analysis.

PubMed

Alakent, Burak; Doruker, Pemra; Camurdan, Mehmet C

2004-09-08

Time series analysis is applied on the collective coordinates obtained from principal component analysis of independent molecular dynamics simulations of alpha-amylase inhibitor tendamistat and immunity protein of colicin E7 based on the Calpha coordinates history. Even though the principal component directions obtained for each run are considerably different, the dynamics information obtained from these runs are surprisingly similar in terms of time series models and parameters. There are two main differences in the dynamics of the two proteins: the higher density of low frequencies and the larger step sizes for the interminima motions of colicin E7 than those of alpha-amylase inhibitor, which may be attributed to the higher number of residues of colicin E7 and/or the structural differences of the two proteins. The cumulative density function of the low frequencies in each run conforms to the expectations from the normal mode analysis. When different runs of alpha-amylase inhibitor are projected on the same set of eigenvectors, it is found that principal components obtained from a certain conformational region of a protein has a moderate explanation power in other conformational regions and the local minima are similar to a certain extent, while the height of the energy barriers in between the minima significantly change. As a final remark, time series analysis tools are further exploited in this study with the motive of explaining the equilibrium fluctuations of proteins. Copyright 2004 American Institute of Physics

Application of time series analysis on molecular dynamics simulations of proteins: A study of different conformational spaces by principal component analysis

NASA Astrophysics Data System (ADS)

Alakent, Burak; Doruker, Pemra; Camurdan, Mehmet C.

2004-09-01

Time series analysis is applied on the collective coordinates obtained from principal component analysis of independent molecular dynamics simulations of α-amylase inhibitor tendamistat and immunity protein of colicin E7 based on the Cα coordinates history. Even though the principal component directions obtained for each run are considerably different, the dynamics information obtained from these runs are surprisingly similar in terms of time series models and parameters. There are two main differences in the dynamics of the two proteins: the higher density of low frequencies and the larger step sizes for the interminima motions of colicin E7 than those of α-amylase inhibitor, which may be attributed to the higher number of residues of colicin E7 and/or the structural differences of the two proteins. The cumulative density function of the low frequencies in each run conforms to the expectations from the normal mode analysis. When different runs of α-amylase inhibitor are projected on the same set of eigenvectors, it is found that principal components obtained from a certain conformational region of a protein has a moderate explanation power in other conformational regions and the local minima are similar to a certain extent, while the height of the energy barriers in between the minima significantly change. As a final remark, time series analysis tools are further exploited in this study with the motive of explaining the equilibrium fluctuations of proteins.

Ultrafast Hydration Dynamics and Coupled Water-Protein Fluctuations in Apomyoglobin

NASA Astrophysics Data System (ADS)

Yang, Yi; Zhang, Luyuan; Wang, Lijuan; Zhong, Dongping

2009-06-01

Protein hydration dynamics are of fundamental importance to its structure and function. Here, we characterize the global solvation dynamics and anisotropy dynamics around the apomyoglobin surface in different conformational states (native and molten globule) by measuring the Stokes shift and anisotropy decay of tryptophan with femtosecond-resolved fluorescence upconversion. With site-directed mutagenesis, we designed sixteen mutants with one tryptophan in each, and placed the probe at a desirable position ranging from buried in the protein core to fully solvent-exposed on the protein surface. In all protein sites studied, two distinct solvation relaxations (1-8 ps and 20-200 ps) were observed, reflecting the initial collective water relaxation and subsequent hydrogen-bond network restructuring, respectively, and both are strongly correlated with protein's local structures and chemical properties. The hydration dynamics of the mutants in molten globule state are faster than those observed in native state, indicating that the protein becomes more flexible and less structured when its conformation is converted from fully-folded native state to partially-folded molten globule state. Complementary, fluorescence anisotropy dynamics of all mutants in native state show an increasing trend of wobbling times (40-260 ps) when the location of the probe is changed from a loop, to a lateral helix, and then, to the compact protein core. Such an increase in wobbling times is related to the local protein structural rigidity, which relates the interaction of water with side chains. The ultrafast hydration dynamics and related side-chain motion around the protein surface unravel the coupled water-protein fluctuations on the picosecond time scales and indicate that the local protein motions are slaved by hydrating water fluctuations.

Single molecule imaging of conformational dynamics in sodium-coupled transporters

NASA Astrophysics Data System (ADS)

Terry, Daniel S.

Neurotransmitter:sodium symporter (NSS) proteins remove neurotransmitters released into the synapse through a transport process driven by the physiological sodium ion (Na+) gradient. NSSs for dopamine, noradrenaline, and serotonin are targeted by the psychostimulants cocaine and amphetamines, as well as by antidepressants. The crystal structure of LeuT, a prokaryotic NSS homologue, revealed the NSS molecular architecture and has been the basis for extensive structural, biochemical, and computational investigations of the mechanism of transporter proteins with a LeuT-like fold. In this dissertation, the conformational states sampled by LeuT are explored using single-molecule fluorescence resonance energy transfer imaging methods, with special focus on the motions of transmembrane helix 1a that lead to inward release of substrate. We also explored how dynamics are modulated by substrate, Na+, and protons to produce efficient transport. These advances represent a first of a kind study of the dynamics of an integral membrane protein at a truly single-molecule scale. Advances in instrumentation, analysis tools, and organic fluorophores were all required to achieve these goals, and such advances are also described. While these experiments were performed with detergent-solubilized protein, preliminary work suggests that imaging of LeuT in proteoliposomes is feasible and a fluorescence sensor assay could be used to simultaneously detect conformational dynamics and transport function.

Evaluation of dose coverage to target volume and normal tissue sparing in the adjuvant radiotherapy of gastric cancers: 3D-CRT compared with dynamic IMRT.

PubMed

Murthy, Kk; Shukeili, Ka; Kumar, Ss; Davis, Ca; Chandran, Rr; Namrata, S

2010-01-01

To assess the potential advantage of intensity-modulated radiotherapy (IMRT) over 3D-conformal radiotherapy (3D-CRT) planning in postoperative adjuvant radiotherapy for patients with gastric carcinoma. In a retrospective study, for plan comparison, dose distribution was recalculated in 15 patients treated with 3D-CRT on the contoured structures of same CT images using an IMRT technique. 3D-conformal plans with three fields and four-fields were compared with seven-field dynamic IMRT plans. The different plans were compared by analyzing the dose coverage of planning target volume using TV(95), D(mean), uniformity index, conformity index and homogeneity index parameters. To assess critical organ sparing, D(mean), D(max), dose to one-third and two-third volumes of the OARs and percentage of volumes receiving more than their tolerance doses were compared. The average dose coverage values of PTV with 3F-CRT and 4F-CRT plans were comparable, where as IMRT plans achieved better target coverage(p<0.001) with higher conformity index value of 0.81±0.07 compared to both the 3D-CRT plans. The doses to the liver and bowel reduced significantly (p<0.001) with IMRT plans compared to other 3D-CRT plans. For all OARs the percentage of volumes receiving more than their tolerance doses were reduced with the IMRT plans. This study showed that a better target coverage and significant dose reduction to OARs could be achieved with the IMRT plans. The IMRT can be preferred with caution for organ motion. The authors are currently studying organ motion in the upper abdomen to use IMRT for patient treatment.

Single-molecule FRET reveals the energy landscape of the full-length SAM-I riboswitch.

PubMed

Manz, Christoph; Kobitski, Andrei Yu; Samanta, Ayan; Keller, Bettina G; Jäschke, Andres; Nienhaus, G Ulrich

2017-11-01

S-adenosyl-L-methionine (SAM) ligand binding induces major structural changes in SAM-I riboswitches, through which gene expression is regulated via transcription termination. Little is known about the conformations and motions governing the function of the full-length Bacillus subtilis yitJ SAM-I riboswitch. Therefore, we have explored its conformational energy landscape as a function of Mg 2+ and SAM ligand concentrations using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling analysis. We resolved four conformational states both in the presence and the absence of SAM and determined their Mg 2+ -dependent fractional populations and conformational dynamics, including state lifetimes, interconversion rate coefficients and equilibration timescales. Riboswitches with terminator and antiterminator folds coexist, and SAM binding only gradually shifts the populations toward terminator states. We observed a pronounced acceleration of conformational transitions upon SAM binding, which may be crucial for off-switching during the brief decision window before expression of the downstream gene.

Dosimetric impact of geometric errors due to respiratory motion prediction on dynamic multileaf collimator-based four-dimensional radiation delivery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vedam, S.; Docef, A.; Fix, M.

2005-06-15

The synchronization of dynamic multileaf collimator (DMLC) response with respiratory motion is critical to ensure the accuracy of DMLC-based four dimensional (4D) radiation delivery. In practice, however, a finite time delay (response time) between the acquisition of tumor position and multileaf collimator response necessitates predictive models of respiratory tumor motion to synchronize radiation delivery. Predicting a complex process such as respiratory motion introduces geometric errors, which have been reported in several publications. However, the dosimetric effect of such errors on 4D radiation delivery has not yet been investigated. Thus, our aim in this work was to quantify the dosimetric effectsmore » of geometric error due to prediction under several different conditions. Conformal and intensity modulated radiation therapy (IMRT) plans for a lung patient were generated for anterior-posterior/posterior-anterior (AP/PA) beam arrangements at 6 and 18 MV energies to provide planned dose distributions. Respiratory motion data was obtained from 60 diaphragm-motion fluoroscopy recordings from five patients. A linear adaptive filter was employed to predict the tumor position. The geometric error of prediction was defined as the absolute difference between predicted and actual positions at each diaphragm position. Distributions of geometric error of prediction were obtained for all of the respiratory motion data. Planned dose distributions were then convolved with distributions for the geometric error of prediction to obtain convolved dose distributions. The dosimetric effect of such geometric errors was determined as a function of several variables: response time (0-0.6 s), beam energy (6/18 MV), treatment delivery (3D/4D), treatment type (conformal/IMRT), beam direction (AP/PA), and breathing training type (free breathing/audio instruction/visual feedback). Dose difference and distance-to-agreement analysis was employed to quantify results. Based on our data, the dosimetric impact of prediction (a) increased with response time, (b) was larger for 3D radiation therapy as compared with 4D radiation therapy, (c) was relatively insensitive to change in beam energy and beam direction, (d) was greater for IMRT distributions as compared with conformal distributions, (e) was smaller than the dosimetric impact of latency, and (f) was greatest for respiration motion with audio instructions, followed by visual feedback and free breathing. Geometric errors of prediction that occur during 4D radiation delivery introduce dosimetric errors that are dependent on several factors, such as response time, treatment-delivery type, and beam energy. Even for relatively small response times of 0.6 s into the future, dosimetric errors due to prediction could approach delivery errors when respiratory motion is not accounted for at all. To reduce the dosimetric impact, better predictive models and/or shorter response times are required.« less

Heterogeneity and dynamics of the ligand recognition mode in purine-sensing riboswitches.

PubMed

Jain, Niyati; Zhao, Liang; Liu, John D; Xia, Tianbing

2010-05-04

High-resolution crystal structures and biophysical analyses of purine-sensing riboswitches have revealed that a network of hydrogen bonding interactions appear to be largey responsible for discrimination of cognate ligands against structurally related compounds. Here we report that by using femtosecond time-resolved fluorescence spectroscopy to capture the ultrafast decay dynamics of the 2-aminopurine base as the ligand, we have detected the presence of multiple conformations of the ligand within the binding pockets of one guanine-sensing and two adenine-sensing riboswitches. All three riboswitches have similar conformational distributions of the ligand-bound state. The known crystal structures represent the global minimum that accounts for 50-60% of the population, where there is no significant stacking interaction between the ligand and bases of the binding pocket, but the hydrogen-bonding cage collectively provides an electronic environment that promotes an ultrafast ( approximately 1 ps) charge transfer pathway. The ligand also samples multiple conformations in which it significantly stacks with either the adenine or the uracil bases of the A21-U75 and A52-U22 base pairs that form the ceiling and floor of the binding pocket, respectively, but favors the larger adenine bases. These alternative conformations with well-defined base stacking interactions are approximately 1-1.5 kcal/mol higher in DeltaG degrees than the global minimum and have distinct charge transfer dynamics within the picosecond to nanosecond time regime. Inside the pocket, the purine ligand undergoes dynamic motion on the low nanosecond time scale, sampling the multiple conformations based on time-resolved anisotropy decay dynamics. These results allowed a description of the energy landscape of the bound ligand with intricate details and demonstrated the elastic nature of the ligand recognition mode by the purine-sensing riboswitches, where there is a dynamic balance between hydrogen bonding and base stacking interactions, yielding the high affinity and specificity by the aptamer domain.

Fluorescence spectroscopic characterization of Humicola lanuginosa lipase dissolved in its substrate.

PubMed

Jutila, Arimatti; Zhu, Keng; Tuominen, Esa K J; Kinnunen, Paavo K J

2004-11-01

The conformational dynamics of Humicola lanuginosa lipases (HLL) and its three mutants were investigated by steady state and time-resolved fluorescence spectroscopy in two different media, aqueous buffer and the substrate triacetin. The fluorescence of the four Trps of the wild-type HLL (wt) reports on the global changes of the whole lipase molecule. In order to monitor conformational changes specifically in the alpha-helical surface loop, the so-called 'lid' of HLL comprised of residues 86-93, the single Trp mutant W89m (W117F, W221H, W260H) was employed. Mutants W89L and W89mN33Q (W117F, W221H, W260H, N33Q) were used to survey the impact of Trp89 and mannose residues, respectively. Based on the data obtained, the following conclusions can be drawn. (i) HLL adapts the 'open' conformation in triacetin, with the alpha-helical surface loop moving so as to expose the active site. (ii) Trp89 contained in the lid plays an unprecedently important role in the structural stability of HLL. (iii) In triacetin, but not in the buffer, the motion of the Trp89 side chain becomes distinguishable from the motion of the lid. (iv) The carbohydrate moiety at Asn33 has only minor effects on the dynamics of Trp89 in the lid as judged from the fluorescence characteristics of the latter residue.

Motions and entropies in proteins as seen in NMR relaxation experiments and molecular dynamics simulations.

PubMed

Allnér, Olof; Foloppe, Nicolas; Nilsson, Lennart

2015-01-22

Molecular dynamics simulations of E. coli glutaredoxin1 in water have been performed to relate the dynamical parameters and entropy obtained in NMR relaxation experiments, with results extracted from simulated trajectory data. NMR relaxation is the most widely used experimental method to obtain data on dynamics of proteins, but it is limited to relatively short timescales and to motions of backbone amides or in some cases (13)C-H vectors. By relating the experimental data to the all-atom picture obtained in molecular dynamics simulations, valuable insights on the interpretation of the experiment can be gained. We have estimated the internal dynamics and their timescales by calculating the generalized order parameters (O) for different time windows. We then calculate the quasiharmonic entropy (S) and compare it to the entropy calculated from the NMR-derived generalized order parameter of the amide vectors. Special emphasis is put on characterizing dynamics that are not expressed through the motions of the amide group. The NMR and MD methods suffer from complementary limitations, with NMR being restricted to local vectors and dynamics on a timescale determined by the rotational diffusion of the solute, while in simulations, it may be difficult to obtain sufficient sampling to ensure convergence of the results. We also evaluate the amount of sampling obtained with molecular dynamics simulations and how it is affected by the length of individual simulations, by clustering of the sampled conformations. We find that two structural turns act as hinges, allowing the α helix between them to undergo large, long timescale motions that cannot be detected in the time window of the NMR dipolar relaxation experiments. We also show that the entropy obtained from the amide vector does not account for correlated motions of adjacent residues. Finally, we show that the sampling in a total of 100 ns molecular dynamics simulation can be increased by around 50%, by dividing the trajectory into 10 replicas with different starting velocities.

New Insights into Active Site Conformation Dynamics of E. coli PNP Revealed by Combined H/D Exchange Approach and Molecular Dynamics Simulations.

PubMed

Kazazić, Saša; Bertoša, Branimir; Luić, Marija; Mikleušević, Goran; Tarnowski, Krzysztof; Dadlez, Michal; Narczyk, Marta; Bzowska, Agnieszka

2016-01-01

The biologically active form of purine nucleoside phosphorylase (PNP) from Escherichia coli (EC 2.4.2.1) is a homohexamer unit, assembled as a trimer of dimers. Upon binding of phosphate, neighboring monomers adopt different active site conformations, described as open and closed. To get insight into the functions of the two distinctive active site conformations, virtually inactive Arg24Ala mutant is complexed with phosphate; all active sites are found to be in the open conformation. To understand how the sites of neighboring monomers communicate with each other, we have combined H/D exchange (H/DX) experiments with molecular dynamics (MD) simulations. Both methods point to the mobility of the enzyme, associated with a few flexible regions situated at the surface and within the dimer interface. Although H/DX provides an average extent of deuterium uptake for all six hexamer active sites, it was able to indicate the dynamic mechanism of cross-talk between monomers, allostery. Using this technique, it was found that phosphate binding to the wild type (WT) causes arrest of the molecular motion in backbone fragments that are flexible in a ligand-free state. This was not the case for the Arg24Ala mutant. Upon nucleoside substrate/inhibitor binding, some release of the phosphate-induced arrest is observed for the WT, whereas the opposite effects occur for the Arg24Ala mutant. MD simulations confirmed that phosphate is bound tightly in the closed active sites of the WT; conversely, in the open conformation of the active site of the WT phosphate is bound loosely moving towards the exit of the active site. In Arg24Ala mutant binary complex Pi is bound loosely, too.

Conformal collineations and anisotropic fluids in general relativity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duggal, K.L.; Sharma, R.

1986-10-01

Recently, Herrera et al. (L. Herrera, J. Jimenez, L. Leal, J. Ponce de Leon, M. Esculpi, and V. Galino, J. Math. Phys. 25, 3274 (1984)) studied the consequences of the existence of a one-parameter group of conformal motions for anisotropic matter. They concluded that for special conformal motions, the stiff equation of state (p = ..mu..) is singled out in a unique way, provided the generating conformal vector field is orthogonal to the four-velocity. In this paper, the same problem is studied by using conformal collineations (which include conformal motions as subgroups). It is shown that, for a special conformalmore » collineation, the stiff equation of state is not singled out. Non-Einstein Ricci-recurrent spaces are considered as physical models for the fluid matter.« less

Dynamics and conformations of PEO chains chemically bonded on silica: comparison between 1H and 2H NMR.

PubMed

Tajouri, T; Hommel, H

2007-06-01

1H NMR was used to study the motion of monomer units in a layer of poly(ethylene oxide) chains grafted on silica. First, the dependence of the relaxation times on the grafting ratios is discussed qualitatively from a phenomenological point of view. Next, the NMR line narrowing effect by high-speed rotation is observed in the same samples with different grafting ratios. The magic angle spinning technique permits determination of two correlation times for each grafting ratio: tau(c) characteristic of an environment with a fast motion and tau(l) characteristic of an environment with a slow motion. In addition, the dynamics of these grafted chains are investigated by deuterium NMR (2H NMR), which is sensitive to the anisotropy of molecular motion. The evolution has been studied for two extreme grafting ratios and each time as a function of temperature. The anisotropy is more marked at low temperatures and for a low grafting ratio. The results are consistent with the 1H NMR relaxation times measured as a function of temperature. Copyright 2007 John Wiley & Sons, Ltd.

MovieMaker: a web server for rapid rendering of protein motions and interactions

PubMed Central

Maiti, Rajarshi; Van Domselaar, Gary H.; Wishart, David S.

2005-01-01

MovieMaker is a web server that allows short (∼10 s), downloadable movies of protein motions to be generated. It accepts PDB files or PDB accession numbers as input and automatically calculates, renders and merges the necessary image files to create colourful animations covering a wide range of protein motions and other dynamic processes. Users have the option of animating (i) simple rotation, (ii) morphing between two end-state conformers, (iii) short-scale, picosecond vibrations, (iv) ligand docking, (v) protein oligomerization, (vi) mid-scale nanosecond (ensemble) motions and (vii) protein folding/unfolding. MovieMaker does not perform molecular dynamics calculations. Instead it is an animation tool that uses a sophisticated superpositioning algorithm in conjunction with Cartesian coordinate interpolation to rapidly and automatically calculate the intermediate structures needed for many of its animations. Users have extensive control over the rendering style, structure colour, animation quality, background and other image features. MovieMaker is intended to be a general-purpose server that allows both experts and non-experts to easily generate useful, informative protein animations for educational and illustrative purposes. MovieMaker is accessible at . PMID:15980488

Imaging of conformational changes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Michl, Josef

2016-03-13

Control of intramolecular conformational change in a small number of molecules or even a single one by an application of an outside electric field defined by potentials on nearby metal or dielectric surfaces has potential applications in both 3-D and 2-D nanotechnology. Specifically, the synthesis, characterization, and understanding of designed solids with controlled built-in internal rotational motion of a dipole promises a new class of materials with intrinsic dielectric, ferroelectric, optical and optoelectronic properties not found in nature. Controlled rotational motion is of great interest due to its expected utility in phenomena as diverse as transport, current flow in molecularmore » junctions, diffusion in microfluidic channels, and rotary motion in molecular machines. A direct time-resolved observation of the dynamics of motion on ps or ns time scale in a single molecule would be highly interesting but is also very difficult and has yet to be accomplished. Much can be learned from an easier but still challenging comparison of directly observed initial and final orientational states of a single molecule, which is the basis of this project. The project also impacts the understanding of surface-enhanced Raman spectroscopy (SERS) and single-molecule spectroscopic detection, as well as the synthesis of solid-state materials with tailored properties from designed precursors.« less

Role of Dynamics in Enzyme Catalysis: Substantial versus Semantic Controversies

PubMed Central

2015-01-01

Conspectus The role of the enzyme’s dynamic motions in catalysis is at the center of heated contemporary debates among both theoreticians and experimentalists. Resolving these apparent disputes is of both intellectual and practical importance: incorporation of enzyme dynamics could be critical for any calculation of enzymatic function and may have profound implications for structure-based drug design and the design of biomimetic catalysts. Analysis of the literature suggests that while part of the dispute may reflect substantial differences between theoretical approaches, much of the debate is semantic. For example, the term “protein dynamics” is often used by some researchers when addressing motions that are in thermal equilibrium with their environment, while other researchers only use this term for nonequilibrium events. The last cases are those in which thermal energy is “stored” in a specific protein mode and “used” for catalysis before it can dissipate to its environment (i.e., “nonstatistical dynamics”). This terminology issue aside, a debate has arisen among theoreticians around the roles of nonstatistical vs statistical dynamics in catalysis. However, the author knows of no experimental findings available today that examined this question in enzyme catalyzed reactions. Another source of perhaps nonsubstantial argument might stem from the varying time scales of enzymatic motions, which range from seconds to femtoseconds. Motions at different time scales play different roles in the many events along the catalytic cascade (reactant binding, reprotonation of reactants, structural rearrangement toward the transition state, product release, etc.). In several cases, when various experimental tools have been used to probe catalytic events at differing time scales, illusory contradictions seem to have emerged. In this Account, recent attempts to sort the merits of those questions are discussed along with possible future directions. A possible summary of current studies could be that enzyme, substrate, and solvent dynamics contribute to enzyme catalyzed reactions in several ways: first via mutual “induced-fit” shifting of their conformational ensemble upon binding; then via thermal search of the conformational space toward the reaction’s transition-state (TS) and the rare event of the barrier crossing toward products, which is likely to be on faster time scales then the first and following events; and finally via the dynamics associated with products release, which are rate-limiting for many enzymatic reactions. From a chemical perspective, close to the TS, enzymatic systems seem to stiffen, restricting motions orthogonal to the chemical coordinate and enabling dynamics along the reaction coordinate to occur selectively. Studies of how enzymes evolved to support those efficient dynamics at various time scales are still in their infancy, and further experiments and calculations are needed to reveal these phenomena in both enzymes and uncatalyzed reactions. PMID:25539442

Probing the structural dynamics of the CRISPR-Cas9 RNA-guided DNA-cleavage system by coarse-grained modeling.

PubMed

Zheng, Wenjun

2017-02-01

In the adaptive immune systems of many bacteria and archaea, the Cas9 endonuclease forms a complex with specific guide/scaffold RNA to identify and cleave complementary target sequences in foreign DNA. This DNA targeting machinery has been exploited in numerous applications of genome editing and transcription control. However, the molecular mechanism of the Cas9 system is still obscure. Recently, high-resolution structures have been solved for Cas9 in different structural forms (e.g., unbound forms, RNA-bound binary complexes, and RNA-DNA-bound tertiary complexes, corresponding to an inactive state, a pre-target-bound state, and a cleavage-competent or product state), which offered key structural insights to the Cas9 mechanism. To further probe the structural dynamics of Cas9 interacting with RNA and DNA at the amino-acid level of details, we have performed systematic coarse-grained modeling using an elastic network model and related analyses. Our normal mode analysis predicted a few key modes of collective motions that capture the observed conformational changes featuring large domain motions triggered by binding of RNA and DNA. Our flexibility analysis identified specific regions with high or low flexibility that coincide with key functional sites (such as DNA/RNA-binding sites, nuclease cleavage sites, and key hinges). We also identified a small set of hotspot residues that control the energetics of functional motions, which overlap with known functional sites and offer promising targets for future mutagenesis efforts to improve the specificity of Cas9. Finally, we modeled the conformational transitions of Cas9 from the unbound form to the binary complex and then the tertiary complex, and predicted a distinct sequence of domain motions. In sum, our findings have offered rich structural and dynamic details relevant to the Cas9 machinery, and will guide future investigation and engineering of the Cas9 systems. Proteins 2017; 85:342-353. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Probing Selection Mechanism of the Most Favorable Conformation of a Dipeptide in Chaotropic and Kosmotropic Solution.

PubMed

Jas, Gouri S; Middaugh, C Russell; Kuczera, Krzysztof

2016-07-21

Chaotropes like urea and guanidinium chloride (GdmCl) tend to destabilize, and kosmotropes like proline tend to stabilize folded structures of peptides and proteins. Here, we combine fluorescence anisotropy decay measurements and molecular dynamics simulations to gain a microscopic understanding of the molecular mechanism for shifting conformational preferences in aqueous, GdmCl, urea, and proline solutions of a simple model dipeptide, N-acetyl-tryptophan-amide (NATA). Measured anisotropy decay of NATA as a function of temperature, pH, and cosolvent concentrations showed reorientations moderately slower in GdmCl and urea and substantially slower in proline compared to those of aqueous environment. A small change in pH significantly slows orientation time in water and GdmCl and less markedly in urea. Computationally, we use molecular dynamics with dihedral restraints to separately analyze the motions and interactions of the representative NATA conformers in the four different solvent environments. This novel analysis provides a dissection of the observed overall diffusion rates into contributions from individual dipeptide conformations. The variation of rotational diffusion rates with conformation are quite large. Population-weighted averaging or using properties of the major cluster reproduces the dynamical features of the full unrestrained dynamics. Additionally, we correlate the observable diffusion rates with microscopic features of conformer size, shape, and solvation. This analysis uncovered underlying differences in detailed atomistic behavior of the three cosolvents-urea, GdmCl, and proline. For both urea and the pure water system we find good agreement with hydrodynamic theory, with diffusion rates primarily correlated with conformer size and shape. In contrast, for GdmCl and proline solutions, the variation in conformer diffusion rates was mostly determined by specific interactions with the cosolvents. We also find preferences for different molecular shapes by the three cosolvents, with increased preferential solvation of smaller and more spherical conformers by urea and larger and more elongated conformers by GdmCl and proline. Additionally, our results provide a basis for a simple approximate model of the effects of pH lowering on dipeptide conformational equilibria. The translational diffusion rates of NATA are less sensitive to conformations, but variation with solvation strength is similar to rotational diffusion. Our results, combining experiment and simulation, show that we can identify the individual peptide conformers with definite microscopic properties of shape, size, and solvation, that are responsible for producing physical observables, such as translational and orientational diffusion in the complex solvent environments of denaturants and osmolytes.

Conformational Fluctuations in G-Protein-Coupled Receptors

NASA Astrophysics Data System (ADS)

Brown, Michael F.

2014-03-01

G-protein-coupled receptors (GPCRs) comprise almost 50% of pharmaceutical drug targets, where rhodopsin is an important prototype and occurs naturally in a lipid membrane. Rhodopsin photoactivation entails 11-cis to all-trans isomerization of the retinal cofactor, yielding an equilibrium between inactive Meta-I and active Meta-II states. Two important questions are: (1) Is rhodopsin is a simple two-state switch? Or (2) does isomerization of retinal unlock an activated conformational ensemble? For an ensemble-based activation mechanism (EAM) a role for conformational fluctuations is clearly indicated. Solid-state NMR data together with theoretical molecular dynamics (MD) simulations detect increased local mobility of retinal after light activation. Resultant changes in local dynamics of the cofactor initiate large-scale fluctuations of transmembrane helices that expose recognition sites for the signal-transducing G-protein. Time-resolved FTIR studies and electronic spectroscopy further show the conformational ensemble is strongly biased by the membrane lipid composition, as well as pH and osmotic pressure. A new flexible surface model (FSM) describes how the curvature stress field of the membrane governs the energetics of active rhodopsin, due to the spontaneous monolayer curvature of the lipids. Furthermore, influences of osmotic pressure dictate that a large number of bulk water molecules are implicated in rhodopsin activation. Around 60 bulk water molecules activate rhodopsin, which is much larger than the number of structural waters seen in X-ray crystallography, or inferred from studies of bulk hydrostatic pressure. Conformational selection and promoting vibrational motions of rhodopsin lead to activation of the G-protein (transducin). Our biophysical data give a paradigm shift in understanding GPCR activation. The new view is: dynamics and conformational fluctuations involve an ensemble of substates that activate the cognate G-protein in the amplified visual response.

Striking Plasticity of CRISPR-Cas9 and Key Role of Non-target DNA, as Revealed by Molecular Simulations.

PubMed

Palermo, Giulia; Miao, Yinglong; Walker, Ross C; Jinek, Martin; McCammon, J Andrew

2016-10-26

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system recently emerged as a transformative genome-editing technology that is innovating basic bioscience and applied medicine and biotechnology. The endonuclease Cas9 associates with a guide RNA to match and cleave complementary sequences in double stranded DNA, forming an RNA:DNA hybrid and a displaced non-target DNA strand. Although extensive structural studies are ongoing, the conformational dynamics of Cas9 and its interplay with the nucleic acids during association and DNA cleavage are largely unclear. Here, by employing multi-microsecond time scale molecular dynamics, we reveal the conformational plasticity of Cas9 and identify key determinants that allow its large-scale conformational changes during nucleic acid binding and processing. We show how the "closure" of the protein, which accompanies nucleic acid binding, fundamentally relies on highly coupled and specific motions of the protein domains, collectively initiating the prominent conformational changes needed for nucleic acid association. We further reveal a key role of the non-target DNA during the process of activation of the nuclease HNH domain, showing how the nontarget DNA positioning triggers local conformational changes that favor the formation of a catalytically competent Cas9. Finally, a remarkable conformational plasticity is identified as an intrinsic property of the HNH domain, constituting a necessary element that allows for the HNH repositioning. These novel findings constitute a reference for future experimental studies aimed at a full characterization of the dynamic features of the CRISPR-Cas9 system, and-more importantly-call for novel structure engineering efforts that are of fundamental importance for the rational design of new genome-engineering applications.

Loss of conformational entropy in protein folding calculated using realistic ensembles and its implications for NMR-based calculations

PubMed Central

Baxa, Michael C.; Haddadian, Esmael J.; Jumper, John M.; Freed, Karl F.; Sosnick, Tobin R.

2014-01-01

The loss of conformational entropy is a major contribution in the thermodynamics of protein folding. However, accurate determination of the quantity has proven challenging. We calculate this loss using molecular dynamic simulations of both the native protein and a realistic denatured state ensemble. For ubiquitin, the total change in entropy is TΔSTotal = 1.4 kcal⋅mol−1 per residue at 300 K with only 20% from the loss of side-chain entropy. Our analysis exhibits mixed agreement with prior studies because of the use of more accurate ensembles and contributions from correlated motions. Buried side chains lose only a factor of 1.4 in the number of conformations available per rotamer upon folding (ΩU/ΩN). The entropy loss for helical and sheet residues differs due to the smaller motions of helical residues (TΔShelix−sheet = 0.5 kcal⋅mol−1), a property not fully reflected in the amide N-H and carbonyl C=O bond NMR order parameters. The results have implications for the thermodynamics of folding and binding, including estimates of solvent ordering and microscopic entropies obtained from NMR. PMID:25313044

Machine Learning and Network Analysis of Molecular Dynamics Trajectories Reveal Two Chains of Red/Ox-specific Residue Interactions in Human Protein Disulfide Isomerase.

PubMed

Karamzadeh, Razieh; Karimi-Jafari, Mohammad Hossein; Sharifi-Zarchi, Ali; Chitsaz, Hamidreza; Salekdeh, Ghasem Hosseini; Moosavi-Movahedi, Ali Akbar

2017-06-16

The human protein disulfide isomerase (hPDI), is an essential four-domain multifunctional enzyme. As a result of disulfide shuffling in its terminal domains, hPDI exists in two oxidation states with different conformational preferences which are important for substrate binding and functional activities. Here, we address the redox-dependent conformational dynamics of hPDI through molecular dynamics (MD) simulations. Collective domain motions are identified by the principal component analysis of MD trajectories and redox-dependent opening-closing structure variations are highlighted on projected free energy landscapes. Then, important structural features that exhibit considerable differences in dynamics of redox states are extracted by statistical machine learning methods. Mapping the structural variations to time series of residue interaction networks also provides a holistic representation of the dynamical redox differences. With emphasizing on persistent long-lasting interactions, an approach is proposed that compiled these time series networks to a single dynamic residue interaction network (DRIN). Differential comparison of DRIN in oxidized and reduced states reveals chains of residue interactions that represent potential allosteric paths between catalytic and ligand binding sites of hPDI.

Structures and transport dynamics of a Campylobacter jejuni multidrug efflux pump

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Chih-Chia; Yin, Linxiang; Kumar, Nitin

2017-08-01

Resistance-nodulation-cell division efflux pumps are integral membrane proteins that catalyze the export of substrates across cell membranes. Within the hydrophobe-amphiphile efflux subfamily, these resistance-nodulation-cell division proteins largely form trimeric efflux pumps. The drug efflux process has been proposed to entail a synchronized motion between subunits of the trimer to advance the transport cycle, leading to the extrusion of drug molecules. Here we use X-ray crystallography and single-molecule fluorescence resonance energy transfer imaging to elucidate the structures and functional dynamics of the Campylobacter jejuni CmeB multidrug efflux pump. We find that the CmeB trimer displays a very unique conformation. A directmore » observation of transport dynamics in individual CmeB trimers embedded in membrane vesicles indicates that each CmeB subunit undergoes conformational transitions uncoordinated and independent of each other. On the basis of our findings and analyses, we propose a model for transport mechanism where CmeB protomers function independently within the trimer.« less

Dynamics simulation on the flexibility and backbone motions of HP1 chromodomain bound to free and lysine 9-methylated histone H3 tails

NASA Astrophysics Data System (ADS)

Jiang, Yanke; Zou, Jianwei; Zeng, Min; Zhang, Na; Yu, Qingsen

Histone methylation has emerged as a central epigenetic modification with both activating and repressive roles in eukaryotic chromatin. Drosophila HP1 (heterochromatin-associated protein 1) is one of the chromodomain proteins that contain the essential aromatic residues as the recognition pocket for lysine methylated histone H3 tail. The aromatic cage indicates that the complex of chromodomain protein binding lysine methylated histone H3 tail can be seen as a typical host-guest system between protein and protein. About 10-ns molecular dynamics simulations have been carried out in this study to examine how the presence of mono-, trimethylated lysine 9 histone H3 tail (Me1K9, Me3K9 H3) influences the motions of HP1 protein receptor. The study shows that the conformation of HP1 protein free of H3 tail easily changes, whereas that of HP1 protein bound to methylated H3 tail does not. But the conformation of inserted Me1K9 H3 changes obviously as the Me1K recognition makes hydrogen-bonded interactions associated with the aromatic cage even more unstable than those in free HP1 protein. The conformational change of Me1K9 H3 is correlated with the motions of HP1 protein. As the recognition factor going from Me1K to Me3K produces a more favorable interaction for aromatic ring, hydrogen-bonded interactions associated with aromatic cage in Me3K9 H3-HP1 complex were observed to be much more stable than those in Me1K9 H3-HP1 complex and free HP1. Because of correlation, the flexibility of Me3K9 H3 decreases. The simulations indicate that both the MeK and the surrounding histone tail sequence are necessary features of recognition which significantly affect the flexibility and backbone motions of HP1 chromodomain. These findings confirm a regulatory mechanism of protein-protein interactions through a trimethylated post-translational modification.

Particle creation phenomenology, Dirac sea and the induced Weyl and Einstein-dilaton gravity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berezin, V.A.; Dokuchaev, V.I.; Eroshenko, Yu.N., E-mail: berezin@inr.ac.ru, E-mail: dokuchaev@inr.ac.ru, E-mail: eroshenko@inr.ac.ru

We constructed the conformally invariant model for scalar particle creation induced by strong gravitational fields. Starting from the 'usual' hydrodynamical description of the particle motion written in the Eulerian coordinates we substituted the particle number conservation law (which enters the formalism) by 'the particle creation law', proportional to the square of the Weyl tensor (following the famous result by Ya.B. Zel'dovich and A.A. Starobinsky). Then, demanding the conformal invariance of the whole dynamical system, we have got both the (Weyl)-conformal gravity and the Einstein-Hilbert gravity action integral with dilaton field. Thus, we obtained something like the induced gravity suggested firstmore » by A.D. Sakharov. It is shown that the resulting system is self-consistent. We considered also the vacuum equations. It is shown that, beside the 'empty vacuum', there may exist the 'dynamical vacuum', which is nothing more but the Dirac sea. The latter is described by the unexpectedly elegant equation which includes both the Bach and Einstein tensors and the cosmological terms.« less

Particle dynamics around time conformal regular black holes via Noether symmetries

NASA Astrophysics Data System (ADS)

Jawad, Abdul; Umair Shahzad, M.

The time conformal regular black hole (RBH) solutions which are admitting the time conformal factor e𝜖g(t), where g(t) is an arbitrary function of time and 𝜖 is the perturbation parameter are being considered. The approximate Noether symmetries technique is being used for finding the function g(t) which leads to t α. The dynamics of particles around RBHs are also being discussed through symmetry generators which provide approximate energy as well as angular momentum of the particles. In addition, we analyze the motion of neutral and charged particles around two well known RBHs such as charged RBH using Fermi-Dirac distribution and Kehagias-Sftesos asymptotically flat RBH. We obtain the innermost stable circular orbit and corresponding approximate energy and angular momentum. The behavior of effective potential, effective force and escape velocity of the particles in the presence/absence of magnetic field for different values of angular momentum near horizons are also being analyzed. The stable and unstable regions of particle near horizons due to the effect of angular momentum and magnetic field are also explained.

An Allosteric Cross-Talk Between the Activation Loop and the ATP Binding Site Regulates the Activation of Src Kinase

NASA Astrophysics Data System (ADS)

Pucheta-Martínez, Encarna; Saladino, Giorgio; Morando, Maria Agnese; Martinez-Torrecuadrada, Jorge; Lelli, Moreno; Sutto, Ludovico; D'Amelio, Nicola; Gervasio, Francesco Luigi

2016-04-01

Phosphorylation of the activation loop is a fundamental step in the activation of most protein kinases. In the case of the Src tyrosine kinase, a prototypical kinase due to its role in cancer and its historic importance, phosphorylation of tyrosine 416 in the activation loop is known to rigidify the structure and contribute to the switch from the inactive to a fully active form. However, whether or not phosphorylation is able per-se to induce a fully active conformation, that efficiently binds ATP and phosphorylates the substrate, is less clear. Here we employ a combination of solution NMR and enhanced-sampling molecular dynamics simulations to fully map the effects of phosphorylation and ATP/ADP cofactor loading on the conformational landscape of Src tyrosine kinase. We find that both phosphorylation and cofactor binding are needed to induce a fully active conformation. What is more, we find a complex interplay between the A-loop and the hinge motion where the phosphorylation of the activation-loop has a significant allosteric effect on the dynamics of the C-lobe.

VP40 of the Ebola Virus as a Target for EboV Therapy: Comprehensive Conformational and Inhibitor Binding Landscape from Accelerated Molecular Dynamics.

PubMed

Balmith, Marissa; Soliman, Mahmoud E S

2017-03-01

The first account of the dynamic features of the loop region of VP40 of the Ebola virus was studied using accelerated molecular dynamics simulations and reported herein. Among the proteins of the Ebola virus, the matrix protein (VP40) plays a significant role in the virus lifecycle thereby making it a promising therapeutic target. Of interest is the newly elucidated N-terminal domain loop region of VP40 comprising residues K127, T129, and N130 which when mutated to alanine have demonstrated an unrecognized role for N-terminal domain-plasma membrane interaction for efficient VP40-plasma membrane localization, oligomerization, matrix assembly, and egress. The molecular understanding of the conformational features of VP40 in complex with a known inhibitor still remains elusive. Using accelerated molecular dynamics approaches, we conducted a comparative study on VP40 apo and bound systems to understand the conformational features of VP40 at the molecular level and to determine the effect of inhibitor binding with the aid of a number of post-dynamic analytical tools. Significant features were seen in the presence of an inhibitor as per molecular mechanics/generalized born surface area binding free energy calculations. Results revealed that inhibitor binding to VP40 reduces the flexibility and mobility of the protein as supported by root mean square fluctuation and root mean square deviation calculations. The study revealed a characteristic "twisting" motion and coiling of the loop region of VP40 accompanied by conformational changes in the dimer interface upon inhibitor binding. We believe that results presented in this study will ultimately provide useful insight into the binding landscape of VP40 which could assist researchers in the discovery of potent Ebola virus inhibitors for anti-Ebola therapies.

Bi-stability in cooperative transport by ants in the presence of obstacles

PubMed Central

Pinkoviezky, Itai; Feinerman, Ofer

2018-01-01

To cooperatively carry large food items to the nest, individual ants conform their efforts and coordinate their motion. Throughout this expedition, collective motion is driven both by internal interactions between the carrying ants and a response to newly arrived informed ants that orient the cargo towards the nest. During the transport process, the carrying group must overcome obstacles that block their path to the nest. Here, we investigate the dynamics of cooperative transport, when the motion of the ants is frustrated by a linear obstacle that obstructs the motion of the cargo. The obstacle contains a narrow opening that serves as the only available passage to the nest, and through which single ants can pass but not with the cargo. We provide an analytical model for the ant-cargo system in the constrained environment that predicts a bi-stable dynamic behavior between an oscillatory mode of motion along the obstacle and a convergent mode of motion near the opening. Using both experiments and simulations, we show how for small cargo sizes, the system exhibits spontaneous transitions between these two modes of motion due to fluctuations in the applied force on the cargo. The bi-stability provides two possible problem solving strategies for overcoming the obstacle, either by attempting to pass through the opening, or take large excursions to circumvent the obstacle. PMID:29746457

Bi-stability in cooperative transport by ants in the presence of obstacles.

PubMed

Ron, Jonathan E; Pinkoviezky, Itai; Fonio, Ehud; Feinerman, Ofer; Gov, Nir S

2018-05-01

To cooperatively carry large food items to the nest, individual ants conform their efforts and coordinate their motion. Throughout this expedition, collective motion is driven both by internal interactions between the carrying ants and a response to newly arrived informed ants that orient the cargo towards the nest. During the transport process, the carrying group must overcome obstacles that block their path to the nest. Here, we investigate the dynamics of cooperative transport, when the motion of the ants is frustrated by a linear obstacle that obstructs the motion of the cargo. The obstacle contains a narrow opening that serves as the only available passage to the nest, and through which single ants can pass but not with the cargo. We provide an analytical model for the ant-cargo system in the constrained environment that predicts a bi-stable dynamic behavior between an oscillatory mode of motion along the obstacle and a convergent mode of motion near the opening. Using both experiments and simulations, we show how for small cargo sizes, the system exhibits spontaneous transitions between these two modes of motion due to fluctuations in the applied force on the cargo. The bi-stability provides two possible problem solving strategies for overcoming the obstacle, either by attempting to pass through the opening, or take large excursions to circumvent the obstacle.

Revealing an outward-facing open conformational state in a CLC Cl –/H + exchange transporter

DOE PAGES

Khantwal, Chandra M.; Abraham, Sherwin J.; Han, Wei; ...

2016-01-22

CLC secondary active transporters exchange Cl - for H + . Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Glu ex ) upon its protonation. Using 19 F NMR, we show that as [H + ] is increased to protonate Glu ex and enrich the outward-facing state, a residue ~20 Å away from Glu ex , near the subunit interface, moves from buried to solvent-exposed. Consistent with functional relevance of this motion, constriction via inter-subunit cross-linking reduces transport. Molecular dynamics simulations indicate that themore » cross-link dampens extracellular gate-opening motions. In support of this model, mutations that decrease steric contact between Helix N (part of the extracellular gate) and Helix P (at the subunit interface) remove the inhibitory effect of the cross-link. Together, these results demonstrate the formation of a previously uncharacterized 'outward-facing open' state, and highlight the relevance of global structural changes in CLC function.« less

Molecular mechanism of Mg2+-dependent gating in CorA

NASA Astrophysics Data System (ADS)

Dalmas, Olivier; Sompornpisut, Pornthep; Bezanilla, Francisco; Perozo, Eduardo

2014-04-01

CorA is the major transport system responsible for Mg2+ uptake in bacteria and can functionally substitute for its homologue Mrs2p in the yeast inner mitochondrial membrane. Although several CorA crystal structures are available, the molecular mechanism of Mg2+ uptake remains to be established. Here we use electron paramagnetic resonance spectroscopy, electrophysiology and molecular dynamic simulations to show that CorA is regulated by cytoplasmic Mg2+ acting as a ligand and elucidate the basic conformational rearrangements responsible for Mg2+-dependent gating. Mg2+ unbinding at the divalent cation sensor triggers a conformational change that leads to the inward motion of the stalk helix, which propagates to the pore-forming transmembrane helix TM1. Helical tilting and rotation in TM1 generates an iris-like motion that increases the diameter of the permeation pathway, triggering ion conduction. This work establishes the molecular basis of a Mg2+-driven negative feedback loop in CorA as the key physiological event controlling Mg2+ uptake and homeostasis in prokaryotes.

Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant.

PubMed

Kamal, Md Zahid; Mohammad, Tabrez Anwar Shamim; Krishnamoorthy, G; Rao, Nalam Madhusudhana

2012-01-01

Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.

Insights into the Hydrogen-Atom Transfer of the Blue Aroxyl.

PubMed

Bächle, Josua; Marković, Marijana; Kelterer, Anne-Marie; Grampp, Günter

2017-10-19

An experimental and theoretical study on hydrogen-atom transfer dynamics in the hydrogen-bonded substituted phenol/phenoxyl complex of the blue aroxyl (2,4,6-tri-tert-butylphenoxyl) is presented. The experimental exchange dynamics is determined in different organic solvents from the temperature-dependent alternating line-width effect in the continuous-wave ESR spectrum. From bent Arrhenius plots, effective tunnelling contributions with parallel heavy-atom motion are concluded. To clarify the transfer mechanism, reaction paths for different conformers of the substituted phenol/phenoxyl complex are modelled theoretically. Various DFT and post-Hartree-Fock methods including multireference methods are applied. From the comparison of experimental and theoretical data it is concluded that the system favours concerted hydrogen-atom transfer along a parabolic reaction path caused by heavy-atom motion. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Animation and radiobiological analysis of 3D motion in conformal radiotherapy.

PubMed

MacKay, R I; Graham, P A; Moore, C J; Logue, J P; Sharrock, P J

1999-07-01

To allow treatment plans to be evaluated against the range of expected organ motion and set up error anticipated during treatment. Planning tools have been developed to allow concurrent animation and radiobiological analysis of three dimensional (3D) target and organ motion in conformal radiotherapy. Surfaces fitted to structures outlined on CT studies are projected onto pre-treatment images or onto megavoltage images collected during the patient treatment. Visual simulation of tumour and normal tissue movement is then performed by the application of three dimensional affine transformations, to the selected surface. Concurrent registration of the surface motion with the 3D dose distribution allows calculation of the change in dose to the volume. Realistic patterns of motion can be applied to the structure to simulate inter-fraction motion and set-up error. The biologically effective dose for the structure is calculated for each fraction as the surface moves over the course of the treatment and is used to calculate the normal tissue complication probability (NTCP) or tumour control probability (TCP) for the moving structure. The tool has been used to evaluate conformal therapy plans against set up measurements recorded during patient treatments. NTCP and TCP were calculated for a patient whose set up had been corrected after systematic deviations from plan geometry were measured during treatment, the effect of not making the correction were also assessed. TCP for the moving tumour was reduced if inadequate margins were set for the treatment. Modelling suggests that smaller margins could have been set for the set up corrected during the course of the treatment. The NTCP for the rectum was also higher for the uncorrected set up due to a more rectal tissue falling in the high dose region. This approach provides a simple way for clinical users to utilise information incrementally collected throughout the whole of a patient's treatment. In particular it is possible to test the robustness of a patient plan against a range of possible motion patterns. The methods described represent a move from the inspection of static pre-treatment plans to a review of the dynamic treatment.

Experimental investigation of a moving averaging algorithm for motion perpendicular to the leaf travel direction in dynamic MLC target tracking.

PubMed

Yoon, Jai-Woong; Sawant, Amit; Suh, Yelin; Cho, Byung-Chul; Suh, Tae-Suk; Keall, Paul

2011-07-01

In dynamic multileaf collimator (MLC) motion tracking with complex intensity-modulated radiation therapy (IMRT) fields, target motion perpendicular to the MLC leaf travel direction can cause beam holds, which increase beam delivery time by up to a factor of 4. As a means to balance delivery efficiency and accuracy, a moving average algorithm was incorporated into a dynamic MLC motion tracking system (i.e., moving average tracking) to account for target motion perpendicular to the MLC leaf travel direction. The experimental investigation of the moving average algorithm compared with real-time tracking and no compensation beam delivery is described. The properties of the moving average algorithm were measured and compared with those of real-time tracking (dynamic MLC motion tracking accounting for both target motion parallel and perpendicular to the leaf travel direction) and no compensation beam delivery. The algorithm was investigated using a synthetic motion trace with a baseline drift and four patient-measured 3D tumor motion traces representing regular and irregular motions with varying baseline drifts. Each motion trace was reproduced by a moving platform. The delivery efficiency, geometric accuracy, and dosimetric accuracy were evaluated for conformal, step-and-shoot IMRT, and dynamic sliding window IMRT treatment plans using the synthetic and patient motion traces. The dosimetric accuracy was quantified via a tgamma-test with a 3%/3 mm criterion. The delivery efficiency ranged from 89 to 100% for moving average tracking, 26%-100% for real-time tracking, and 100% (by definition) for no compensation. The root-mean-square geometric error ranged from 3.2 to 4.0 mm for moving average tracking, 0.7-1.1 mm for real-time tracking, and 3.7-7.2 mm for no compensation. The percentage of dosimetric points failing the gamma-test ranged from 4 to 30% for moving average tracking, 0%-23% for real-time tracking, and 10%-47% for no compensation. The delivery efficiency of moving average tracking was up to four times higher than that of real-time tracking and approached the efficiency of no compensation for all cases. The geometric accuracy and dosimetric accuracy of the moving average algorithm was between real-time tracking and no compensation, approximately half the percentage of dosimetric points failing the gamma-test compared with no compensation.

Population shuffling between ground and high energy excited states

PubMed Central

Sabo, T Michael; Trent, John O; Lee, Donghan

2015-01-01

Stochastic processes powered by thermal energy lead to protein motions traversing time-scales from picoseconds to seconds. Fundamental to protein functionality is the utilization of these dynamics for tasks such as catalysis, folding, and allostery. A hierarchy of motion is hypothesized to connect and synergize fast and slow dynamics toward performing these essential activities. Population shuffling predicts a “top-down” temporal hierarchy, where slow time-scale conformational interconversion leads to a shuffling of the free energy landscape for fast time-scale events. Until now, population shuffling was only applied to interconverting ground states. Here, we extend the framework of population shuffling to be applicable for a system interconverting between low energy ground and high energy excited states, such as the SH3 domain mutants G48M and A39V/N53P/V55L from the Fyn tyrosine kinase, providing another tool for accessing the structural dynamics of high energy excited states. Our results indicate that the higher energy gauche− rotameric state for the leucine χ2 dihedral angle contributes significantly to the distribution of rotameric states in both the major and minor forms of the SH3 domain. These findings are corroborated with unrestrained molecular dynamics (MD) simulations on both the major and minor states of the SH3 domain demonstrating high correlations between experimental and back-calculated leucine χ2 rotameric populations. Taken together, we demonstrate how fast time-scale rotameric side-chain population distributions can be extracted from slow time-scale conformational exchange data further extending the scope and the applicability of the population shuffling model. PMID:26316263

Population shuffling between ground and high energy excited states.

PubMed

Sabo, T Michael; Trent, John O; Lee, Donghan

2015-11-01

Stochastic processes powered by thermal energy lead to protein motions traversing time-scales from picoseconds to seconds. Fundamental to protein functionality is the utilization of these dynamics for tasks such as catalysis, folding, and allostery. A hierarchy of motion is hypothesized to connect and synergize fast and slow dynamics toward performing these essential activities. Population shuffling predicts a "top-down" temporal hierarchy, where slow time-scale conformational interconversion leads to a shuffling of the free energy landscape for fast time-scale events. Until now, population shuffling was only applied to interconverting ground states. Here, we extend the framework of population shuffling to be applicable for a system interconverting between low energy ground and high energy excited states, such as the SH3 domain mutants G48M and A39V/N53P/V55L from the Fyn tyrosine kinase, providing another tool for accessing the structural dynamics of high energy excited states. Our results indicate that the higher energy gauche - rotameric state for the leucine χ2 dihedral angle contributes significantly to the distribution of rotameric states in both the major and minor forms of the SH3 domain. These findings are corroborated with unrestrained molecular dynamics (MD) simulations on both the major and minor states of the SH3 domain demonstrating high correlations between experimental and back-calculated leucine χ2 rotameric populations. Taken together, we demonstrate how fast time-scale rotameric side-chain population distributions can be extracted from slow time-scale conformational exchange data further extending the scope and the applicability of the population shuffling model. © 2015 The Protein Society.

High energy conformers of M(+)(APE)(H2O)(0-1)Ar(0-1) clusters revealed by combined IR-PD and DFT-MD anharmonic vibrational spectroscopy.

PubMed

Brites, V; Nicely, A L; Sieffert, N; Gaigeot, M-P; Lisy, J M

2014-07-14

IR-PD vibrational spectroscopy and DFT-based molecular dynamics simulations are combined in order to unravel the structures of M(+)(APE)(H2O)0-1 ionic clusters (M = Na, K), where APE (2-amino-1-phenyl ethanol) is commonly used as an analogue for the noradrenaline neurotransmitter. The strength of the synergy between experiments and simulations presented here is that DFT-MD provides anharmonic vibrational spectra that unambiguously help assign the ionic clusters structures. Depending on the interacting cation, we have found that the lowest energy conformers of K(+)(APE)(H2O)0-1 clusters are formed, while the lowest energy conformers of Na(+)(APE)(H2O)0-1 clusters can only be observed through water loss channel (i.e. without argon tagged to the clusters). Trapping of higher energy conformers is observed when the argon loss channel is recorded in the experiment. This has been rationalized by transition state energies. The dynamical anharmonic vibrational spectra unambiguously provide the prominent OH stretch due to the OH···NH2 H-bond, within 10 cm(-1) of the experiment, hence reproducing the 240-300 cm(-1) red-shift (depending on the interacting cation) from bare neutral APE. When this H-bond is not present, the dynamical anharmonic spectra provide the water O-H stretches as well as the rotational motion of the water molecule at finite temperature, as observed in the experiment.

Ubiquitin in Motion: Structural Studies of the Ubiquitin-Conjugating Enzyme~Ubiquitin Conjugate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pruneda, Jonathan N.; Stoll, Kate E.; Bolton, Laura J.

2011-03-15

Ubiquitination of proteins provides a powerful and versatile post-translational signal in the eukaryotic cell. The formation of a thioester bond between ubiquitin (Ub) and the active site of a ubiquitin-conjugating enzyme (E2) is critical for the transfer of Ub to substrates. Assembly of a functional ubiquitin ligase (E3) complex poised for Ub transfer involves recognition and binding of an E2~Ub conjugate. Therefore, full characterization of the structure and dynamics of E2~Ub conjugates is required for further mechanistic understanding of Ub transfer reactions. Here we present characterization of the dynamic behavior of E2~Ub conjugates of two human enzymes, UbcH5c~Ub and Ubc13~Ub,more » in solution as determined by nuclear magnetic resonance and small-angle X-ray scattering. Within each conjugate, Ub retains great flexibility with respect to the E2, indicative of highly dynamic species that adopt manifold orientations. The population distribution of Ub conformations is dictated by the identity of the E2: the UbcH5c~Ub conjugate populates an array of extended conformations, and the population of Ubc13~Ub conjugates favors a closed conformation in which the hydrophobic surface of Ub faces helix 2 of Ubc13. Finally, we propose that the varied conformations adopted by Ub represent available binding modes of the E2~Ub species and thus provide insight into the diverse E2~Ub protein interactome, particularly with regard to interaction with Ub ligases.« less

Analysis of accelerated motion in the theory of relativity

NASA Technical Reports Server (NTRS)

Jones, R. T.

1976-01-01

Conventional treatments of accelerated motion in the theory of relativity have led to certain difficulties of interpretation. Certain reversals in the apparent gravitational field of an accelerated body may be avoided by simpler analysis based on the use of restricted conformal transformations. In the conformal theory the velocity of light remains constant even for experimenters in accelerated motion. The problem considered is that of rectilinear motion with a variable velocity. The motion takes place along the x or x' axis of two coordinate systems.

Multi-scale dynamics and relaxation of a tethered membrane in a solvent by Monte Carlo simulations

NASA Astrophysics Data System (ADS)

Pandey, Ras; Anderson, Kelly; Farmer, Barry

2006-03-01

A tethered membrane modeled by a flexible sheet dissipates entropy as it wrinkles and crumples. Nodes of a coarse grained membrane are connected via multiple pathways for dynamical modes to propagate. We consider a sheet with nodes connected by fluctuating bonds on a cubic lattice. The empty lattice sites constitute an effective solvent medium via node-solvent interaction. Each node execute its stochastic motion with the Metropolis algorithm subject to bond fluctuations, excluded volume constraints, and interaction energy. Dynamics and conformation of the sheet are examined at a low and a high temperature with attractive and repulsive node-node interactions for the contrast in an attractive solvent medium. Variations of the mean square displacement of the center node of the sheet and that of its center of mass with the time steps are examined in detail which show different power-law motion from short to long time regimes. Relaxation of the gyration radius and scaling of its asymptotic value with the molecular weight are examined.

The application of tailor-made force fields and molecular dynamics for NMR crystallography: a case study of free base cocaine

PubMed Central

Neumann, Marcus A.

2017-01-01

Motional averaging has been proven to be significant in predicting the chemical shifts in ab initio solid-state NMR calculations, and the applicability of motional averaging with molecular dynamics has been shown to depend on the accuracy of the molecular mechanical force field. The performance of a fully automatically generated tailor-made force field (TMFF) for the dynamic aspects of NMR crystallography is evaluated and compared with existing benchmarks, including static dispersion-corrected density functional theory calculations and the COMPASS force field. The crystal structure of free base cocaine is used as an example. The results reveal that, even though the TMFF outperforms the COMPASS force field for representing the energies and conformations of predicted structures, it does not give significant improvement in the accuracy of NMR calculations. Further studies should direct more attention to anisotropic chemical shifts and development of the method of solid-state NMR calculations. PMID:28250956

Watching proteins function with picosecond X-ray crystallography and molecular dynamics simulations.

NASA Astrophysics Data System (ADS)

Anfinrud, Philip

2006-03-01

Time-resolved electron density maps of myoglobin, a ligand-binding heme protein, have been stitched together into movies that unveil with < 2-å spatial resolution and 150-ps time-resolution the correlated protein motions that accompany and/or mediate ligand migration within the hydrophobic interior of a protein. A joint analysis of all-atom molecular dynamics (MD) calculations and picosecond time-resolved X-ray structures provides single-molecule insights into mechanisms of protein function. Ensemble-averaged MD simulations of the L29F mutant of myoglobin following ligand dissociation reproduce the direction, amplitude, and timescales of crystallographically-determined structural changes. This close agreement with experiments at comparable resolution in space and time validates the individual MD trajectories, which identify and structurally characterize a conformational switch that directs dissociated ligands to one of two nearby protein cavities. This unique combination of simulation and experiment unveils functional protein motions and illustrates at an atomic level relationships among protein structure, dynamics, and function. In collaboration with Friedrich Schotte and Gerhard Hummer, NIH.

Motion of spin label side chains in cellular retinol-binding protein: correlation with structure and nearest-neighbor interactions in an antiparallel beta-sheet.

PubMed

Lietzow, Michael A; Hubbell, Wayne L

2004-03-23

A goal in the development of site-directed spin labeling in proteins is to correlate the motion of a nitroxide side chain with local structure, interactions, and dynamics. Significant progress toward this goal has been made using alpha-helical proteins of known structure, and the present study is the first step in a similar exploration of a beta-sheet protein, cellular retinol-binding protein (CRBP). Nitroxide side chains were introduced along both interior and edge strands. At sites in interior strands, the side-chain motion is strongly influenced by interactions with side chains of neighboring strands, giving rise to a rich variety of dynamic modes (weakly ordered, strongly ordered, immobilized) and complex electron paramagnetic resonance spectra that are modulated by strand twist. The interactions giving rise to the dynamic modes are explored using mutagenesis, and the results demonstrate the particular importance of the non-hydrogen-bonded neighbor residue in giving rise to highly ordered states. Along edge strands of the beta-sheet, the motion of the side chain is simple and weakly ordered, resembling that at solvent-exposed surfaces of an alpha-helix. A simple working model is proposed that can account for the wide variety of dynamic modes encountered. Collectively, the results suggest that the nitroxide side chain is an effective probe of side-chain interactions, and that site-directed spin labeling should be a powerful means of monitoring conformational changes that involve changes in beta-sheet topology.

Backbone dynamics and global effects of an activating mutation in minimized Mtu RecA inteins.

PubMed

Du, Zhenming; Liu, Yangzhong; Ban, David; Lopez, Maria M; Belfort, Marlene; Wang, Chunyu

2010-07-23

Inteins mediate protein splicing, which has found many applications in biotechnology and protein engineering. A single valine-to-leucine mutation (V67L) can globally enhance splicing and related cleavage reactions in minimized Mycobacterium tuberculosis RecA inteins. However, V67L mutation causes little change in crystal structures. To test whether protein dynamics contribute to activity enhancement in the V67L mutation, we have studied the conformations and dynamics of the minimized and engineered intein DeltaDeltaIhh-V67CM and a single V67L mutant, DeltaDeltaIhh-L67CM, by solution NMR. Chemical shift perturbations established that the V67L mutation causes global changes, including changes at the N-terminus and C-terminus of the intein, which are active sites for protein splicing. The single V67L mutation significantly slows hydrogen-exchange rates globally, indicating a shift to more stable conformations and reduction in ensemble distribution. Whereas the V67L mutation causes little change for motions on the picosecond-to-nanosecond timescale, motions on the microsecond-to-millisecond timescale affect a region involving the conserved F-block histidine and C-terminal asparagine, which are residues important for C-terminal cleavage. The V67L mutation is proposed to activate splicing by reducing the ensemble distribution of the intein structure and by modifying the active sites. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Metadynamics in the conformational space nonlinearly dimensionally reduced by Isomap

NASA Astrophysics Data System (ADS)

Spiwok, Vojtěch; Králová, Blanka

2011-12-01

Atomic motions in molecules are not linear. This infers that nonlinear dimensionality reduction methods can outperform linear ones in analysis of collective atomic motions. In addition, nonlinear collective motions can be used as potentially efficient guides for biased simulation techniques. Here we present a simulation with a bias potential acting in the directions of collective motions determined by a nonlinear dimensionality reduction method. Ad hoc generated conformations of trans,trans-1,2,4-trifluorocyclooctane were analyzed by Isomap method to map these 72-dimensional coordinates to three dimensions, as described by Brown and co-workers [J. Chem. Phys. 129, 064118 (2008)]. Metadynamics employing the three-dimensional embeddings as collective variables was applied to explore all relevant conformations of the studied system and to calculate its conformational free energy surface. The method sampled all relevant conformations (boat, boat-chair, and crown) and corresponding transition structures inaccessible by an unbiased simulation. This scheme allows to use essentially any parameter of the system as a collective variable in biased simulations. Moreover, the scheme we used for mapping out-of-sample conformations from the 72D to 3D space can be used as a general purpose mapping for dimensionality reduction, beyond the context of molecular modeling.

MovieMaker: a web server for rapid rendering of protein motions and interactions.

PubMed

Maiti, Rajarshi; Van Domselaar, Gary H; Wishart, David S

2005-07-01

MovieMaker is a web server that allows short ( approximately 10 s), downloadable movies of protein motions to be generated. It accepts PDB files or PDB accession numbers as input and automatically calculates, renders and merges the necessary image files to create colourful animations covering a wide range of protein motions and other dynamic processes. Users have the option of animating (i) simple rotation, (ii) morphing between two end-state conformers, (iii) short-scale, picosecond vibrations, (iv) ligand docking, (v) protein oligomerization, (vi) mid-scale nanosecond (ensemble) motions and (vii) protein folding/unfolding. MovieMaker does not perform molecular dynamics calculations. Instead it is an animation tool that uses a sophisticated superpositioning algorithm in conjunction with Cartesian coordinate interpolation to rapidly and automatically calculate the intermediate structures needed for many of its animations. Users have extensive control over the rendering style, structure colour, animation quality, background and other image features. MovieMaker is intended to be a general-purpose server that allows both experts and non-experts to easily generate useful, informative protein animations for educational and illustrative purposes. MovieMaker is accessible at http://wishart.biology.ualberta.ca/moviemaker.

Principal component analysis for protein folding dynamics.

PubMed

Maisuradze, Gia G; Liwo, Adam; Scheraga, Harold A

2009-01-09

Protein folding is considered here by studying the dynamics of the folding of the triple beta-strand WW domain from the Formin-binding protein 28. Starting from the unfolded state and ending either in the native or nonnative conformational states, trajectories are generated with the coarse-grained united residue (UNRES) force field. The effectiveness of principal components analysis (PCA), an already established mathematical technique for finding global, correlated motions in atomic simulations of proteins, is evaluated here for coarse-grained trajectories. The problems related to PCA and their solutions are discussed. The folding and nonfolding of proteins are examined with free-energy landscapes. Detailed analyses of many folding and nonfolding trajectories at different temperatures show that PCA is very efficient for characterizing the general folding and nonfolding features of proteins. It is shown that the first principal component captures and describes in detail the dynamics of a system. Anomalous diffusion in the folding/nonfolding dynamics is examined by the mean-square displacement (MSD) and the fractional diffusion and fractional kinetic equations. The collisionless (or ballistic) behavior of a polypeptide undergoing Brownian motion along the first few principal components is accounted for.

Insight into structural rearrangements and interdomain interactions related to electron transfer between flavin mononucleotide and heme in nitric oxide synthase: A molecular dynamics study.

PubMed

Sheng, Yinghong; Zhong, Linghao; Guo, Dahai; Lau, Gavin; Feng, Changjian

2015-12-01

Calmodulin (CaM) binding to nitric oxide synthase (NOS) enables a conformational change, in which the FMN domain shuttles between the FAD and heme domains to deliver electrons to the active site heme center. A clear understanding of this large conformational change is critical, since this step is the rate-limiting in NOS catalysis. Herein molecular dynamics simulations were conducted on a model of an oxygenase/FMN (oxyFMN) construct of human inducible NOS (iNOS). This is to investigate the structural rearrangements and the domain interactions related to the FMN-heme interdomain electron transfer (IET). We carried out simulations on the iNOS oxyFMN·CaM complex models in [Fe(III)][FMNH(-)] and [Fe(II)][FMNH] oxidation states, the pre- and post-IET states. The comparison of the dynamics and conformations of the iNOS construct at the two oxidation states has allowed us to identify key factors related to facilitating the FMN-heme IET process. The computational results demonstrated, for the first time, that the conformational change is redox-dependent. Predictions of the key interacting sites in optimal interdomain FMN/heme docking are well supported by experimental data in the literature. An intra-subunit pivot region is predicted to modulate the FMN domain motion and correlate with existence of a bottleneck in the conformational sampling that leads to the electron transfer-competent state. Interactions of the residues identified in this work are proposed to ensure that the FMN domain moves with appropriate degrees of freedom and docks to proper positions at the heme domain, resulting in efficient IET and nitric oxide production. Copyright © 2015 Elsevier Inc. All rights reserved.

Perspectives on electrostatics and conformational motions in enzyme catalysis.

PubMed

Hanoian, Philip; Liu, C Tony; Hammes-Schiffer, Sharon; Benkovic, Stephen

2015-02-17

CONSPECTUS: Enzymes are essential for all living organisms, and their effectiveness as chemical catalysts has driven more than a half century of research seeking to understand the enormous rate enhancements they provide. Nevertheless, a complete understanding of the factors that govern the rate enhancements and selectivities of enzymes remains elusive, due to the extraordinary complexity and cooperativity that are the hallmarks of these biomolecules. We have used a combination of site-directed mutagenesis, pre-steady-state kinetics, X-ray crystallography, nuclear magnetic resonance (NMR), vibrational and fluorescence spectroscopies, resonance energy transfer, and computer simulations to study the implications of conformational motions and electrostatic interactions on enzyme catalysis in the enzyme dihydrofolate reductase (DHFR). We have demonstrated that modest equilibrium conformational changes are functionally related to the hydride transfer reaction. Results obtained for mutant DHFRs illustrated that reductions in hydride transfer rates are correlated with altered conformational motions, and analysis of the evolutionary history of DHFR indicated that mutations appear to have occurred to preserve both the hydride transfer rate and the associated conformational changes. More recent results suggested that differences in local electrostatic environments contribute to finely tuning the substrate pKa in the initial protonation step. Using a combination of primary and solvent kinetic isotope effects, we demonstrated that the reaction mechanism is consistent across a broad pH range, and computer simulations suggested that deprotonation of the active site Tyr100 may play a crucial role in substrate protonation at high pH. Site-specific incorporation of vibrational thiocyanate probes into the ecDHFR active site provided an experimental tool for interrogating these microenvironments and for investigating changes in electrostatics along the DHFR catalytic cycle. Complementary molecular dynamics simulations in conjunction with mixed quantum mechanical/molecular mechanical calculations accurately reproduced the vibrational frequency shifts in these probes and provided atomic-level insight into the residues influencing these changes. Our findings indicate that conformational and electrostatic changes are intimately related and functionally essential. This approach can be readily extended to the study of other enzyme systems to identify more general trends in the relationship between conformational fluctuations and electrostatic interactions. These results are relevant to researchers seeking to design novel enzymes as well as those seeking to develop therapeutic agents that function as enzyme inhibitors.

Perspectives on Electrostatics and Conformational Motions in Enzyme Catalysis

PubMed Central

2016-01-01

Conspectus Enzymes are essential for all living organisms, and their effectiveness as chemical catalysts has driven more than a half century of research seeking to understand the enormous rate enhancements they provide. Nevertheless, a complete understanding of the factors that govern the rate enhancements and selectivities of enzymes remains elusive, due to the extraordinary complexity and cooperativity that are the hallmarks of these biomolecules. We have used a combination of site-directed mutagenesis, pre-steady-state kinetics, X-ray crystallography, nuclear magnetic resonance (NMR), vibrational and fluorescence spectroscopies, resonance energy transfer, and computer simulations to study the implications of conformational motions and electrostatic interactions on enzyme catalysis in the enzyme dihydrofolate reductase (DHFR). We have demonstrated that modest equilibrium conformational changes are functionally related to the hydride transfer reaction. Results obtained for mutant DHFRs illustrated that reductions in hydride transfer rates are correlated with altered conformational motions, and analysis of the evolutionary history of DHFR indicated that mutations appear to have occurred to preserve both the hydride transfer rate and the associated conformational changes. More recent results suggested that differences in local electrostatic environments contribute to finely tuning the substrate pKa in the initial protonation step. Using a combination of primary and solvent kinetic isotope effects, we demonstrated that the reaction mechanism is consistent across a broad pH range, and computer simulations suggested that deprotonation of the active site Tyr100 may play a crucial role in substrate protonation at high pH. Site-specific incorporation of vibrational thiocyanate probes into the ecDHFR active site provided an experimental tool for interrogating these microenvironments and for investigating changes in electrostatics along the DHFR catalytic cycle. Complementary molecular dynamics simulations in conjunction with mixed quantum mechanical/molecular mechanical calculations accurately reproduced the vibrational frequency shifts in these probes and provided atomic-level insight into the residues influencing these changes. Our findings indicate that conformational and electrostatic changes are intimately related and functionally essential. This approach can be readily extended to the study of other enzyme systems to identify more general trends in the relationship between conformational fluctuations and electrostatic interactions. These results are relevant to researchers seeking to design novel enzymes as well as those seeking to develop therapeutic agents that function as enzyme inhibitors. PMID:25565178

The Dynameomics Entropy Dictionary: A Large-Scale Assessment of Conformational Entropy across Protein Fold Space.

PubMed

Towse, Clare-Louise; Akke, Mikael; Daggett, Valerie

2017-04-27

Molecular dynamics (MD) simulations contain considerable information with regard to the motions and fluctuations of a protein, the magnitude of which can be used to estimate conformational entropy. Here we survey conformational entropy across protein fold space using the Dynameomics database, which represents the largest existing data set of protein MD simulations for representatives of essentially all known protein folds. We provide an overview of MD-derived entropies accounting for all possible degrees of dihedral freedom on an unprecedented scale. Although different side chains might be expected to impose varying restrictions on the conformational space that the backbone can sample, we found that the backbone entropy and side chain size are not strictly coupled. An outcome of these analyses is the Dynameomics Entropy Dictionary, the contents of which have been compared with entropies derived by other theoretical approaches and experiment. As might be expected, the conformational entropies scale linearly with the number of residues, demonstrating that conformational entropy is an extensive property of proteins. The calculated conformational entropies of folding agree well with previous estimates. Detailed analysis of specific cases identifies deviations in conformational entropy from the average values that highlight how conformational entropy varies with sequence, secondary structure, and tertiary fold. Notably, α-helices have lower entropy on average than do β-sheets, and both are lower than coil regions.

Arginine Kinase. Joint Crystallographic & NMR RDC Analyses link Substrate-Associated Motions to Intrinsic Flexibility

PubMed Central

Niu, Xiaogang; Brüschweiler-Li, Lei; Davulcu, Omar; Skalicky, Jack J.; Brüschweiler, Rafael; Chapman, Michael S.

2010-01-01

The phosphagen kinase family, including creatine and arginine kinases, catalyze the reversible transfer of a “high energy” phosphate between ATP and a phospho-guanidino substrate. They have become a model for the study of both substrate-induced conformational change and intrinsic protein dynamics. Prior crystallographic studies indicated large substrate-induced domain rotations, but differences among a recent set of arginine kinase structures was interpreted as a plastic deformation. Here, the structure of Limulus substrate-free arginine kinase is refined against high resolution crystallographic data and compared quantitatively with NMR chemical shifts and residual dipolar couplings (RDCs). This demonstrates the feasibility of this type of RDC analysis of proteins that are large by NMR standards (42 kDa), and illuminates the solution structure, free from crystal-packing constraints. Detailed comparison of the 1.7 Å resolution substrate-free crystal structure against the 1.2 Å transition state analog complex shows large substrate-induced domain motions which can be broken down into movements of smaller quasi-rigid bodies. The solution state structure of substrate-free arginine kinase is most consistent with an equilibrium of substrate-free and –bound structures, with the substrate-free form dominating, but with varying displacements of the quasi-rigid groups. Rigid-group rotations evident from the crystal structures are about axes previously associated with intrinsic millisecond dynamics using NMR relaxation dispersion. Thus, “substrate-induced” motions are along modes that are intrinsically flexible in the substrate-free enzyme, and likely involve some degree of conformational selection. PMID:21075117

Brownian motion in non-equilibrium systems and the Ornstein-Uhlenbeck stochastic process.

PubMed

Donado, F; Moctezuma, R E; López-Flores, L; Medina-Noyola, M; Arauz-Lara, J L

2017-10-03

The Ornstein-Uhlenbeck stochastic process is an exact mathematical model providing accurate representations of many real dynamic processes in systems in a stationary state. When applied to the description of random motion of particles such as that of Brownian particles, it provides exact predictions coinciding with those of the Langevin equation but not restricted to systems in thermal equilibrium but only conditioned to be stationary. Here, we investigate experimentally single particle motion in a two-dimensional granular system in a stationary state, consisting of 1 mm stainless balls on a plane circular surface. The motion of the particles is produced by an alternating magnetic field applied perpendicular to the surface of the container. The mean square displacement of the particles is measured for a range of low concentrations and it is found that following an appropriate scaling of length and time, the short-time experimental curves conform a master curve covering the range of particle motion from ballistic to diffusive in accordance with the description of the Ornstein-Uhlenbeck model.

Hierarchical structure of the energy landscape of proteins revisited by time series analysis. II. Investigation of explicit solvent effects

NASA Astrophysics Data System (ADS)

Alakent, Burak; Camurdan, Mehmet C.; Doruker, Pemra

2005-10-01

Time series analysis tools are employed on the principal modes obtained from the Cα trajectories from two independent molecular-dynamics simulations of α-amylase inhibitor (tendamistat). Fluctuations inside an energy minimum (intraminimum motions), transitions between minima (interminimum motions), and relaxations in different hierarchical energy levels are investigated and compared with those encountered in vacuum by using different sampling window sizes and intervals. The low-frequency low-indexed mode relationship, established in vacuum, is also encountered in water, which shows the reliability of the important dynamics information offered by principal components analysis in water. It has been shown that examining a short data collection period (100ps) may result in a high population of overdamped modes, while some of the low-frequency oscillations (<10cm-1) can be captured in water by using a longer data collection period (1200ps). Simultaneous analysis of short and long sampling window sizes gives the following picture of the effect of water on protein dynamics. Water makes the protein lose its memory: future conformations are less dependent on previous conformations due to the lowering of energy barriers in hierarchical levels of the energy landscape. In short-time dynamics (<10ps), damping factors extracted from time series model parameters are lowered. For tendamistat, the friction coefficient in the Langevin equation is found to be around 40-60cm-1 for the low-indexed modes, compatible with literature. The fact that water has increased the friction and that on the other hand has lubrication effect at first sight contradicts. However, this comes about because water enhances the transitions between minima and forces the protein to reduce its already inherent inability to maintain oscillations observed in vacuum. Some of the frequencies lower than 10cm-1 are found to be overdamped, while those higher than 20cm-1 are slightly increased. As for the long-time dynamics in water, it is found that random-walk motion is maintained for approximately 200ps (about five times of that in vacuum) in the low-indexed modes, showing the lowering of energy barriers between the higher-level minima.

Modeling Conformational Transitions and Energetics of Ligand Binding with the Glutamate Receptor Ligand Binding Domain

NASA Astrophysics Data System (ADS)

Kurnikova, Maria

2009-03-01

Understanding of protein motion and energetics of conformational transitions is crucial to understanding protein function. The glutamate receptor ligand binding domain (GluR2 S1S2) is a two lobe protein, which binds ligand at the interface of two lobes and undergoes conformational transition. The cleft closure conformational transition of S1S2 has been implicated in gating of the ion channel formed by the transmembrane domain of the receptor. In this study we present a composite multi-faceted theoretical analysis of the detailed mechanism of this conformational transition based on rigid cluster decomposition of the protein structure [1] and identifying hydrogen bonds that are responsible for stabilizing the closed conformation [2]. Free energy of the protein reorganization upon ligand binding was calculated using combined Thermodynamic Integration (TI) and Umbrella Sampling (US) simulations [3]. Ligand -- protein interactions in the binding cleft were analyzed using Molecular Dynamics, continuum electrostatics and QM/MM models [4]. All model calculations compare well with corresponding experimental measurements. [4pt] [1] Protein Flexibility using Constraints from Molecular Dynamics Simulations T. Mamonova, B. Hespenheide, R. Straub, M. F. Thorpe, M. G. Kurnikova , Phys. Biol., 2, S137 (2005)[0pt] [2] Theoretical Study of the Glutamate Receptor Ligand Binding Domain Flexibility and Conformational Reorganization T. Mamonova, K. Speranskiy, and M. Kurnikova , Prot.: Struct., Func., Bioinf., 73,656 (2008)[0pt] [3] Energetics of the cleft closing transition and glutamate binding in the Glutamate Receptor ligand Binding Domain T. Mamonova, M. Yonkunas, and M. Kurnikova Biochemistry 47, 11077 (2008)[0pt] [4] On the Binding Determinants of the Glutamate Agonist with the Glutamate Receptor Ligand Binding Domain K. Speranskiy and M. Kurnikova Biochemistry 44, 11208 (2005)

Molecular dynamics simulations of viral RNA polymerases link conserved and correlated motions of functional elements to fidelity

PubMed Central

Moustafa, Ibrahim M.; Shen, Hujun; Morton, Brandon; Colina, Coray M.; Cameron, Craig E.

2011-01-01

The viral RNA-dependent RNA polymerase (RdRp) is essential for multiplication of all RNA viruses. The sequence diversity of an RNA virus population contributes to its ability to infect the host. This diversity emanates from errors made by the RdRp during RNA synthesis. The physical basis for RdRp fidelity is unclear but is linked to conformational changes occurring during the nucleotide-addition cycle. To understand RdRp dynamics that might influence RdRp function, we have analyzed all-atom molecular dynamics (MD) simulations on the nanosecond timescale of four RdRps from the picornavirus family that exhibit 30–74% sequence identity. Principal component analysis showed that the major motions observed during the simulations derived from conserved structural motifs and regions of known function. Dynamics of residues participating in the same biochemical property, for example RNA binding, nucleotide binding or catalysis, were correlated even when spatially distant on the RdRp structure. The conserved and correlated dynamics of functional, structural elements suggest co-evolution of dynamics with structure and function of the RdRp. Crystal structures of all picornavirus RdRps exhibit a template-nascent RNA duplex channel too small to fully accommodate duplex RNA. Simulations revealed opening and closing motions of the RNA and NTP channels, which might be relevant to NTP entry, PPi exit and translocation. A role for nanosecond timescale dynamics in RdRp fidelity is supported by altered dynamics of the high-fidelity G64S derivative of PV RdRp relative to wild-type enzyme. PMID:21575642

Allosteric Regulation of the Hsp90 Dynamics and Stability by Client Recruiter Cochaperones: Protein Structure Network Modeling

PubMed Central

Blacklock, Kristin; Verkhivker, Gennady M.

2014-01-01

The fundamental role of the Hsp90 chaperone in supporting functional activity of diverse protein clients is anchored by specific cochaperones. A family of immune sensing client proteins is delivered to the Hsp90 system with the aid of cochaperones Sgt1 and Rar1 that act cooperatively with Hsp90 to form allosterically regulated dynamic complexes. In this work, functional dynamics and protein structure network modeling are combined to dissect molecular mechanisms of Hsp90 regulation by the client recruiter cochaperones. Dynamic signatures of the Hsp90-cochaperone complexes are manifested in differential modulation of the conformational mobility in the Hsp90 lid motif. Consistent with the experiments, we have determined that targeted reorganization of the lid dynamics is a unifying characteristic of the client recruiter cochaperones. Protein network analysis of the essential conformational space of the Hsp90-cochaperone motions has identified structurally stable interaction communities, interfacial hubs and key mediating residues of allosteric communication pathways that act concertedly with the shifts in conformational equilibrium. The results have shown that client recruiter cochaperones can orchestrate global changes in the dynamics and stability of the interaction networks that could enhance the ATPase activity and assist in the client recruitment. The network analysis has recapitulated a broad range of structural and mutagenesis experiments, particularly clarifying the elusive role of Rar1 as a regulator of the Hsp90 interactions and a stability enhancer of the Hsp90-cochaperone complexes. Small-world organization of the interaction networks in the Hsp90 regulatory complexes gives rise to a strong correspondence between highly connected local interfacial hubs, global mediator residues of allosteric interactions and key functional hot spots of the Hsp90 activity. We have found that cochaperone-induced conformational changes in Hsp90 may be determined by specific interaction networks that can inhibit or promote progression of the ATPase cycle and thus control the recruitment of client proteins. PMID:24466147

Allosteric regulation of the Hsp90 dynamics and stability by client recruiter cochaperones: protein structure network modeling.

PubMed

Blacklock, Kristin; Verkhivker, Gennady M

2014-01-01

The fundamental role of the Hsp90 chaperone in supporting functional activity of diverse protein clients is anchored by specific cochaperones. A family of immune sensing client proteins is delivered to the Hsp90 system with the aid of cochaperones Sgt1 and Rar1 that act cooperatively with Hsp90 to form allosterically regulated dynamic complexes. In this work, functional dynamics and protein structure network modeling are combined to dissect molecular mechanisms of Hsp90 regulation by the client recruiter cochaperones. Dynamic signatures of the Hsp90-cochaperone complexes are manifested in differential modulation of the conformational mobility in the Hsp90 lid motif. Consistent with the experiments, we have determined that targeted reorganization of the lid dynamics is a unifying characteristic of the client recruiter cochaperones. Protein network analysis of the essential conformational space of the Hsp90-cochaperone motions has identified structurally stable interaction communities, interfacial hubs and key mediating residues of allosteric communication pathways that act concertedly with the shifts in conformational equilibrium. The results have shown that client recruiter cochaperones can orchestrate global changes in the dynamics and stability of the interaction networks that could enhance the ATPase activity and assist in the client recruitment. The network analysis has recapitulated a broad range of structural and mutagenesis experiments, particularly clarifying the elusive role of Rar1 as a regulator of the Hsp90 interactions and a stability enhancer of the Hsp90-cochaperone complexes. Small-world organization of the interaction networks in the Hsp90 regulatory complexes gives rise to a strong correspondence between highly connected local interfacial hubs, global mediator residues of allosteric interactions and key functional hot spots of the Hsp90 activity. We have found that cochaperone-induced conformational changes in Hsp90 may be determined by specific interaction networks that can inhibit or promote progression of the ATPase cycle and thus control the recruitment of client proteins.

Allosteric Pathways in the PPARγ-RXRα nuclear receptor complex

NASA Astrophysics Data System (ADS)

Ricci, Clarisse G.; Silveira, Rodrigo L.; Rivalta, Ivan; Batista, Victor S.; Skaf, Munir S.

2016-01-01

Understanding the nature of allostery in DNA-nuclear receptor (NR) complexes is of fundamental importance for drug development since NRs regulate the transcription of a myriad of genes in humans and other metazoans. Here, we investigate allostery in the peroxisome proliferator-activated/retinoid X receptor heterodimer. This important NR complex is a target for antidiabetic drugs since it binds to DNA and functions as a transcription factor essential for insulin sensitization and lipid metabolism. We find evidence of interdependent motions of Ω-loops and PPARγ-DNA binding domain with contacts susceptible to conformational changes and mutations, critical for regulating transcriptional functions in response to sequence-dependent DNA dynamics. Statistical network analysis of the correlated motions, observed in molecular dynamics simulations, shows preferential allosteric pathways with convergence centers comprised of polar amino acid residues. These findings are particularly relevant for the design of allosteric modulators of ligand-dependent transcription factors.

A comparison between elastic network interpolation and MD simulation of 16S ribosomal RNA.

PubMed

Kim, Moon K; Li, Wen; Shapiro, Bruce A; Chirikjian, Gregory S

2003-12-01

In this paper a coarse-grained method called elastic network interpolation (ENI) is used to generate feasible transition pathways between two given conformations of the core central domain of 16S Ribosomal RNA (16S rRNA). The two given conformations are the extremes generated by a molecular dynamics (MD) simulation, which differ from each other by 10A in root-mean-square deviation (RMSD). It takes only several hours to build an ENI pathway on a 1.5GHz Pentium with 512 MB memory, while the MD takes several weeks on high-performance multi-processor servers such as the SGI ORIGIN 2000/2100. It is shown that multiple ENI pathways capture the essential anharmonic motions of millions of timesteps in a particular MD simulation. A coarse-grained normal mode analysis (NMA) is performed on each intermediate ENI conformation, and the lowest 1% of the normal modes (representing about 40 degrees of freedom (DOF)) are used to parameterize fluctuations. This combined ENI/NMA method captures all intermediate conformations in the MD run with 1.5A RMSD on average. In addition, if we restrict attention to the time interval of the MD run between the two extreme conformations, the RMSD between the closest ENI/NMA pathway and the MD results is about 1A. These results may serve as a paradigm for reduced-DOF dynamic simulations of large biological macromolecules as well as a method for the reduced-parameter interpretation of massive amounts of MD data.

Disturbance of DNA conformation by the binding of testosterone-based platinum drugs via groove-face and intercalative interactions: a molecular dynamics simulation study

PubMed Central

2013-01-01

Background To explore novel platinum-based anticancer agents that are distinct from the structure and interaction mode of the traditional cisplatin by forming the bifunctional intrastrand 1,2 GpG adduct, the monofunctional platinum + DNA adducts with extensive non-covalent interactions had been studied. It was reported that the monofunctional testosterone-based platinum(II) agents present the high anticancer activity. Moreover, it was also found that the testosterone-based platinum agents could cause the DNA helix to undergo significant unwinding and bending over the non-testosterone-based platinum agents. However, the interaction mechanisms of these platinum agents with DNA at the atomic level are not yet clear so far. Results In the present work, we used molecular dynamics (MD) simulations and DNA conformational dynamics calculations to study the DNA distortion properties of the testosterone-based platinum + DNA, the improved testosterone-based platinum + DNA and the non-testosterone-based platinum + DNA adducts. The results show that the intercalative interaction of the improved flexible testosterone-based platinum agent with DNA molecule could cause larger DNA conformational distortion than the groove-face interaction of the rigid testosterone-based platinum agent with DNA molecule. Further investigations for the non-testosterone-based platinum agent reveal the occurrence of insignificant change of DNA conformation due to the absence of testosterone ligand in such agent. Based on the DNA dynamics analysis, the DNA base motions relating to DNA groove parameter changes and hydrogen bond destruction of DNA base pairs were also discussed in this work. Conclusions The flexible linker in the improved testosterone-based platinum agent causes an intercalative interaction with DNA in the improved testosterone-based platinum + DNA adduct, which is different from the groove-face interaction caused by a rigid linker in the testosterone-based platinum agent. The present investigations provide useful information of DNA conformation affected by a testosterone-based platinum complex at the atomic level. PMID:23517640

Nuclear magnetic resonance study of the conformation and dynamics of beta-casein at the oil/water interface in emulsions.

PubMed Central

ter Beek, L C; Ketelaars, M; McCain, D C; Smulders, P E; Walstra, P; Hemminga, M A

1996-01-01

A (13)C and (31)P nuclear magnetic resonance (NMR) study has been carried out on beta-casein adsorbed at the interface of a tetradecane/water emulsion. (13)C NMR spectra show signals from the carbonyl, carboxyl, aromatic, and C alpha carbons in beta-casein, well resolved from solvent resonances. Only a small fraction of all carbon atoms in beta-casein contribute to detectable signals; intensity measurements show that the observable spectrum is derived from about 30 to 40 amino acid residues.(31)P NMR spectra show signals from the five phosphoserines on the hydrophilic N-terminal part of the protein. Analysis of T(1) relaxation times of these nuclei, using the model free approach for the spectral density function and the line shape of the alpha-carbon region, indicates that a large part of the protein is in a random coil conformation with restricted motion and a relatively long internal correlation time. The NMR results show that the conformation and dynamics of the N-terminal part of beta-casein are not strongly altered at the oil/water interface, as compared to beta-casein in micelle-like aggregates in aqueous solution. PMID:9172765

Binding Site Configurations Probe the Structure and Dynamics of the Zinc Finger of NEMO (NF-κB Essential Modulator).

PubMed

Godwin, Ryan C; Melvin, Ryan L; Gmeiner, William H; Salsbury, Freddie R

2017-01-31

Zinc-finger proteins are regulators of critical signaling pathways for various cellular functions, including apoptosis and oncogenesis. Here, we investigate how binding site protonation states and zinc coordination influence protein structure, dynamics, and ultimately function, as these pivotal regulatory proteins are increasingly important for protein engineering and therapeutic discovery. To better understand the thermodynamics and dynamics of the zinc finger of NEMO (NF-κB essential modulator), as well as the role of zinc, we present results of 20 μs molecular dynamics trajectories, 5 μs for each of four active site configurations. Consistent with experimental evidence, the zinc ion is essential for mechanical stabilization of the functional, folded conformation. Hydrogen bond motifs are unique for deprotonated configurations yet overlap in protonated cases. Correlated motions and principal component analysis corroborate the similarity of the protonated configurations and highlight unique relationships of the zinc-bound configuration. We hypothesize a potential mechanism for zinc binding from results of the thiol configurations. The deprotonated, zinc-bound configuration alone predominantly maintains its tertiary structure throughout all 5 μs and alludes rare conformations potentially important for (im)proper zinc-finger-related protein-protein or protein-DNA interactions.

Hydrogen/deuterium exchange studies of native rabbit MM-CK dynamics.

PubMed

Mazon, Hortense; Marcillat, Olivier; Forest, Eric; Vial, Christian

2004-02-01

Creatine kinase (CK) isoenzymes catalyse the reversible transfer of a phosphoryl group from ATP onto creatine. This reaction plays a very important role in the regulation of intracellular ATP concentrations in excitable tissues. CK isoenzymes are highly resistant to proteases in native conditions. To appreciate localized backbone dynamics, kinetics of amide hydrogen exchange with deuterium was measured by pulse-labeling the dimeric cytosolic muscle CK isoenzyme. Upon exchange, the protein was digested with pepsin, and the deuterium content of the resulting peptides was determined by liquid chromatography coupled to mass spectrometry (MS). The deuteration kinetics of 47 peptides identified by MS/MS and covering 96% of the CK backbone were analyzed. Four deuteration patterns have been recognized: The less deuterated peptides are located in the saddle-shaped core of CK, whereas most of the highly deuterated peptides are close to the surface and located around the entrance to the active site. Their exchange kinetics are discussed by comparison with the known secondary and tertiary structures of CK with the goal to reveal the conformational dynamics of the protein. Some of the observed dynamic motions may be linked to the conformational changes associated with substrate binding and catalytic mechanism.

Metadynamics in the conformational space nonlinearly dimensionally reduced by Isomap.

PubMed

Spiwok, Vojtěch; Králová, Blanka

2011-12-14

Atomic motions in molecules are not linear. This infers that nonlinear dimensionality reduction methods can outperform linear ones in analysis of collective atomic motions. In addition, nonlinear collective motions can be used as potentially efficient guides for biased simulation techniques. Here we present a simulation with a bias potential acting in the directions of collective motions determined by a nonlinear dimensionality reduction method. Ad hoc generated conformations of trans,trans-1,2,4-trifluorocyclooctane were analyzed by Isomap method to map these 72-dimensional coordinates to three dimensions, as described by Brown and co-workers [J. Chem. Phys. 129, 064118 (2008)]. Metadynamics employing the three-dimensional embeddings as collective variables was applied to explore all relevant conformations of the studied system and to calculate its conformational free energy surface. The method sampled all relevant conformations (boat, boat-chair, and crown) and corresponding transition structures inaccessible by an unbiased simulation. This scheme allows to use essentially any parameter of the system as a collective variable in biased simulations. Moreover, the scheme we used for mapping out-of-sample conformations from the 72D to 3D space can be used as a general purpose mapping for dimensionality reduction, beyond the context of molecular modeling. © 2011 American Institute of Physics

Dynamics of Hydrophobic Core Phenylalanine Residues Probed by Solid-State Deuteron NMR

PubMed Central

Vugmeyster, Liliya; Ostrovsky, Dmitry; Villafranca, Toni; Sharp, Janelle; Xu, Wei; Lipton, Andrew S.; Hoatson, Gina L.; Vold, Robert L.

2016-01-01

We conducted a detailed investigation of the dynamics of two phenylalanine side chains in the hydrophobic core of the villin headpiece subdomain protein (HP36) in the hydrated powder state over the 298–80 K temperature range. Our main tools were static deuteron NMR measurements of longitudinal relaxation and line shapes supplemented with computational modeling. The temperature dependence of the relaxation times reveals the presence of two main mechanisms that can be attributed to the ring-flips, dominating at high temperatures, and small-angle fluctuations, dominating at low temperatures. The relaxation is non-exponential at all temperatures with the extent of non-exponentiality increasing from higher to lower temperatures. This behavior suggests a distribution of conformers with unique values of activation energies. The central values of the activation energies for the ring-flipping motions are among the smallest reported for aromatic residues in peptides and proteins and point to a very mobile hydrophobic core. The analysis of the widths of the distributions, in combination with the earlier results on the dynamics of flanking methyl groups (Vugmeyster et al., J. Phys. Chem. B 2013, 117, 6129–6137), suggests that the hydrophobic core undergoes slow concerted fluctuations. There is a pronounced effect of dehydration on the ring-flipping motions, which shifts the distribution toward more rigid conformers. The cross-over temperature between the regions of dominance of the small-angle fluctuations and ring-flips shifts from 195 K in the hydrated protein to 278 K in the dry one. This result points to the role of solvent in softening the core and highlights aromatic residues as markers of the protein dynamical transitions. PMID:26529128

Dynamics of Hydrophobic Core Phenylalanine Residues Probed by Solid-State Deuteron NMR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vugmeyster, Liliya; Ostrovsky, Dmitry; Villafranca, Toni

We conducted a detailed investigation of the dynamics of two phenylalanine side chains in the hydrophobic core of the villin headpiece subdomain protein (HP36) in the hydrated powder state over the 298–80 K temperature range. We utilized static deuteron NMR measurements of longitudinal relaxation and line shapes supplemented with computational modeling. The temperature dependence of the relaxation times reveals the presence of two main mechanisms that can be attributed to the ring-flips, dominating at high temperatures, and small-angle fluctuations, dominating at low temperatures. The relaxation is non- exponential at all temperatures with the extent of non-exponentiality increasing from higher tomore » lower temperatures. This behavior suggests a distribution of conformers with unique values of activation energies. The central values of the activation energies for the ring-flipping motions are among the smallest reported for aromatic residues in peptides and proteins and point to a very mobile hydrophobic core. The analysis of the widths of the distributions, in combination with the earlier results on the dynamics of flanking methyl groups (Vugmeyster et al., J. Phys. Chem. 2013, 117, 6129–6137), suggests that the hydrophobic core undergoes concerted fluctuations. There is a pronounced effect of dehydration on the ring-flipping motions, which shifts the distribution toward more rigid conformers. The cross-over temperature between the regions of dominance of the small-angle fluctuations and ring-flips shifts from 195 K in the hydrated protein to 278 K in the dry one. This result points to the role of solvent in the onset of the concerted fluctuations of the core and highlights aromatic residues as markers of the protein dynamical transitions.« less

Mechanisms of Intramolecular Communication in a Hyperthermophilic Acylaminoacyl Peptidase: A Molecular Dynamics Investigation

PubMed Central

Papaleo, Elena; Renzetti, Giulia; Tiberti, Matteo

2012-01-01

Protein dynamics and the underlying networks of intramolecular interactions and communicating residues within the three-dimensional (3D) structure are known to influence protein function and stability, as well as to modulate conformational changes and allostery. Acylaminoacyl peptidase (AAP) subfamily of enzymes belongs to a unique class of serine proteases, the prolyl oligopeptidase (POP) family, which has not been thoroughly investigated yet. POPs have a characteristic multidomain three-dimensional architecture with the active site at the interface of the C-terminal catalytic domain and a β-propeller domain, whose N-terminal region acts as a bridge to the hydrolase domain. In the present contribution, protein dynamics signatures of a hyperthermophilic acylaminoacyl peptidase (AAP) of the prolyl oligopeptidase (POP) family, as well as of a deletion variant and alanine mutants (I12A, V13A, V16A, L19A, I20A) are reported. In particular, we aimed at identifying crucial residues for long range communications to the catalytic site or promoting the conformational changes to switch from closed to open ApAAP conformations. Our investigation shows that the N-terminal α1-helix mediates structural intramolecular communication to the catalytic site, concurring to the maintenance of a proper functional architecture of the catalytic triad. Main determinants of the effects induced by α1-helix are a subset of hydrophobic residues (V16, L19 and I20). Moreover, a subset of residues characterized by relevant interaction networks or coupled motions have been identified, which are likely to modulate the conformational properties at the interdomain interface. PMID:22558199

Possible Dynamically Gated Conductance along Heme Wires in Bacterial Multiheme Cytochromes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Dayle MA; Rosso, Kevin M.

2014-07-24

The staggered cross decaheme configuration of electron transfer co-factors in the outer-membrane cytochrome MtrF may serve as a prototype for conformationally-gated multi-heme electron transport. Derived from the bacterium Shewanella oneidensis, the staggered cross configuration reveals intersecting c-type octaheme and tetraheme “wires” containing thermodynamic “hills” and “valleys”, suggesting that the protein structure may include a dynamical mechanism for conductance and pathway switching depending on enzymatic functional need. Recent molecular simulations have established the pair-wise electronic couplings, redox potentials, and reorganization energies to predict the maximum conductance along the various heme wire pathways by sequential hopping of a single electron (PNAS (2014)more » 11,611-616). Here, we expand this information with classical molecular and statistical mechanics calculations of large-amplitude protein dynamics in MtrF, to address its potential to modulate pathway conductance, including assessment of the effect of the total charge state. Explicit solvent molecular dynamics simulations of fully oxidized and fully reduced MtrF employing ten independent 50-ns simulations at 300 K and 1 atm showed that reduced MtrF is more expanded and explores more conformational space than oxidized MtrF, and that heme reduction leads to increased heme solvent exposure. The slowest mode of collective decaheme motion is 90% similar between the oxidized and reduced states, and consists primarily of inter-heme separation with minor rotational contributions. The frequency of this motion is 1.7×107 s 1 for fully-oxidized and fully-reduced MtrF, respectively, slower than the downhill electron transfer rates between stacked heme pairs at the octaheme termini and faster than the electron transfer rates between parallel hemes in the tetraheme chain. This implies that MtrF uses slow conformational fluctuations to modulate electron flow along the octaheme pathway, apparently for the purpose of increasing the residence time of electrons on lowest potential hemes 4 and 9. This apparent gating mechanism should increase the success rate of electron transfer from MtrF to low potential environmental acceptors via these two solvent-exposed hemes.« less

Applications of AdS-CFT to problems in black hole physics and hydrodynamics

NASA Astrophysics Data System (ADS)

Hansen, James Michael

The work contained in this thesis is divided naturally into two parts. In the first part we present a systematic treatment of angular momentum charges in asymptotically AdS spacetimes. This treatment, motivated by AdS-CFT, explains the puzzle of charges generated by coordinate transformations in a manifestly diffeomorphism invariant theory. In the second part we explore a promising connection between the dynamics of dyonic black holes and 2+1 dimensional conformal fluids in the presence of a strong magnetic field. We explicitly demonstrate that long-wavelength perturbations of the dyonic black hole satisfy the equations of motion of a conformal fluid and derive the stress tensor, currents, and transport properties of this fluid.

Investigations of the Binding of [Pt2(DTBPA)Cl2](II) and [Pt2(TPXA)Cl2](II) to DNA via Various Cross-Linking Modes

PubMed Central

Yue, Hongwei; Yang, Bo; Wang, Yan; Chen, Guangju

2013-01-01

We have constructed models for a series of platinum-DNA adducts that represent the binding of two agents, [Pt2(DTBPA)Cl2](II) and [Pt2(TPXA)Cl2](II), to DNA via inter- and intra-strand cross-linking, and carried out molecular dynamics simulations and DNA conformational dynamics calculations. The effects of trans- and cis-configurations of the centers of these di-nuclear platinum agents, and of different bridging linkers, have been investigated on the conformational distortions of platinum-DNA adducts formed via inter- and intra-strand cross-links. The results demonstrate that the DNA conformational distortions for the various platinum-DNA adducts with differing cross-linking modes are greatly influenced by the difference between the platinum-platinum distance for the platinum agent and the platinum-bound N7–N7 distance for the DNA molecule, and by the flexibility of the bridging linkers in the platinum agent. However, the effects of trans/cis-configurations of the platinum-centers on the DNA conformational distortions in the platinum-DNA adducts depend on the inter- and intra-strand cross-linking modes. In addition, we discuss the relevance of DNA base motions, including opening, shift and roll, to the changes in the parameters of the DNA major and minor grooves caused by binding of the platinum agent. PMID:24077126

Energetics and Structural Characterization of the large-scale Functional Motion of Adenylate Kinase

PubMed Central

Formoso, Elena; Limongelli, Vittorio; Parrinello, Michele

2015-01-01

Adenylate Kinase (AK) is a signal transducing protein that regulates cellular energy homeostasis balancing between different conformations. An alteration of its activity can lead to severe pathologies such as heart failure, cancer and neurodegenerative diseases. A comprehensive elucidation of the large-scale conformational motions that rule the functional mechanism of this enzyme is of great value to guide rationally the development of new medications. Here using a metadynamics-based computational protocol we elucidate the thermodynamics and structural properties underlying the AK functional transitions. The free energy estimation of the conformational motions of the enzyme allows characterizing the sequence of events that regulate its action. We reveal the atomistic details of the most relevant enzyme states, identifying residues such as Arg119 and Lys13, which play a key role during the conformational transitions and represent druggable spots to design enzyme inhibitors. Our study offers tools that open new areas of investigation on large-scale motion in proteins. PMID:25672826

Energetics and Structural Characterization of the large-scale Functional Motion of Adenylate Kinase

NASA Astrophysics Data System (ADS)

Formoso, Elena; Limongelli, Vittorio; Parrinello, Michele

2015-02-01

Adenylate Kinase (AK) is a signal transducing protein that regulates cellular energy homeostasis balancing between different conformations. An alteration of its activity can lead to severe pathologies such as heart failure, cancer and neurodegenerative diseases. A comprehensive elucidation of the large-scale conformational motions that rule the functional mechanism of this enzyme is of great value to guide rationally the development of new medications. Here using a metadynamics-based computational protocol we elucidate the thermodynamics and structural properties underlying the AK functional transitions. The free energy estimation of the conformational motions of the enzyme allows characterizing the sequence of events that regulate its action. We reveal the atomistic details of the most relevant enzyme states, identifying residues such as Arg119 and Lys13, which play a key role during the conformational transitions and represent druggable spots to design enzyme inhibitors. Our study offers tools that open new areas of investigation on large-scale motion in proteins.

Principal Component Analysis reveals correlation of cavities evolution and functional motions in proteins.

PubMed

Desdouits, Nathan; Nilges, Michael; Blondel, Arnaud

2015-02-01

Protein conformation has been recognized as the key feature determining biological function, as it determines the position of the essential groups specifically interacting with substrates. Hence, the shape of the cavities or grooves at the protein surface appears to drive those functions. However, only a few studies describe the geometrical evolution of protein cavities during molecular dynamics simulations (MD), usually with a crude representation. To unveil the dynamics of cavity geometry evolution, we developed an approach combining cavity detection and Principal Component Analysis (PCA). This approach was applied to four systems subjected to MD (lysozyme, sperm whale myoglobin, Dengue envelope protein and EF-CaM complex). PCA on cavities allows us to perform efficient analysis and classification of the geometry diversity explored by a cavity. Additionally, it reveals correlations between the evolutions of the cavities and structures, and can even suggest how to modify the protein conformation to induce a given cavity geometry. It also helps to perform fast and consensual clustering of conformations according to cavity geometry. Finally, using this approach, we show that both carbon monoxide (CO) location and transfer among the different xenon sites of myoglobin are correlated with few cavity evolution modes of high amplitude. This correlation illustrates the link between ligand diffusion and the dynamic network of internal cavities. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Dynamical Coupling of Intrinsically Disordered Proteins and Their Hydration Water: Comparison with Folded Soluble and Membrane Proteins

PubMed Central

Gallat, F.-X.; Laganowsky, A.; Wood, K.; Gabel, F.; van Eijck, L.; Wuttke, J.; Moulin, M.; Härtlein, M.; Eisenberg, D.; Colletier, J.-P.; Zaccai, G.; Weik, M.

2012-01-01

Hydration water is vital for various macromolecular biological activities, such as specific ligand recognition, enzyme activity, response to receptor binding, and energy transduction. Without hydration water, proteins would not fold correctly and would lack the conformational flexibility that animates their three-dimensional structures. Motions in globular, soluble proteins are thought to be governed to a certain extent by hydration-water dynamics, yet it is not known whether this relationship holds true for other protein classes in general and whether, in turn, the structural nature of a protein also influences water motions. Here, we provide insight into the coupling between hydration-water dynamics and atomic motions in intrinsically disordered proteins (IDP), a largely unexplored class of proteins that, in contrast to folded proteins, lack a well-defined three-dimensional structure. We investigated the human IDP tau, which is involved in the pathogenic processes accompanying Alzheimer disease. Combining neutron scattering and protein perdeuteration, we found similar atomic mean-square displacements over a large temperature range for the tau protein and its hydration water, indicating intimate coupling between them. This is in contrast to the behavior of folded proteins of similar molecular weight, such as the globular, soluble maltose-binding protein and the membrane protein bacteriorhodopsin, which display moderate to weak coupling, respectively. The extracted mean square displacements also reveal a greater motional flexibility of IDP compared with globular, folded proteins and more restricted water motions on the IDP surface. The results provide evidence that protein and hydration-water motions mutually affect and shape each other, and that there is a gradient of coupling across different protein classes that may play a functional role in macromolecular activity in a cellular context. PMID:22828339

Dynamic features of carboxy cytoglobin distal mutants investigated by molecular dynamics simulations.

PubMed

Zhao, Cong; Du, Weihong

2016-04-01

Cytoglobin (Cgb) is a member of hemoprotein family with roles in NO metabolism, fibrosis, and tumourigenesis. Similar to other hemoproteins, Cgb structure and functions are markedly influenced by distal key residues. The sixth ligand His(81) (E7) is crucial to exogenous ligand binding, heme pocket conformation, and physiological roles of this protein. However, the effects of other key residues on heme pocket and protein biological functions are not well known. In this work, a molecular dynamics (MD) simulation study of two single mutants in CO-ligated Cgb (L46FCgbCO and L46VCgbCO) and two double mutants (L46FH81QCgbCO and L46VH81QCgbCO) was conducted to explore the effects of the key distal residues Leu(46)(B10) and His(81)(E7) on Cgb structure and functions. Results indicated that the distal mutation of B10 and E7 affected CgbCO dynamic properties on loop region fluctuation, internal cavity rearrangement, and heme motion. The distal conformation change was reflected by the distal key residues Gln(62) (CD3) and Arg(84)(E10). The hydrogen bond between heme propionates with CD3 or E10 residues were evidently influenced by B10/E7 mutation. Furthermore, heme pocket rearrangement was also observed based on the distal pocket volume and occurrence rate of inner cavities. The mutual effects of B10 and E7 residues on protein conformational rearrangement and other dynamic features were expressed in current MD studies of CgbCO and its distal mutants, suggesting their crucial role in heme pocket stabilization, ligand binding, and Cgb biological functions. The mutation of distal B10 and E7 residues affects the dynamic features of carboxy cytoglobin.

In silico strategies toward enzyme function and dynamics.

PubMed

Estácio, Sílvia G

2012-01-01

Enzymes are outstanding biocatalysts involved in a plethora of chemical reactions occurring in the cell. Despite their incommensurable importance, a comprehensive understanding of enzyme catalysis is still missing. This task becomes more laborious given the unavoidability of including the inherent dynamic nature of enzymes into that description. As such, it is essential to ascertain the nature and contribution of enzyme conformational changes to catalysis and to evaluate the adequacy of the proposal associating protein internal motions to the rate enhancement achieved. Dynamic events in enzymes span a wide range of time- and length-scales which have led to a surge in multiscale methodologies targeting enzyme function and dynamics. Computational strategies assume a preponderant role in such studies by allowing the atomic detail investigation of the fundamental mechanisms of enzyme catalysis thus surpassing what is achievable through experiments. While high-accuracy quantum mechanical methods are indicated to uncover the details of the chemical reaction occurring at the active site, molecular mechanical force fields and molecular dynamics approaches provide powerful means to access the conformational energy landscape accessible to enzymes. This review outlines some of the most important in silico methodologies in this area, highlighting examples of problems tackled and the insights obtained. Copyright © 2012 Elsevier Inc. All rights reserved.

Molecular dynamics simulations reveal the conformational dynamics of Arabidopsis thaliana BRI1 and BAK1 receptor-like kinases.

PubMed

Moffett, Alexander S; Bender, Kyle W; Huber, Steven C; Shukla, Diwakar

2017-07-28

The structural motifs responsible for activation and regulation of eukaryotic protein kinases in animals have been studied extensively in recent years, and a coherent picture of their activation mechanisms has begun to emerge. In contrast, non-animal eukaryotic protein kinases are not as well understood from a structural perspective, representing a large knowledge gap. To this end, we investigated the conformational dynamics of two key Arabidopsis thaliana receptor-like kinases, brassinosteroid-insensitive 1 (BRI1) and BRI1-associated kinase 1 (BAK1), through extensive molecular dynamics simulations of their fully phosphorylated kinase domains. Molecular dynamics simulations calculate the motion of each atom in a protein based on classical approximations of interatomic forces, giving researchers insight into protein function at unparalleled spatial and temporal resolutions. We found that in an otherwise "active" BAK1 the αC helix is highly disordered, a hallmark of deactivation, whereas the BRI1 αC helix is moderately disordered and displays swinging behavior similar to numerous animal kinases. An analysis of all known sequences in the A. thaliana kinome found that αC helix disorder may be a common feature of plant kinases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Equivalent equations of motion for gravity and entropy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Czech, Bartlomiej; Lamprou, Lampros; McCandlish, Samuel

We demonstrate an equivalence between the wave equation obeyed by the entanglement entropy of CFT subregions and the linearized bulk Einstein equation in Anti-de Sitter space. In doing so, we make use of the formalism of kinematic space and fields on this space. We show that the gravitational dynamics are equivalent to a gauge invariant wave-equation on kinematic space and that this equation arises in natural correspondence to the conformal Casimir equation in the CFT.

Equivalent equations of motion for gravity and entropy

DOE PAGES

Czech, Bartlomiej; Lamprou, Lampros; McCandlish, Samuel; ...

2017-02-01

We demonstrate an equivalence between the wave equation obeyed by the entanglement entropy of CFT subregions and the linearized bulk Einstein equation in Anti-de Sitter space. In doing so, we make use of the formalism of kinematic space and fields on this space. We show that the gravitational dynamics are equivalent to a gauge invariant wave-equation on kinematic space and that this equation arises in natural correspondence to the conformal Casimir equation in the CFT.

Mechanism of mRNA-STAR domain interaction: Molecular dynamics simulations of Mammalian Quaking STAR protein.

PubMed

Sharma, Monika; Anirudh, C R

2017-10-03

STAR proteins are evolutionary conserved mRNA-binding proteins that post-transcriptionally regulate gene expression at all stages of RNA metabolism. These proteins possess conserved STAR domain that recognizes identical RNA regulatory elements as YUAAY. Recently reported crystal structures show that STAR domain is composed of N-terminal QUA1, K-homology domain (KH) and C-terminal QUA2, and mRNA binding is mediated by KH-QUA2 domain. Here, we present simulation studies done to investigate binding of mRNA to STAR protein, mammalian Quaking protein (QKI). We carried out conventional MD simulations of STAR domain in presence and absence of mRNA, and studied the impact of mRNA on the stability, dynamics and underlying allosteric mechanism of STAR domain. Our unbiased simulations results show that presence of mRNA stabilizes the overall STAR domain by reducing the structural deviations, correlating the 'within-domain' motions, and maintaining the native contacts information. Absence of mRNA not only influenced the essential modes of motion of STAR domain, but also affected the connectivity of networks within STAR domain. We further explored the dissociation of mRNA from STAR domain using umbrella sampling simulations, and the results suggest that mRNA binding to STAR domain occurs in multi-step: first conformational selection of mRNA backbone conformations, followed by induced fit mechanism as nucleobases interact with STAR domain.

D Animation Reconstruction from Multi-Camera Coordinates Transformation

NASA Astrophysics Data System (ADS)

Jhan, J. P.; Rau, J. Y.; Chou, C. M.

2016-06-01

Reservoir dredging issues are important to extend the life of reservoir. The most effective and cost reduction way is to construct a tunnel to desilt the bottom sediment. Conventional technique is to construct a cofferdam to separate the water, construct the intake of tunnel inside and remove the cofferdam afterwards. In Taiwan, the ZengWen reservoir dredging project will install an Elephant-trunk Steel Pipe (ETSP) in the water to connect the desilting tunnel without building the cofferdam. Since the installation is critical to the whole project, a 1:20 model was built to simulate the installation steps in a towing tank, i.e. launching, dragging, water injection, and sinking. To increase the construction safety, photogrammetry technic is adopted to record images during the simulation, compute its transformation parameters for dynamic analysis and reconstruct the 4D animations. In this study, several Australiscoded targets are fixed on the surface of ETSP for auto-recognition and measurement. The cameras orientations are computed by space resection where the 3D coordinates of coded targets are measured. Two approaches for motion parameters computation are proposed, i.e. performing 3D conformal transformation from the coordinates of cameras and relative orientation computation by the orientation of single camera. Experimental results show the 3D conformal transformation can achieve sub-mm simulation results, and relative orientation computation shows the flexibility for dynamic motion analysis which is easier and more efficiency.

Folding of the four-helix bundle FF domain from a compact on-pathway intermediate state is governed predominantly by water motion.

PubMed

Sekhar, Ashok; Vallurupalli, Pramodh; Kay, Lewis E

2012-11-20

Friction plays a critical role in protein folding. Frictional forces originating from random solvent and protein fluctuations both retard motion along the folding pathway and activate protein molecules to cross free energy barriers. Studies of friction thus may provide insights into the driving forces underlying protein conformational dynamics. However, the molecular origin of friction in protein folding remains poorly understood because, with the exception of the native conformer, there generally is little detailed structural information on the other states participating in the folding process. Here, we study the folding of the four-helix bundle FF domain that proceeds via a transiently formed, sparsely populated compact on-pathway folding intermediate whose structure was elucidated previously. Because the intermediate is stabilized by both native and nonnative interactions, friction in the folding transition between intermediate and folded states is expected to arise from intrachain reorganization in the protein. However, the viscosity dependencies of rates of folding from or unfolding to the intermediate, as established by relaxation dispersion NMR spectroscopy, clearly indicate that contributions from internal friction are small relative to those from solvent, so solvent frictional forces drive the folding process. Our results emphasize the importance of solvent dynamics in mediating the interconversion between protein configurations, even those that are highly compact, and in equilibrium folding/unfolding fluctuations in general.

Revealing an outward-facing open conformational state in a CLC Cl–/H+ exchange transporter

PubMed Central

Khantwal, Chandra M; Abraham, Sherwin J; Han, Wei; Jiang, Tao; Chavan, Tanmay S; Cheng, Ricky C; Elvington, Shelley M; Liu, Corey W; Mathews, Irimpan I; Stein, Richard A; Mchaourab, Hassane S; Tajkhorshid, Emad; Maduke, Merritt

2016-01-01

CLC secondary active transporters exchange Cl- for H+. Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its protonation. Using 19F NMR, we show that as [H+] is increased to protonate Gluex and enrich the outward-facing state, a residue ~20 Å away from Gluex, near the subunit interface, moves from buried to solvent-exposed. Consistent with functional relevance of this motion, constriction via inter-subunit cross-linking reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gate-opening motions. In support of this model, mutations that decrease steric contact between Helix N (part of the extracellular gate) and Helix P (at the subunit interface) remove the inhibitory effect of the cross-link. Together, these results demonstrate the formation of a previously uncharacterized 'outward-facing open' state, and highlight the relevance of global structural changes in CLC function. DOI: http://dx.doi.org/10.7554/eLife.11189.001 PMID:26799336

New insights into the molecular characteristics behind the function of Renilla luciferase.

PubMed

Fanaei-Kahrani, Zahra; Ganjalikhany, Mohamad Reza; Rasa, Seyed Mohammad Mahdi; Emamzadeh, Rahman

2018-02-01

Renilla Luciferase (RLuc) is a blue light emitter protein which can be applied as a valuable tool in medical diagnosis. But due to lack of the crystal structure of RLuc-ligand complex, the functional motions and catalytic mechanism of this enzyme remain largely unknown. In the present study, the active site properties and the ligand-receptor interactions of the native RLuc and its red-shifted light emitting variant (Super RLuc 8) were investigated using molecular docking approach, molecular dynamics (MD) analysis, and MM-PBSA method. The detailed analysis of the main clusters led to identifying a lid-like structure and its functional motions. Furthermore, an induced-fit mechanism is proposed where ligand-binding induces conformational changes of the active site. Our findings give an insight into the deeper understanding of RLuc conformational changes during binding steps and ligand-receptor pattern. Moreover, our work broaden our understanding of how active site geometry is adjusted to support the catalytic activity and red-shifted light emission in Super RLuc 8. © 2017 Wiley Periodicals, Inc.

Domain motions of Argonaute, the catalytic engine of RNA interference

PubMed Central

Ming, Dengming; Wall, Michael E; Sanbonmatsu, Kevin Y

2007-01-01

Background The Argonaute protein is the core component of the RNA-induced silencing complex, playing the central role of cleaving the mRNA target. Visual inspection of static crystal structures already has enabled researchers to suggest conformational changes of Argonaute that might occur during RNA interference. We have taken the next step by performing an all-atom normal mode analysis of the Pyrococcus furiosus and Aquifex aeolicus Argonaute crystal structures, allowing us to quantitatively assess the feasibility of these conformational changes. To perform the analysis, we begin with the energy-minimized X-ray structures. Normal modes are then calculated using an all-atom molecular mechanics force field. Results The analysis reveals low-frequency vibrations that facilitate the accommodation of RNA duplexes – an essential step in target recognition. The Pyrococcus furiosus and Aquifex aeolicus Argonaute proteins both exhibit low-frequency torsion and hinge motions; however, differences in the overall architecture of the proteins cause the detailed dynamics to be significantly different. Conclusion Overall, low-frequency vibrations of Argonaute are consistent with mechanisms within the current reaction cycle model for RNA interference. PMID:18053142

Domain motions of Argonaute, the catalytic engine of RNA interference.

PubMed

Ming, Dengming; Wall, Michael E; Sanbonmatsu, Kevin Y

2007-11-30

The Argonaute protein is the core component of the RNA-induced silencing complex, playing the central role of cleaving the mRNA target. Visual inspection of static crystal structures already has enabled researchers to suggest conformational changes of Argonaute that might occur during RNA interference. We have taken the next step by performing an all-atom normal mode analysis of the Pyrococcus furiosus and Aquifex aeolicus Argonaute crystal structures, allowing us to quantitatively assess the feasibility of these conformational changes. To perform the analysis, we begin with the energy-minimized X-ray structures. Normal modes are then calculated using an all-atom molecular mechanics force field. The analysis reveals low-frequency vibrations that facilitate the accommodation of RNA duplexes - an essential step in target recognition. The Pyrococcus furiosus and Aquifex aeolicus Argonaute proteins both exhibit low-frequency torsion and hinge motions; however, differences in the overall architecture of the proteins cause the detailed dynamics to be significantly different. Overall, low-frequency vibrations of Argonaute are consistent with mechanisms within the current reaction cycle model for RNA interference.

Fourier transformation microwave spectroscopy of the methyl glycolate-H2O complex

NASA Astrophysics Data System (ADS)

Fujitake, Masaharu; Tanaka, Toshihiro; Ohashi, Nobukimi

2018-01-01

The rotational spectrum of one conformer of the methyl glycolate-H2O complex has been measured by means of the pulsed jet Fourier transform microwave spectrometer. The observed a- and b-type transitions exhibit doublet splittings due to the internal rotation of the methyl group. On the other hand, most of the c-type transitions exhibit quartet splittings arising from the methyl internal rotation and the inversion motion between two equivalent conformations. The spectrum was analyzed using parameterized expressions of the Hamiltonian matrix elements derived by applying the tunneling matrix formalism. Based on the results obtained from ab initio calculation, the observed complex of methyl glycolate-H2O was assigned to the most stable conformer of the insertion complex, in which a non-planer seven membered-ring structure is formed by the intermolecular hydrogen bonds between methyl glycolate and H2O subunits. The inversion motion observed in the c-type transitions is therefore a kind of ring-inversion motion between two equivalent conformations. Conformational flexibility, which corresponds to the ring-inversion between two equivalent conformations and to the isomerization between two possible conformers of the insertion complex, was investigated with the help of the ab initio calculation.

Direct observation of the myosin Va recovery stroke that contributes to unidirectional stepping along actin.

PubMed

Shiroguchi, Katsuyuki; Chin, Harvey F; Hannemann, Diane E; Muneyuki, Eiro; De La Cruz, Enrique M; Kinosita, Kazuhiko

2011-04-01

Myosins are ATP-driven linear molecular motors that work as cellular force generators, transporters, and force sensors. These functions are driven by large-scale nucleotide-dependent conformational changes, termed "strokes"; the "power stroke" is the force-generating swinging of the myosin light chain-binding "neck" domain relative to the motor domain "head" while bound to actin; the "recovery stroke" is the necessary initial motion that primes, or "cocks," myosin while detached from actin. Myosin Va is a processive dimer that steps unidirectionally along actin following a "hand over hand" mechanism in which the trailing head detaches and steps forward ∼72 nm. Despite large rotational Brownian motion of the detached head about a free joint adjoining the two necks, unidirectional stepping is achieved, in part by the power stroke of the attached head that moves the joint forward. However, the power stroke alone cannot fully account for preferential forward site binding since the orientation and angle stability of the detached head, which is determined by the properties of the recovery stroke, dictate actin binding site accessibility. Here, we directly observe the recovery stroke dynamics and fluctuations of myosin Va using a novel, transient caged ATP-controlling system that maintains constant ATP levels through stepwise UV-pulse sequences of varying intensity. We immobilized the neck of monomeric myosin Va on a surface and observed real time motions of bead(s) attached site-specifically to the head. ATP induces a transient swing of the neck to the post-recovery stroke conformation, where it remains for ∼40 s, until ATP hydrolysis products are released. Angle distributions indicate that the post-recovery stroke conformation is stabilized by ≥ 5 k(B)T of energy. The high kinetic and energetic stability of the post-recovery stroke conformation favors preferential binding of the detached head to a forward site 72 nm away. Thus, the recovery stroke contributes to unidirectional stepping of myosin Va.

DNA Scrunching in the Packaging of Viral Genomes.

PubMed

Waters, James T; Kim, Harold D; Gumbart, James C; Lu, Xiang-Jun; Harvey, Stephen C

2016-07-07

The motors that drive double-stranded DNA (dsDNA) genomes into viral capsids are among the strongest of all biological motors for which forces have been measured, but it is not known how they generate force. We previously proposed that the DNA is not a passive substrate but that it plays an active role in force generation. This "scrunchworm hypothesis" holds that the motor proteins repeatedly dehydrate and rehydrate the DNA, which then undergoes cyclic shortening and lengthening motions. These are captured by a coupled protein-DNA grip-and-release cycle to rectify the motion and translocate the DNA into the capsid. In this study, we examined the interactions of dsDNA with the dodecameric connector protein of bacteriophage ϕ29, using molecular dynamics simulations on four different DNA sequences, starting from two different conformations (A-DNA and B-DNA). In all four simulations starting with the protein equilibrated with A-DNA in the channel, we observed transitions to a common, metastable, highly scrunched conformation, designated A*. This conformation is very similar to one recently reported by Kumar and Grubmüller in much longer MD simulations on B-DNA docked into the ϕ29 connector. These results are significant for four reasons. First, the scrunched conformations occur spontaneously, without requiring lever-like protein motions often believed to be necessary for DNA translocation. Second, the transition takes place within the connector, providing the location of the putative "dehydrator". Third, the protein has more contacts with one strand of the DNA than with the other; the former was identified in single-molecule laser tweezer experiments as the "load-bearing strand". Finally, the spontaneity of the DNA-protein interaction suggests that it may play a role in the initial docking of DNA in motors like that of T4 that can load and package any sequence.

A planning comparison of 3-dimensional conformal multiple static field, conformal arc, and volumetric modulated arc therapy for the delivery of stereotactic body radiotherapy for early stage lung cancer.

PubMed

Dickey, Mike; Roa, Wilson; Drodge, Suzanne; Ghosh, Sunita; Murray, Brad; Scrimger, Rufus; Gabos, Zsolt

2015-01-01

The primary objective of this study was to compare dosimetric variables as well as treatment times of multiple static fields (MSFs), conformal arcs (CAs), and volumetric modulated arc therapy (VMAT) techniques for the treatment of early stage lung cancer using stereotactic body radiotherapy (SBRT). Treatments of 23 patients previously treated with MSF of 48Gy to 95% of the planning target volume (PTV) in 4 fractions were replanned using CA and VMAT techniques. Dosimetric parameters of the Radiation Therapy Oncology Group (RTOG) 0915 trial were evaluated, along with the van׳t Riet conformation number (CN), monitor units (MUs), and actual and calculated treatment times. Paired t-tests for noninferiority were used to compare the 3 techniques. CA had significant dosimetric improvements over MSF for the ratio of the prescription isodose volume to PTV (R100%, p < 0.0001), the maximum dose 2cm away from the PTV (D2cm, p = 0.005), and van׳t Riet CN (p < 0.0001). CA was not statistically inferior to MSF for the 50% prescription isodose volume to PTV (R50%, p = 0.05). VMAT was significantly better than CA for R100% (p < 0.0001), R50% (p < 0.0001), D2cm (p = 0.006), and CN (p < 0.0001). CA plans had significantly shorter treatment times than those of VMAT (p < 0.0001). Both CA and VMAT planning showed significant dosimetric improvements and shorter treatment times over those of MSF. VMAT showed the most favorable dosimetry of all 3 techniques; however, the dosimetric effect of tumor motion was not evaluated. CA plans were significantly faster to treat, and minimize the interplay of tumor motion and dynamic multileaf collimator (MLC) motion effects. Given these results, CA has become the treatment technique of choice at our facility. Copyright © 2015 American Association of Medical Dosimetrists. Published by Elsevier Inc. All rights reserved.

Strand swapping regulates the iron-sulfur cluster in the diabetes drug target mitoNEET

PubMed Central

Baxter, Elizabeth Leigh; Jennings, Patricia A.; Onuchic, José N.

2012-01-01

MitoNEET is a recently identified diabetes drug target that coordinates a transferable 2Fe-2S cluster, and additionally contains an unusual strand swap. In this manuscript, we use a dual basin structure-based model to predict and characterize the folding and functionality of strand swapping in mitoNEET. We demonstrate that a strand unswapped conformation is kinetically accessible and that multiple levels of control are employed to regulate the conformational dynamics of the system. Environmental factors such as temperature can shift route preference toward the unswapped pathway. Additionally we see that a region recently identified as contributing to frustration in folding acts as a regulatory hinge loop that modulates conformational balance. Interestingly, strand unswapping transfers strain specifically to cluster-coordinating residues, opening the cluster-coordinating pocket. Strengthening contacts within the cluster-coordinating pocket opens a new pathway between the swapped and unswapped conformation that utilizes cracking to bypass the unfolded basin. These results suggest that local control within distinct regions affect motions important in regulating mitoNEET’s 2Fe-2S clusters. PMID:22308404

Decomposition of total solvation energy into core, side-chains and water contributions: Role of cross correlations and protein conformational fluctuations in dynamics of hydration layer

NASA Astrophysics Data System (ADS)

Mondal, Sayantan; Mukherjee, Saumyak; Bagchi, Biman

2017-09-01

Dynamical coupling between water and amino acid side-chain residues in solvation dynamics is investigated by selecting residues often used as natural probes, namely tryptophan, tyrosine and histidine, located at different positions on protein surface. Such differently placed residues are found to exhibit different timescales of relaxation. The total solvation response measured by the probe is decomposed in terms of its interactions with (i) protein core, (ii) side-chain and (iii) water. Significant anti cross-correlation among these contributions are observed. When the motion of the protein side-chains is quenched, solvation either becomes faster or slower depending on the location of the probe.

Effects of Mutations on Structure-Function Relationships of Matrix Metalloproteinase-1.

PubMed

Singh, Warispreet; Fields, Gregg B; Christov, Christo Z; Karabencheva-Christova, Tatyana G

2016-10-14

Matrix metalloproteinase-1 (MMP-1) is one of the most widely studied enzymes involved in collagen degradation. Mutations of specific residues in the MMP-1 hemopexin-like (HPX) domain have been shown to modulate activity of the MMP-1 catalytic (CAT) domain. In order to reveal the structural and conformational effects of such mutations, a molecular dynamics (MD) study was performed of in silico mutated residues in the X-ray crystallographic structure of MMP-1 complexed with a collagen-model triple-helical peptide (THP). The results indicate an important role of the mutated residues in MMP-1 interactions with the THP and communication between the CAT and the HPX domains. Each mutation has a distinct impact on the correlated motions in the MMP-1•THP. An increased collagenase activity corresponded to the appearance of a unique anti-correlated motion and decreased correlated motions, while decreased collagenase activity corresponded both to increased and decreased anti-correlated motions.

Experimental validation of the van Herk margin formula for lung radiation therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ecclestone, Gillian; Heath, Emily; Bissonnette, Jean-Pierre

2013-11-15

Purpose: To validate the van Herk margin formula for lung radiation therapy using realistic dose calculation algorithms and respiratory motion modeling. The robustness of the margin formula against variations in lesion size, peak-to-peak motion amplitude, tissue density, treatment technique, and plan conformity was assessed, along with the margin formula assumption of a homogeneous dose distribution with perfect plan conformity.Methods: 3DCRT and IMRT lung treatment plans were generated within the ORBIT treatment planning platform (RaySearch Laboratories, Sweden) on 4DCT datasets of virtual phantoms. Random and systematic respiratory motion induced errors were simulated using deformable registration and dose accumulation tools available withinmore » ORBIT for simulated cases of varying lesion sizes, peak-to-peak motion amplitudes, tissue densities, and plan conformities. A detailed comparison between the margin formula dose profile model, the planned dose profiles, and penumbra widths was also conducted to test the assumptions of the margin formula. Finally, a correction to account for imperfect plan conformity was tested as well as a novel application of the margin formula that accounts for the patient-specific motion trajectory.Results: The van Herk margin formula ensured full clinical target volume coverage for all 3DCRT and IMRT plans of all conformities with the exception of small lesions in soft tissue. No dosimetric trends with respect to plan technique or lesion size were observed for the systematic and random error simulations. However, accumulated plans showed that plan conformity decreased with increasing tumor motion amplitude. When comparing dose profiles assumed in the margin formula model to the treatment plans, discrepancies in the low dose regions were observed for the random and systematic error simulations. However, the margin formula respected, in all experiments, the 95% dose coverage required for planning target volume (PTV) margin derivation, as defined by the ICRU; thus, suitable PTV margins were estimated. The penumbra widths calculated in lung tissue for each plan were found to be very similar to the 6.4 mm value assumed by the margin formula model. The plan conformity correction yielded inconsistent results which were largely affected by image and dose grid resolution while the trajectory modified PTV plans yielded a dosimetric benefit over the standard internal target volumes approach with up to a 5% decrease in the V20 value.Conclusions: The margin formula showed to be robust against variations in tumor size and motion, treatment technique, plan conformity, as well as low tissue density. This was validated by maintaining coverage of all of the derived PTVs by 95% dose level, as required by the formal definition of the PTV. However, the assumption of perfect plan conformity in the margin formula derivation yields conservative margin estimation. Future modifications to the margin formula will require a correction for plan conformity. Plan conformity can also be improved by using the proposed trajectory modified PTV planning approach. This proves especially beneficial for tumors with a large anterior–posterior component of respiratory motion.« less

Minimum Free Energy Path of Ligand-Induced Transition in Adenylate Kinase

PubMed Central

Matsunaga, Yasuhiro; Fujisaki, Hiroshi; Terada, Tohru; Furuta, Tadaomi; Moritsugu, Kei; Kidera, Akinori

2012-01-01

Large-scale conformational changes in proteins involve barrier-crossing transitions on the complex free energy surfaces of high-dimensional space. Such rare events cannot be efficiently captured by conventional molecular dynamics simulations. Here we show that, by combining the on-the-fly string method and the multi-state Bennett acceptance ratio (MBAR) method, the free energy profile of a conformational transition pathway in Escherichia coli adenylate kinase can be characterized in a high-dimensional space. The minimum free energy paths of the conformational transitions in adenylate kinase were explored by the on-the-fly string method in 20-dimensional space spanned by the 20 largest-amplitude principal modes, and the free energy and various kinds of average physical quantities along the pathways were successfully evaluated by the MBAR method. The influence of ligand binding on the pathways was characterized in terms of rigid-body motions of the lid-shaped ATP-binding domain (LID) and the AMP-binding (AMPbd) domains. It was found that the LID domain was able to partially close without the ligand, while the closure of the AMPbd domain required the ligand binding. The transition state ensemble of the ligand bound form was identified as those structures characterized by highly specific binding of the ligand to the AMPbd domain, and was validated by unrestrained MD simulations. It was also found that complete closure of the LID domain required the dehydration of solvents around the P-loop. These findings suggest that the interplay of the two different types of domain motion is an essential feature in the conformational transition of the enzyme. PMID:22685395

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ono, Tomohiro; Miyabe, Yuki, E-mail: miyabe@kuhp.kyoto-u.ac.jp; Yamada, Masahiro

Purpose: The Vero4DRT system has the capability for dynamic tumor-tracking (DTT) stereotactic irradiation using a unique gimbaled x-ray head. The purposes of this study were to develop DTT conformal arc irradiation and to estimate its geometric and dosimetric accuracy. Methods: The gimbaled x-ray head, supported on an O-ring gantry, was moved in the pan and tilt directions during O-ring gantry rotation. To evaluate the mechanical accuracy, the gimbaled x-ray head was moved during the gantry rotating according to input command signals without a target tracking, and a machine log analysis was performed. The difference between a command and a measuredmore » position was calculated as mechanical error. To evaluate beam-positioning accuracy, a moving phantom, which had a steel ball fixed at the center, was driven based on a sinusoidal wave (amplitude [A]: 20 mm, time period [T]: 4 s), a patient breathing motion with a regular pattern (A: 16 mm, average T: 4.5 s), and an irregular pattern (A: 7.2–23.0 mm, T: 2.3–10.0 s), and irradiated with DTT during gantry rotation. The beam-positioning error was evaluated as the difference between the centroid position of the irradiated field and the steel ball on images from an electronic portal imaging device. For dosimetric accuracy, dose distributions in static and moving targets were evaluated with DTT conformal arc irradiation. Results: The root mean squares (RMSs) of the mechanical error were up to 0.11 mm for pan motion and up to 0.14 mm for tilt motion. The RMSs of the beam-positioning error were within 0.23 mm for each pattern. The dose distribution in a moving phantom with tracking arc irradiation was in good agreement with that in static conditions. Conclusions: The gimbal positional accuracy was not degraded by gantry motion. As in the case of a fixed port, the Vero4DRT system showed adequate accuracy of DTT conformal arc irradiation.« less

Trifluoperazine Regulation of Calmodulin Binding to Fas: A Computational Study

PubMed Central

Pan, Di; Yan, Qi; Chen, Yabing; McDonald, Jay M; Song, Yuhua

2011-01-01

Death-inducing signaling complex (DISC) formation is a critical step in Fas-mediated signaling for apoptosis. Previous experiments have demonstrated that the calmodulin (CaM) antagonist, trifluoperazine (TFP) regulates CaM-Fas binding and affects Fas-mediated DISC formation. In this study, we investigated the anti-cooperative characteristics of TFP binding to CaM and the effect of TFP on the CaM-Fas interaction from both structural and thermodynamic perspectives using combined molecular dynamics simulations and binding free energy analyses. We studied the interactions of different numbers of TFP molecules with CaM and explored the effects of the resulting conformational changes in CaM on CaM-Fas binding. Results from these analyses showed that the number of TFP molecules bound to CaM directly influenced α-helix formation and hydrogen bond occupancy within the α-helices of CaM, contributing to the conformational and motion changes in CaM. These changes affected CaM binding to Fas, resulting in secondary structural changes in Fas and conformational and motion changes of Fas in CaM-Fas complexes, potentially perturbing the recruitment of Fas-associated death domain (FADD) for DISC formation. The computational results from this study reveal the structural and molecular mechanisms that underlie the role of the CaM antagonist, TFP, in regulation of CaM-Fas binding and Fas-mediated DISC formation in a concentration-dependent manner. PMID:21656570

Methods for modeling cytoskeletal and DNA filaments

NASA Astrophysics Data System (ADS)

Andrews, Steven S.

2014-02-01

This review summarizes the models that researchers use to represent the conformations and dynamics of cytoskeletal and DNA filaments. It focuses on models that address individual filaments in continuous space. Conformation models include the freely jointed, Gaussian, angle-biased chain (ABC), and wormlike chain (WLC) models, of which the first three bend at discrete joints and the last bends continuously. Predictions from the WLC model generally agree well with experiment. Dynamics models include the Rouse, Zimm, stiff rod, dynamic WLC, and reptation models, of which the first four apply to isolated filaments and the last to entangled filaments. Experiments show that the dynamic WLC and reptation models are most accurate. They also show that biological filaments typically experience strong hydrodynamic coupling and/or constrained motion. Computer simulation methods that address filament dynamics typically compute filament segment velocities from local forces using the Langevin equation and then integrate these velocities with explicit or implicit methods; the former are more versatile and the latter are more efficient. Much remains to be discovered in biological filament modeling. In particular, filament dynamics in living cells are not well understood, and current computational methods are too slow and not sufficiently versatile. Although primarily a review, this paper also presents new statistical calculations for the ABC and WLC models. Additionally, it corrects several discrepancies in the literature about bending and torsional persistence length definitions, and their relations to flexural and torsional rigidities.

Probe conformational dynamics of proteins in aqueous solutions by terahertz spectroscopy

NASA Astrophysics Data System (ADS)

Vinh, Nguyen Q.

2016-10-01

Proteins solvated in their biologically milieu are expected to exhibit strong absorption in the terahertz frequencies, that contain information on their global and sub-global collective vibrational modes (conformational dynamics) and global dynamic correlations among solvent water and proteins. The dynamics play an important role in enzymatic activities of proteins, but obtaining an accurate and quantitative pictures of these activities, however, is challenging due to the strong absorption of water. In response, we have developed the world's highest precision, highest sensitivity terahertz-frequency domain spectrometer and a standard terahertz-time domain system to probe the collective dynamics of proteins in aqueous solutions. Operating over the frequency range from 5 GHz up to 3 THz, our spectrometers provide an unparalleled ability to probe directly such questions as the hydration level, the dynamics of water and hydrated proteins over the 100 fs to 1 ns timescale. Employing an effective medium approximation to describe the complex dielectric response of the solvated proteins in solution we find that proteins are surrounded by a loosely and tightly held layers of water molecules that behave as if they are an integral part of the protein. The number of water molecules in the protein hydration shells varies with proteins, which can tell us the average surface structure of proteins. These measurements shed light on the macromolecular motions of proteins in their biologically relevant environment.

Perpetual Motion with Maxwell's Demon

NASA Astrophysics Data System (ADS)

Gordon, Lyndsay G. M.

2002-11-01

A method for producing a temperature gradient by Brownian motion in an equilibrated isolated system composed of two fluid compartments and a separating adiabatic membrane is discussed. This method requires globular protein molecules, partially embedded in the membrane, to alternate between two conformations which lie on opposite sides of the membrane. The greater part of each conformer is bathed by one of the fluids and rotates in Brownian motion around its axis, perpendicular to the membrane. Rotational energy is transferred through the membrane during conformational changes. Angular momentum is conserved during the transitions. The energy flow becomes asymmetrical when the conformational changes of the protein are sterically hindered by two of its side-chains, the positions of which are affected by the angular velocity of the rotor. The heat flow increases the temperature gradient in contravention of the Second Law. A second hypothetical model which illustrates solute transfer at variance with the Second Law is also discussed.

Identifying Slow Molecular Motions in Complex Chemical Reactions.

PubMed

Piccini, GiovanniMaria; Polino, Daniela; Parrinello, Michele

2017-09-07

We have studied the cyclization reaction of deprotonated 4-chloro-1-butanethiol to tetrahydrothiophene by means of well-tempered metadynamics. To properly select the collective variables, we used the recently proposed variational approach to conformational dynamics within the framework of metadyanmics. This allowed us to select the appropriate linear combinations from a set of collective variables representing the slow degrees of freedom that best describe the slow modes of the reaction. We performed our calculations at three different temperatures, namely, 300, 350, and 400 K. We show that the choice of such collective variables allows one to easily interpret the complex free-energy surface of such a reaction by univocal identification of the conformers belonging to reactants and product states playing a fundamental role in the reaction mechanism.

Conformational Entropy of FK506 Binding to FKBP12 Determined by Nuclear Magnetic Resonance Relaxation and Molecular Dynamics Simulations.

PubMed

Solomentsev, Gleb; Diehl, Carl; Akke, Mikael

2018-03-06

FKBP12 (FK506 binding protein 12 kDa) is an important drug target. Nuclear magnetic resonance (NMR) order parameters, describing amplitudes of motion on the pico- to nanosecond time scale, can provide estimates of changes in conformational entropy upon ligand binding. Here we report backbone and methyl-axis order parameters of the apo and FK506-bound forms of FKBP12, based on 15 N and 2 H NMR relaxation. Binding of FK506 to FKBP12 results in localized changes in order parameters, notably for the backbone of residues E54 and I56 and the side chains of I56, I90, and I91, all positioned in the binding site. The order parameters increase slightly upon FK506 binding, indicating an unfavorable entropic contribution to binding of TΔ S = -18 ± 2 kJ/mol at 293 K. Molecular dynamics simulations indicate a change in conformational entropy, associated with all dihedral angles, of TΔ S = -26 ± 9 kJ/mol. Both these values are significant compared to the total entropy of binding determined by isothermal titration calorimetry and referenced to a reactant concentration of 1 mM ( TΔ S = -29 ± 1 kJ/mol). Our results reveal subtle differences in the response to ligand binding compared to that of the previously studied rapamycin-FKBP12 complex, despite the high degree of structural homology between the two complexes and their nearly identical ligand-FKBP12 interactions. These results highlight the delicate dependence of protein dynamics on drug interactions, which goes beyond the view provided by static structures, and reinforce the notion that protein conformational entropy can make important contributions to the free energy of ligand binding.

Single DNA molecules on freestanding and supported cationic lipid bilayers: diverse conformational dynamics controlled by the local bilayer properties

NASA Astrophysics Data System (ADS)

Herold, Christoph; Schwille, Petra; Petrov, Eugene P.

2016-02-01

We present experimental results on the interaction of DNA macromolecules with cationic lipid membranes with different properties, including freestanding membranes in the fluid and gel state, and supported lipid membranes in the fluid state and under conditions of fluid-gel phase coexistence. We observe diverse conformational dynamics of membrane-bound DNA molecules controlled by the local properties of the lipid bilayer. In case of fluid-state freestanding lipid membranes, the behaviour of DNA on the membrane is controlled by the membrane charge density: whereas DNA bound to weakly charged membranes predominantly behaves as a 2D random coil, an increase in the membrane charge density leads to membrane-driven irreversible DNA collapse and formation of subresolution-sized DNA globules. On the other hand, electrostatic binding of DNA macromolecules to gel-state freestanding membranes leads to completely arrested diffusion and conformational dynamics of membrane-adsorbed DNA. A drastically different picture is observed in case of DNA interaction with supported cationic lipid bilayers: When the supported bilayer is in the fluid state, membrane-bound DNA molecules undergo 2D translational Brownian motion and conformational fluctuations, irrespectively of the charge density of the supported bilayer. At the same time, when the supported cationic membrane shows fluid-gel phase coexistence, membrane-bound DNA molecules are strongly attracted to micrometre-sized gel-phase domains enriched with the cationic lipid, which results in 2D compaction of the membrane-bound macromolecules. This DNA compaction, however, is fully reversible, and disappears as soon as the membrane is heated above the fluid-gel coexistence. We also discuss possible biological implications of our experimental findings.

Fluctuation Dynamics Analysis of gp120 Envelope Protein Reveals a Topologically Based Communication Network

PubMed Central

Shrivastava, Indira; LaLonde, Judith M.

2012-01-01

HIV infection is initiated by binding of the viral glycoprotein gp120, to the cellular receptor CD4. Upon CD4 binding, gp120 undergoes conformational change, permitting binding to the chemokine receptor. Crystal structures of gp120 ternary complex reveal the CD4 bound conformation of gp120. We report here the application of Gaussian Network Model (GNM) to the crystal structures of gp120 bound to CD4 or CD4 mimic and 17b, to study the collective motions of the gp120 core and determine the communication propensities of the residue network. The GNM fluctuation profiles identify residues in the inner domain and outer domain that may facilitate conformational change or stability, respectively. Communication propensities delineate a residue network that is topologically suited for signal propagation from the Phe43 cavity throughout the gp120 outer domain. . These results provide a new context for interpreting gp120 core envelope structure-function relationships. PMID:20718047

Damped-Dynamics Flexible Fitting

PubMed Central

Kovacs, Julio A.; Yeager, Mark; Abagyan, Ruben

2008-01-01

In fitting atomic structures into EM maps, it often happens that the map corresponds to a different conformation of the structure. We have developed a new methodology to handle these situations that preserves the covalent geometry of the structure and allows the modeling of large deformations. The first goal is achieved by working in generalized coordinates (positional and internal coordinates), and the second by avoiding harmonic potentials. Instead, we use dampers (shock absorbers) between every pair of atoms, combined with a force field that attracts the atomic structure toward incompletely occupied regions of the EM map. The trajectory obtained by integrating the resulting equations of motion converges to a conformation that, in our validation cases, was very close to the target atomic structure. Compared to current methods, our approach is more efficient and robust against wrong solutions and to overfitting, and does not require user intervention or subjective decisions. Applications to the computation of transition pathways between known conformers, homology and loop modeling, as well as protein docking, are also discussed. PMID:18586844

Damped-dynamics flexible fitting.

PubMed

Kovacs, Julio A; Yeager, Mark; Abagyan, Ruben

2008-10-01

In fitting atomic structures into EM maps, it often happens that the map corresponds to a different conformation of the structure. We have developed a new methodology to handle these situations that preserves the covalent geometry of the structure and allows the modeling of large deformations. The first goal is achieved by working in generalized coordinates (positional and internal coordinates), and the second by avoiding harmonic potentials. Instead, we use dampers (shock absorbers) between every pair of atoms, combined with a force field that attracts the atomic structure toward incompletely occupied regions of the EM map. The trajectory obtained by integrating the resulting equations of motion converges to a conformation that, in our validation cases, was very close to the target atomic structure. Compared to current methods, our approach is more efficient and robust against wrong solutions and to overfitting, and does not require user intervention or subjective decisions. Applications to the computation of transition pathways between known conformers, homology and loop modeling, as well as protein docking, are also discussed.

Increasing the sampling efficiency of protein conformational transition using velocity-scaling optimized hybrid explicit/implicit solvent REMD simulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Yuqi; Wang, Jinan; Shao, Qiang, E-mail: qshao@mail.shcnc.ac.cn, E-mail: Jiye.Shi@ucb.com, E-mail: wlzhu@mail.shcnc.ac.cn

2015-03-28

The application of temperature replica exchange molecular dynamics (REMD) simulation on protein motion is limited by its huge requirement of computational resource, particularly when explicit solvent model is implemented. In the previous study, we developed a velocity-scaling optimized hybrid explicit/implicit solvent REMD method with the hope to reduce the temperature (replica) number on the premise of maintaining high sampling efficiency. In this study, we utilized this method to characterize and energetically identify the conformational transition pathway of a protein model, the N-terminal domain of calmodulin. In comparison to the standard explicit solvent REMD simulation, the hybrid REMD is much lessmore » computationally expensive but, meanwhile, gives accurate evaluation of the structural and thermodynamic properties of the conformational transition which are in well agreement with the standard REMD simulation. Therefore, the hybrid REMD could highly increase the computational efficiency and thus expand the application of REMD simulation to larger-size protein systems.« less

The ribosome as a molecular machine: the mechanism of tRNA-mRNA movement in translocation.

PubMed

Rodnina, Marina V; Wintermeyer, Wolfgang

2011-04-01

Translocation of tRNA and mRNA through the ribosome is one of the most dynamic events during protein synthesis. In the cell, translocation is catalysed by EF-G (elongation factor G) and driven by GTP hydrolysis. Major unresolved questions are: how the movement is induced and what the moving parts of the ribosome are. Recent progress in time-resolved cryoelectron microscopy revealed trajectories of tRNA movement through the ribosome. Driven by thermal fluctuations, the ribosome spontaneously samples a large number of conformational states. The spontaneous movement of tRNAs through the ribosome is loosely coupled to the motions within the ribosome. EF-G stabilizes conformational states prone to translocation and promotes a conformational rearrangement of the ribosome (unlocking) that accelerates the rate-limiting step of translocation: the movement of the tRNA anticodons on the small ribosomal subunit. EF-G acts as a Brownian ratchet providing directional bias for movement at the cost of GTP hydrolysis.

Understanding the impact of Fc glycosylation on its conformational changes by molecular dynamics simulations and bioinformatics.

PubMed

Zhang, Yubo

2015-12-01

N-linked glycosylation of Fc at N297 plays an important role in its effector function, aberrance of which would cause disease pathogenesis. Here, we performed all-atom molecular dynamics simulations to explore the effects of Fc glycosylation on its dynamics behaviors. Firstly, equilibrium simulations suggested that Fc deglycosylation was able to induce residual flexibility in its CH2 domain. Besides, the free energy landscape revealed three minimum energy wells in deglycosylated Fc, representing its "open", "semi-closed" and "closed" states. However, we could only observe the "open" state of glycosylated Fc. Supportively, principal component analysis emphasized the prominent motion of delyclosylated Fc and dynamically depicted how it changed from the "open" state to its "closed" state. Secondly, we studied the recognition mechanism of the Fc binding to its partners. Energy decomposition analysis identified key residues of Fc to recognize its two partners P13 and P34. Evidently, electrostatic potential surfaces showed that electrostatic attraction helped to stabilize the interaction between Fc and its partners. Also, relative binding free energies explained different binding affinities in Fc-P13 and Fc-P34. Collectively, these results together provided the structural basis for understanding conformational changes of deglycosylated Fc and the recognition mechanism of the Fc binding to its partners.

Anti-Brownian ELectrokinetic (ABEL) trapping of single β2-adrenergic receptors in the absence and presence of agonist

NASA Astrophysics Data System (ADS)

Bockenhauer, Samuel; Fuerstenberg, Alexandre; Yao, Xiao Jie; Kobilka, Brian K.; Moerner, W. E.

2012-02-01

The ABEL trap allows trapping of single biomolecules in solution for extended observation without immobilization. The essential idea combines fluorescence-based position estimation with fast electrokinetic feedback in a microfluidic geometry to counter the Brownian motion of a single nanoscale object, hence maintaining its position in the field of view for hundreds of milliseconds to seconds. Such prolonged observation of single proteins allows access to slow dynamics, as probed by any available photophysical observables. We have used the ABEL trap to study conformational dynamics of the β2-adrenergic receptor, a key G-protein coupled receptor and drug target, in the absence and presence of agonist. A single environment-sensitive dye reports on the receptor microenvironment, providing a real-time readout of conformational change for each trapped receptor. The focus of this paper will be a quantitative comparison of the ligandfree and agonist-bound receptor data from our ABEL trap experiments. We observe a small but clearly detectable shift in conformational equilibria and a lengthening of fluctuation timescales upon binding of agonist. In order to quantify the shift in state distributions and timescales, we apply nonparametric statistical tests to place error bounds on the resulting single-molecule distributions.

O-H anharmonic vibrational motions in Cl(-)···(CH3OH)(1-2) ionic clusters. Combined IRPD experiments and AIMD simulations.

PubMed

Beck, Jordan P; Cimas, Alvaro; Lisy, James M; Gaigeot, Marie-Pierre

2014-02-05

The structures of Cl(-)-(Methanol)1,2 clusters have been unraveled combining Infrared Predissociation (IR-PD) experiments and DFT-based molecular dynamics simulations (DFT-MD) at 100 K. The dynamical IR spectra extracted from DFT-MD provide the initial 600 cm(-1) large anharmonic red-shift of the O-H stretch from uncomplexed methanol (3682 cm(-1)) to Cl(-)-(Methanol)1 complex (3085 cm(-1)) as observed in the IR-PD experiment, as well as the subtle supplementary blue- and red-shifts of the O-H stretch in Cl(-)-(Methanol)2 depending on the structure. The anharmonic vibrational calculations remarkably provide the 100 cm(-1) O-H blue-shift when the two methanol molecules are simultaneously organized in the anion first hydration shell (conformer 2A), while they provide the 240 cm(-1) O-H red-shift when the second methanol is in the second hydration shell of Cl(-) (conformer 2B). RRKM calculations have also shown that 2A/2B conformers interconvert on a nanosecond time-scale at the estimated 100 K temperature of the clusters formed by evaporative cooling of argon prior to the IR-PD process. Copyright © 2013 Elsevier B.V. All rights reserved.

Modeling meiotic chromosome pairing: nuclear envelope attachment, telomere-led active random motion, and anomalous diffusion

PubMed Central

Marshall, Wallace F.; Fung, Jennifer C.

2016-01-01

The recognition and pairing of homologous chromosomes during meiosis is a complex physical and molecular process involving a combination of polymer dynamics and molecular recognition events. Two highly conserved features of meiotic chromosome behavior are the attachment of telomeres to the nuclear envelope and the active random motion of telomeres driven by their interaction with cytoskeletal motor proteins. Both of these features have been proposed to facilitate the process of homolog pairing, but exactly what role these features play in meiosis remains poorly understood. Here we investigate the roles of active motion and nuclear envelope tethering using a Brownian dynamics simulation in which meiotic chromosomes are represented by a Rouse polymer model subjected to tethering and active forces at the telomeres. We find that tethering telomeres to the nuclear envelope slows down pairing relative to the rates achieved by un-attached chromosomes, but that randomly-directed active forces applied to the telomeres speeds up pairing dramatically in a manner that depends on the statistical properties of the telomere force fluctuations. The increased rate of initial pairing cannot be explained by stretching out of the chromosome conformation but instead seems to correlate with anomalous diffusion of sub-telomeric regions. PMID:27046097

Modeling meiotic chromosome pairing: nuclear envelope attachment, telomere-led active random motion, and anomalous diffusion

NASA Astrophysics Data System (ADS)

Marshall, Wallace F.; Fung, Jennifer C.

2016-04-01

The recognition and pairing of homologous chromosomes during meiosis is a complex physical and molecular process involving a combination of polymer dynamics and molecular recognition events. Two highly conserved features of meiotic chromosome behavior are the attachment of telomeres to the nuclear envelope and the active random motion of telomeres driven by their interaction with cytoskeletal motor proteins. Both of these features have been proposed to facilitate the process of homolog pairing, but exactly what role these features play in meiosis remains poorly understood. Here we investigate the roles of active motion and nuclear envelope tethering using a Brownian dynamics simulation in which meiotic chromosomes are represented by a Rouse polymer model subjected to tethering and active forces at the telomeres. We find that tethering telomeres to the nuclear envelope slows down pairing relative to the rates achieved by unattached chromosomes, but that randomly directed active forces applied to the telomeres speed up pairing dramatically in a manner that depends on the statistical properties of the telomere force fluctuations. The increased rate of initial pairing cannot be explained by stretching out of the chromosome conformation but instead seems to correlate with anomalous diffusion of sub-telomeric regions.

The flexibility and dynamics of protein disulfide isomerase

PubMed Central

Wells, Stephen A.; Emilio Jimenez‐Roldan, J.; Bhattacharyya, Moitrayee; Vishweshwara, Saraswathi; Freedman, Robert B.

2016-01-01

ABSTRACT We have studied the mobility of the multidomain folding catalyst, protein disulfide isomerase (PDI), by a coarse‐graining approach based on flexibility. We analyze our simulations of yeast PDI (yPDI) using measures of backbone movement, relative positions and orientations of domains, and distances between functional sites. We find that there is interdomain flexibility at every interdomain junction but these show very different characteristics. The extent of interdomain flexibility is such that yPDI's two active sites can approach much more closely than is found in crystal structures—and indeed hinge motion to bring these sites into proximity is the lowest energy normal mode of motion of the protein. The flexibility predicted for yPDI (based on one structure) includes the other known conformation of yPDI and is consistent with (i) the mobility observed experimentally for mammalian PDI and (ii) molecular dynamics. We also observe intradomain flexibility and clear differences between the domains in their propensity for internal motion. Our results suggest that PDI flexibility enables it to interact with many different partner molecules of widely different sizes and shapes, and highlights considerable similarities of yPDI and mammalian PDI. Proteins 2016; 84:1776–1785. © 2016 Wiley Periodicals, Inc. PMID:27616289

TH-EF-BRB-04: 4π Dynamic Conformal Arc Therapy Dynamic Conformal Arc Therapy (DCAT) for SBRT

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chiu, T; Long, T; Tian, Z.

2016-06-15

Purpose: To develop an efficient and effective trajectory optimization methodology for 4π dynamic conformal arc treatment (4π DCAT) with synchronized gantry and couch motion; and to investigate potential clinical benefits for stereotactic body radiation therapy (SBRT) to breast, lung, liver and spine tumors. Methods: The entire optimization framework for 4π DCAT inverse planning consists of two parts: 1) integer programming algorithm and 2) particle swarm optimization (PSO) algorithm. The integer programming is designed to find an optimal solution for arc delivery trajectory with both couch and gantry rotation, while PSO minimize a non-convex objective function based on the selected trajectorymore » and dose-volume constraints. In this study, control point interaction is explicitly taken into account. Beam trajectory was modeled as a series of control points connected together to form a deliverable path. With linear treatment planning objectives, a mixed-integer program (MIP) was formulated. Under mild assumptions, the MIP is tractable. Assigning monitor units to control points along the path can be integrated into the model and done by PSO. The developed 4π DCAT inverse planning strategy is evaluated on SBRT cases and compared to clinically treated plans. Results: The resultant dose distribution of this technique was evaluated between 3D conformal treatment plan generated by Pinnacle treatment planning system and 4π DCAT on a lung SBRT patient case. Both plans share the same scale of MU, 3038 and 2822 correspondingly to 3D conformal plan and 4π DCAT. The mean doses for most of OARs were greatly reduced at 32% (cord), 70% (esophagus), 2.8% (lung) and 42.4% (stomach). Conclusion: Initial results in this study show the proposed 4π DCAT treatment technique can achieve better OAR sparing and lower MUs, which indicates that the developed technique is promising for high dose SBRT to reduce the risk of secondary cancer.« less

Models of Voltage-Dependent Conformational Changes in NaChBac Channels

PubMed Central

Shafrir, Yinon; Durell, Stewart R.; Guy, H. Robert

2008-01-01

Models of the transmembrane region of the NaChBac channel were developed in two open/inactivated and several closed conformations. Homology models of NaChBac were developed using crystal structures of Kv1.2 and a Kv1.2/2.1 chimera as templates for open conformations, and MlotiK and KcsA channels as templates for closed conformations. Multiple molecular-dynamic simulations were performed to refine and evaluate these models. A striking difference between the S4 structures of the Kv1.2-like open models and MlotiK-like closed models is the secondary structure. In the open model, the first part of S4 forms an α-helix, and the last part forms a 310 helix, whereas in the closed model, the first part of S4 forms a 310 helix, and the last part forms an α-helix. A conformational change that involves this type of transition in secondary structure should be voltage-dependent. However, this transition alone is not sufficient to account for the large gating charge movement reported for NaChBac channels and for experimental results in other voltage-gated channels. To increase the magnitude of the motion of S4, we developed another model of an open/inactivated conformation, in which S4 is displaced farther outward, and a number of closed models in which S4 is displaced farther inward. A helical screw motion for the α-helical part of S4 and a simple axial translation for the 310 portion were used to develop models of these additional conformations. In our models, four positively charged residues of S4 moved outwardly during activation, across a transition barrier formed by highly conserved hydrophobic residues on S1, S2, and S3. The S4 movement was coupled to an opening of the activation gate formed by S6 through interactions with the segment linking S4 to S5. Consistencies of our models with experimental studies of NaChBac and Kv channels are discussed. PMID:18641074

The Role of Flexibility and Conformational Selection in the Binding Promiscuity of PDZ Domains

PubMed Central

Münz, Márton; Hein, Jotun; Biggin, Philip C.

2012-01-01

In molecular recognition, it is often the case that ligand binding is coupled to conformational change in one or both of the binding partners. Two hypotheses describe the limiting cases involved; the first is the induced fit and the second is the conformational selection model. The conformational selection model requires that the protein adopts conformations that are similar to the ligand-bound conformation in the absence of ligand, whilst the induced-fit model predicts that the ligand-bound conformation of the protein is only accessible when the ligand is actually bound. The flexibility of the apo protein clearly plays a major role in these interpretations. For many proteins involved in signaling pathways there is the added complication that they are often promiscuous in that they are capable of binding to different ligand partners. The relationship between protein flexibility and promiscuity is an area of active research and is perhaps best exemplified by the PDZ domain family of proteins. In this study we use molecular dynamics simulations to examine the relationship between flexibility and promiscuity in five PDZ domains: the human Dvl2 (Dishevelled-2) PDZ domain, the human Erbin PDZ domain, the PDZ1 domain of InaD (inactivation no after-potential D protein) from fruit fly, the PDZ7 domain of GRIP1 (glutamate receptor interacting protein 1) from rat and the PDZ2 domain of PTP-BL (protein tyrosine phosphatase) from mouse. We show that despite their high structural similarity, the PDZ binding sites have significantly different dynamics. Importantly, the degree of binding pocket flexibility was found to be closely related to the various characteristics of peptide binding specificity and promiscuity of the five PDZ domains. Our findings suggest that the intrinsic motions of the apo structures play a key role in distinguishing functional properties of different PDZ domains and allow us to make predictions that can be experimentally tested. PMID:23133356

Protein flexibility: coordinate uncertainties and interpretation of structural differences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rashin, Alexander A., E-mail: alexander-rashin@hotmail.com; LH Baker Center for Bioinformatics and Department of Biochemistry, Biophysics and Molecular Biology, 112 Office and Lab Building, Iowa State University, Ames, IA 50011-3020; Rashin, Abraham H. L.

2009-11-01

Criteria for the interpretability of coordinate differences and a new method for identifying rigid-body motions and nonrigid deformations in protein conformational changes are developed and applied to functionally induced and crystallization-induced conformational changes. Valid interpretations of conformational movements in protein structures determined by X-ray crystallography require that the movement magnitudes exceed their uncertainty threshold. Here, it is shown that such thresholds can be obtained from the distance difference matrices (DDMs) of 1014 pairs of independently determined structures of bovine ribonuclease A and sperm whale myoglobin, with no explanations provided for reportedly minor coordinate differences. The smallest magnitudes of reportedly functionalmore » motions are just above these thresholds. Uncertainty thresholds can provide objective criteria that distinguish between true conformational changes and apparent ‘noise’, showing that some previous interpretations of protein coordinate changes attributed to external conditions or mutations may be doubtful or erroneous. The use of uncertainty thresholds, DDMs, the newly introduced CDDMs (contact distance difference matrices) and a novel simple rotation algorithm allows a more meaningful classification and description of protein motions, distinguishing between various rigid-fragment motions and nonrigid conformational deformations. It is also shown that half of 75 pairs of identical molecules, each from the same asymmetric crystallographic cell, exhibit coordinate differences that range from just outside the coordinate uncertainty threshold to the full magnitude of large functional movements. Thus, crystallization might often induce protein conformational changes that are comparable to those related to or induced by the protein function.« less

SU-E-T-562: Motion Tracking Optimization for Conformal Arc Radiotherapy Plans: A QUASAR Phantom Based Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Z; Wang, I; Yao, R

Purpose: This study is to use plan parameters optimization (Dose rate, collimator angle, couch angle, initial starting phase) to improve the performance of conformal arc radiotherapy plans with motion tracking by increasing the plan performance score (PPS). Methods: Two types of 3D conformal arc plans were created based on QUASAR respiratory motion phantom with spherical and cylindrical targets. Sinusoidal model was applied to the MLC leaves to generate motion tracking plans. A MATLAB program was developed to calculate PPS of each plan (ranges from 0–1) and optimize plan parameters. We first selected the dose rate for motion tracking plans andmore » then used simulated annealing algorithm to search for the combination of the other parameters that resulted in the plan of the maximal PPS. The optimized motion tracking plan was delivered by Varian Truebeam Linac. In-room cameras and stopwatch were used for starting phase selection and synchronization between phantom motion and plan delivery. Gaf-EBT2 dosimetry films were used to measure the dose delivered to the target in QUASAR phantom. Dose profiles and Truebeam trajectory log files were used for plan delivery performance evaluation. Results: For spherical target, the maximal PPS (PPSsph) of the optimized plan was 0.79: (Dose rate: 500MU/min, Collimator: 90°, Couch: +10°, starting phase: 0.83π). For cylindrical target, the maximal PPScyl was 0.75 (Dose rate: 300MU/min, Collimator: 87°, starting phase: 0.97π) with couch at 0°. Differences of dose profiles between motion tracking plans (with the maximal and the minimal PPS) and 3D conformal plans were as follows: PPSsph=0.79: %ΔFWHM: 8.9%, %Dmax: 3.1%; PPSsph=0.52: %ΔFWHM: 10.4%, %Dmax: 6.1%. PPScyl=0.75: %ΔFWHM: 4.7%, %Dmax: 3.6%; PPScyl=0.42: %ΔFWHM: 12.5%, %Dmax: 9.6%. Conclusion: By achieving high plan performance score through parameters optimization, we can improve target dose conformity of motion tracking plan by decreasing total MLC leaf travel distance and leaf speed.« less

The structure of free L11 and functional dynamics of L11 in free, L11-rRNA(58 nt) binary and L11-rRNA(58 nt)-thiostrepton ternary complexes.

PubMed

Lee, Donghan; Walsh, Joseph D; Yu, Ping; Markus, Michelle A; Choli-Papadopoulou, Theodora; Schwieters, Charles D; Krueger, Susan; Draper, David E; Wang, Yun-Xing

2007-04-06

The L11 binding site is one of the most important functional sites in the ribosome. The N-terminal domain of L11 has been implicated as a "reversible switch" in facilitating the coordinated movements associated with EF-G-driven GTP hydrolysis. The reversible switch mechanism has been hypothesized to require conformational flexibility involving re-orientation and re-positioning of the two L11 domains, and warrants a close examination of the structure and dynamics of L11. Here we report the solution structure of free L11, and relaxation studies of free L11, L11 complexed to its 58 nt RNA recognition site, and L11 in a ternary complex with the RNA and thiostrepton antibiotic. The binding site of thiostrepton on L11 was also defined by analysis of structural and dynamics data and chemical shift mapping. The conclusions of this work are as follows: first, the binding of L11 to RNA leads to sizable conformation changes in the regions flanking the linker and in the hinge area that links a beta-sheet and a 3(10)-helix-turn-helix element in the N terminus. Concurrently, the change in the relative orientation may lead to re-positioning of the N terminus, as implied by a decrease of radius of gyration from 18.5 A to 16.2 A. Second, the regions, which undergo large conformation changes, exhibit motions on milliseconds-microseconds or nanoseconds-picoseconds time scales. Third, binding of thiostrepton results in more rigid conformations near the linker (Thr71) and near its putative binding site (Leu12). Lastly, conformational changes in the putative thiostrepton binding site are implicated by the re-emergence of cross-correlation peaks in the spectrum of the ternary complex, which were missing in that of the binary complex. Our combined analysis of both the chemical shift perturbation and dynamics data clearly indicates that thiostrepton binds to a pocket involving residues in the 3(10)-helix in L11.

The Structure of Free L11 and Functional Dynamics of L11 in Free, L11-rRNA(58nt) Binary and L11-rRNA(58nt)-thiostrepton Ternary Complexes

PubMed Central

Lee, Donghan; Walsh, Joseph D.; Yu, Ping; Markus, Michelle A.; Choli-Papadopoulou, Theodora; Schwieters, Charles D.; Krueger, Susan; Draper, David E.; Wang, Yun-Xing

2007-01-01

Summary The L11 binding site is one of the most important functional sites in the ribosome. The N-terminal domain of L11 has been implicated as a “reversible switch” in facilitating the coordinated movements associated with EF-G–driven GTP hydrolysis. The “reversible switch” mechanism has been hypothesized to require conformational flexibility involving re-orientation and re-positioning of the two L11 domains, and warrants a close examination of the structure and dynamics of L11. Here we report the solution structure of free L11, and relaxation studies of free L11, L11complexed to its 58 nt RNA recognition site, and L11 in a ternary complex with the RNA and thiostrepton antibiotic. The binding site of thiostrepton on L11 was also defined by analysis of structural and dynamics data and chemical shift mapping. The conclusions of this work are as follows: First, the binding of L11 to RNA leads to sizable conformation changes in the regions flanking the linker and in the hinge area that links a β-sheets and a 310-helix-turn-helix element in the N-terminus. Concurrently, the change in the relative orientation may lead to re-positioning of the N-terminus, as implied by a decrease of radius of gyration from 18.5 Å to 16.2 Å. Second, the regions, which undergo large conformation changes, exhibit motions on ms-μs or ns-ps time scales. Third, binding of thiostrepton results in more rigid conformations near the linker (Thr71) and near its putative binding site (Leu12). Lastly, conformational changes in the putative thiostrepton binding site are implicated by the re-emergence of cross-correlation peaks in the spectrum of the ternary complex, which were missing in that of the binary complex. Our combined analysis of both the chemical shift perturbation and dynamics data clearly indicates that thiostrepton binds to a pocket involving residues in the 310-helix in L11. PMID:17292917

On the null trajectories in conformal Weyl gravity

NASA Astrophysics Data System (ADS)

Villanueva, J. R.; Olivares, Marco

2013-06-01

In this work we find analytical solutions to the null geodesics around a black hole in the conformal Weyl gravity. Exact expressions for the horizons are found, and they depend on the cosmological constant and the coupling constants of the conformal Weyl gravity. Then, we study the radial motion from the point of view of the proper and coordinate frames, and compare it with that found in spacetimes of general relativity. The angular motion is also examined qualitatively by means of an effective potential; quantitatively, the equation of motion is solved in terms of wp-Weierstrass elliptic function. Thus, we find the deflection angle for photons without using any approximation, which is a novel result for this kind of gravity.

Protein dynamics and enzyme catalysis: insights from simulations.

PubMed

McGeagh, John D; Ranaghan, Kara E; Mulholland, Adrian J

2011-08-01

The role of protein dynamics in enzyme catalysis is one of the most active and controversial areas in enzymology today. Some researchers claim that protein dynamics are at the heart of enzyme catalytic efficiency, while others state that dynamics make no significant contribution to catalysis. What is the biochemist - or student - to make of the ferocious arguments in this area? Protein dynamics are complex and fascinating, as molecular dynamics simulations and experiments have shown. The essential question is: do these complex motions have functional significance? In particular, how do they affect or relate to chemical reactions within enzymes, and how are chemical and conformational changes coupled together? Biomolecular simulations can analyse enzyme reactions and dynamics in atomic detail, beyond that achievable in experiments: accurate atomistic modelling has an essential part to play in clarifying these issues. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches. Copyright © 2010 Elsevier B.V. All rights reserved.

Collective helicity switching of a DNA-coat assembly

NASA Astrophysics Data System (ADS)

Kim, Yongju; Li, Huichang; He, Ying; Chen, Xi; Ma, Xiaoteng; Lee, Myongsoo

2017-07-01

Hierarchical assemblies of biomolecular subunits can carry out versatile tasks at the cellular level with remarkable spatial and temporal precision. As an example, the collective motion and mutual cooperation between complex protein machines mediate essential functions for life, such as replication, synthesis, degradation, repair and transport. Nucleic acid molecules are far less dynamic than proteins and need to bind to specific proteins to form hierarchical structures. The simplest example of these nucleic acid-based structures is provided by a rod-shaped tobacco mosaic virus, which consists of genetic material surrounded by coat proteins. Inspired by the complexity and hierarchical assembly of viruses, a great deal of effort has been devoted to design similarly constructed artificial viruses. However, such a wrapping approach makes nucleic acid dynamics insensitive to environmental changes. This limitation generally restricts, for example, the amplification of the conformational dynamics between the right-handed B form to the left-handed Z form of double-stranded deoxyribonucleic acid (DNA). Here we report a virus-like hierarchical assembly in which the native DNA and a synthetic coat undergo repeated collective helicity switching triggered by pH change under physiological conditions. We also show that this collective helicity inversion occurs during translocation of the DNA-coat assembly into intracellular compartments. Translating DNA conformational dynamics into a higher level of hierarchical dynamics may provide an approach to create DNA-based nanomachines.

An energy function for dynamics simulations of polypeptides in torsion angle space

NASA Astrophysics Data System (ADS)

Sartori, F.; Melchers, B.; Böttcher, H.; Knapp, E. W.

1998-05-01

Conventional simulation techniques to model the dynamics of proteins in atomic detail are restricted to short time scales. A simplified molecular description, in which high frequency motions with small amplitudes are ignored, can overcome this problem. In this protein model only the backbone dihedrals φ and ψ and the χi of the side chains serve as degrees of freedom. Bond angles and lengths are fixed at ideal geometry values provided by the standard molecular dynamics (MD) energy function CHARMM. In this work a Monte Carlo (MC) algorithm is used, whose elementary moves employ cooperative rotations in a small window of consecutive amide planes, leaving the polypeptide conformation outside of this window invariant. A single of these window MC moves generates local conformational changes only. But, the application of many such moves at different parts of the polypeptide backbone leads to global conformational changes. To account for the lack of flexibility in the protein model employed, the energy function used to evaluate conformational energies is split into sequentially neighbored and sequentially distant contributions. The sequentially neighbored part is represented by an effective (φ,ψ)-torsion potential. It is derived from MD simulations of a flexible model dipeptide using a conventional MD energy function. To avoid exaggeration of hydrogen bonding strengths, the electrostatic interactions involving hydrogen atoms are scaled down at short distances. With these adjustments of the energy function, the rigid polypeptide model exhibits the same equilibrium distributions as obtained by conventional MD simulation with a fully flexible molecular model. Also, the same temperature dependence of the stability and build-up of α helices of 18-alanine as found in MD simulations is observed using the adapted energy function for MC simulations. Analyses of transition frequencies demonstrate that also dynamical aspects of MD trajectories are faithfully reproduced. Finally, it is demonstrated that even for high temperature unfolded polypeptides the MC simulation is more efficient by a factor of 10 than conventional MD simulations.

Recognition of the 3′ splice site RNA by the U2AF heterodimer involves a dynamic population shift

PubMed Central

Voith von Voithenberg, Lena; Sánchez-Rico, Carolina; Kang, Hyun-Seo; Madl, Tobias; Zanier, Katia; Barth, Anders; Warner, Lisa R.; Sattler, Michael; Lamb, Don C.

2016-01-01

An essential early step in the assembly of human spliceosomes onto pre-mRNA involves the recognition of regulatory RNA cis elements in the 3′ splice site by the U2 auxiliary factor (U2AF). The large (U2AF65) and small (U2AF35) subunits of the U2AF heterodimer contact the polypyrimidine tract (Py-tract) and the AG-dinucleotide, respectively. The tandem RNA recognition motif domains (RRM1,2) of U2AF65 adopt closed/inactive and open/active conformations in the free form and when bound to bona fide Py-tract RNA ligands. To investigate the molecular mechanism and dynamics of 3′ splice site recognition by U2AF65 and the role of U2AF35 in the U2AF heterodimer, we have combined single-pair FRET and NMR experiments. In the absence of RNA, the RRM1,2 domain arrangement is highly dynamic on a submillisecond time scale, switching between closed and open conformations. The addition of Py-tract RNA ligands with increasing binding affinity (strength) gradually shifts the equilibrium toward an open conformation. Notably, the protein–RNA complex is rigid in the presence of a strong Py-tract but exhibits internal motion with weak Py-tracts. Surprisingly, the presence of U2AF35, whose UHM domain interacts with U2AF65 RRM1, increases the population of the open arrangement of U2AF65 RRM1,2 in the absence and presence of a weak Py-tract. These data indicate that the U2AF heterodimer promotes spliceosome assembly by a dynamic population shift toward the open conformation of U2AF65 to facilitate the recognition of weak Py-tracts at the 3′ splice site. The structure and RNA binding of the heterodimer was unaffected by cancer-linked myelodysplastic syndrome mutants. PMID:27799531

Inward open characterization of EmrD transporter with molecular dynamics simulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Xianwei; Wang, Boxiong, E-mail: boxiong_wang@yahoo.com

EmrD is a member of the multidrug resistance exporter family. Up to now, little is known about the structural dynamics that underline the function of the EmrD protein in inward-facing open state and how the EmrD transits from an occluded state to an inward open state. For the first time the article applied the AT simulation to investigate the membrane transporter protein EmrD, and described the dynamic features of the whole protein, the domain, the helices, and the amino acid residues during an inward-open process from its occluded state. The gradual inward-open process is different from the current model ofmore » rigid-body domain motion in alternating-access mechanism. Simulation results show that the EmrD inward-open conformational fluctuation propagates from a C-terminal domain to an N-terminal domain via the linker region during the transition from its occluded state. The conformational fluctuation of the C-terminal domain is larger than that of the N-terminal domain. In addition, it is observed that the helices exposed to the surrounding membrane show a higher level of flexibility than the other regions, and the protonated E227 plays a key role in the transition from the occluded to the open state. -- Highlights: •This study described the dynamic features of the whole EmrD protein, during an inward-open process from its occluded state. •The EmrD inward-open conformational fluctuation propagates from a C-terminal domain to an N-terminal domain via the linker region during the transition from its occluded state. •The conformational fluctuation of the C-terminal domain is larger than that of the N-terminal domain. •The protonated E227 plays a key role in the transition from the occluded to the open state.« less

Structural, molecular motions, and free-energy landscape of Leishmania sterol-14α-demethylase wild type and drug resistant mutant: a comparative molecular dynamics study.

PubMed

Vijayakumar, Saravanan; Das, Pradeep

2018-04-18

Sterol-14α-demethylase (CYP51) is an ergosterol pathway enzyme crucial for the survival of infectious Leishmania parasite. Recent high-throughput metabolomics and whole genome sequencing study revealed amphotericin B resistance in Leishmania is indeed due to mutation in CYP51. The residue of mutation (asparagine 176) is conserved across the kinetoplastidae and not in yeast or humans, portraying its functional significance. In order to understand the possible cause for the resistance, knowledge of structural changes due to mutation is of high importance. To shed light on the structural changes of wild and mutant CYP51, we conducted comparative molecular dynamics simulation study. The active site, substrate biding cavity, substrate channel entrance (SCE), and cavity involving the mutated site were studied based on basic parameters and large concerted molecular motions derived from essential dynamics analyses of 100 ns simulation. Results indicated that mutant CYP51 is stable and less compact than the wild type. Correspondingly, the solvent accessible surface area (SASA) of the mutant was found to be increased, especially in active site and cavities not involving the mutation site. Free-energy landscape analysis disclosed mutant to have a rich conformational diversity than wild type, with various free-energy conformations of mutant having SASA greater than wild type with SCE open. More residues were found to interact with the mutant CYP51 upon docking of substrate to both the wild and mutant CYP51. These results indicate that, relative to wild type, the N176I mutation of CYP51 in Leishmania mexicana could possibly favor increased substrate binding efficiency.

An experimental comparison of conventional two-bank and novel four-bank dynamic MLC tracking.

PubMed

Davies, G A; Clowes, P; McQuaid, D; Evans, P M; Webb, S; Poludniowski, G

2013-03-07

The AccuLeaf mMLC featuring four multileaf-collimator (MLC) banks has been used for the first time for an experimental comparison of conventional two-bank with novel four-bank dynamic MLC tracking of a two-dimensional sinusoidal respiratory motion. This comparison was performed for a square aperture, and for three conformal treatment apertures from clinical radiotherapy lung cancer patients. The system latency of this prototype tracking system was evaluated and found to be 1.0 s and the frequency at which MLC positions could be updated, 1 Hz, and therefore accurate MLC tracking of irregular patient motion would be difficult with the system in its current form. The MLC leaf velocity required for two-bank-MLC and four-bank-MLC tracking was evaluated for the apertures studied and a substantial decrease was found in the maximum MLC velocity required when four-banks were used for tracking rather than two. A dosimetric comparison of the two techniques was also performed and minimal difference was found between two-bank-MLC and four-bank-MLC tracking. The use of four MLC banks for dynamic MLC tracking is shown to be potentially advantageous for increasing the delivery efficiency compared with two-bank-MLC tracking where difficulties are encountered if large leaf shifts are required to track motion perpendicular to the direction of leaf travel.

Quantification of the uncertainty in coronary CTA plaque measurements using dynamic cardiac phantom and 3D-printed plaque models

NASA Astrophysics Data System (ADS)

Richards, Taylor; Sturgeon, Gregory M.; Ramirez-Giraldo, Juan Carlos; Rubin, Geoffrey; Segars, Paul; Samei, Ehsan

2017-03-01

The purpose of this study was to quantify the accuracy of coronary computed tomography angiography (CTA) stenosis measurements using newly developed physical coronary plaque models attached to a base dynamic cardiac phantom (Shelley Medical DHP-01). Coronary plaque models (5 mm diameter, 50% stenosis, and 32 mm long) were designed and 3D-printed with tissue equivalent materials (calcified plaque with iodine enhanced lumen). Realistic cardiac motion was achieved by fitting known cardiac motion vectors to left ventricle volume-time curves to create synchronized heart motion profiles executed by the base cardiac phantom. Realistic coronary CTA acquisition was accomplished by synthesizing corresponding ECG waveforms for gating and reconstruction purposes. All scans were acquired using a retrospective gating technique on a dual-source CT system (Siemens SOMATOM FLASH) with 75ms temporal resolution. Multi-planar reformatted images were reconstructed along vessel centerlines and the enhanced lumens were manually segmented by 5 independent operators. On average, the stenosis measurement accuracy was 0.9% positive bias for the motion free condition (0 bpm). The measurement accuracy monotonically decreased to 18.5% negative bias at 90 bpm. Contrast-tonoise (CNR), vessel circularity, and segmentation conformity also decreased monotonically with increasing heart rate. These results demonstrate successful implementation of the base cardiac phantom with 3D-printed coronary plaque models, adjustable motion profiles, and coordinated ECG waveforms. They further show the utility of the model to ascertain metrics of coronary CT accuracy and image quality under a variety of plaque, motion, and acquisition conditions.

SURVEY AND SUMMARY: The morph server: a standardized system for analyzing and visualizing macromolecular motions in a database framework

PubMed Central

Krebs, Werner G.; Gerstein, Mark

2000-01-01

The number of solved structures of macromolecules that have the same fold and thus exhibit some degree of conformational variability is rapidly increasing. It is consequently advantageous to develop a standardized terminology for describing this variability and automated systems for processing protein structures in different conformations. We have developed such a system as a ‘front-end’ server to our database of macromolecular motions. Our system attempts to describe a protein motion as a rigid-body rotation of a small ‘core’ relative to a larger one, using a set of hinges. The motion is placed in a standardized coordinate system so that all statistics between any two motions are directly comparable. We find that while this model can accommodate most protein motions, it cannot accommodate all; the degree to which a motion can be accommodated provides an aid in classifying it. Furthermore, we perform an adiabatic mapping (a restrained interpolation) between every two conformations. This gives some indication of the extent of the energetic barriers that need to be surmounted in the motion, and as a by-product results in a ‘morph movie’. We make these movies available over the Web to aid in visualization. Many instances of conformational variability occur between proteins with somewhat different sequences. We can accommodate these differences in a rough fashion, generating an ‘evolutionary morph’. Users have already submitted hundreds of examples of protein motions to our server, producing a comprehensive set of statistics. So far the statistics show that the median submitted motion has a rotation of ~10° and a maximum Cα displacement of 17 Å. Almost all involve at least one large torsion angle change of >140°. The server is accessible at http://bioinfo.mbb.yale.edu/MolMovDB PMID:10734184

Unbiased, scalable sampling of protein loop conformations from probabilistic priors.

PubMed

Zhang, Yajia; Hauser, Kris

2013-01-01

Protein loops are flexible structures that are intimately tied to function, but understanding loop motion and generating loop conformation ensembles remain significant computational challenges. Discrete search techniques scale poorly to large loops, optimization and molecular dynamics techniques are prone to local minima, and inverse kinematics techniques can only incorporate structural preferences in adhoc fashion. This paper presents Sub-Loop Inverse Kinematics Monte Carlo (SLIKMC), a new Markov chain Monte Carlo algorithm for generating conformations of closed loops according to experimentally available, heterogeneous structural preferences. Our simulation experiments demonstrate that the method computes high-scoring conformations of large loops (>10 residues) orders of magnitude faster than standard Monte Carlo and discrete search techniques. Two new developments contribute to the scalability of the new method. First, structural preferences are specified via a probabilistic graphical model (PGM) that links conformation variables, spatial variables (e.g., atom positions), constraints and prior information in a unified framework. The method uses a sparse PGM that exploits locality of interactions between atoms and residues. Second, a novel method for sampling sub-loops is developed to generate statistically unbiased samples of probability densities restricted by loop-closure constraints. Numerical experiments confirm that SLIKMC generates conformation ensembles that are statistically consistent with specified structural preferences. Protein conformations with 100+ residues are sampled on standard PC hardware in seconds. Application to proteins involved in ion-binding demonstrate its potential as a tool for loop ensemble generation and missing structure completion.

Unbiased, scalable sampling of protein loop conformations from probabilistic priors

PubMed Central

2013-01-01

Background Protein loops are flexible structures that are intimately tied to function, but understanding loop motion and generating loop conformation ensembles remain significant computational challenges. Discrete search techniques scale poorly to large loops, optimization and molecular dynamics techniques are prone to local minima, and inverse kinematics techniques can only incorporate structural preferences in adhoc fashion. This paper presents Sub-Loop Inverse Kinematics Monte Carlo (SLIKMC), a new Markov chain Monte Carlo algorithm for generating conformations of closed loops according to experimentally available, heterogeneous structural preferences. Results Our simulation experiments demonstrate that the method computes high-scoring conformations of large loops (>10 residues) orders of magnitude faster than standard Monte Carlo and discrete search techniques. Two new developments contribute to the scalability of the new method. First, structural preferences are specified via a probabilistic graphical model (PGM) that links conformation variables, spatial variables (e.g., atom positions), constraints and prior information in a unified framework. The method uses a sparse PGM that exploits locality of interactions between atoms and residues. Second, a novel method for sampling sub-loops is developed to generate statistically unbiased samples of probability densities restricted by loop-closure constraints. Conclusion Numerical experiments confirm that SLIKMC generates conformation ensembles that are statistically consistent with specified structural preferences. Protein conformations with 100+ residues are sampled on standard PC hardware in seconds. Application to proteins involved in ion-binding demonstrate its potential as a tool for loop ensemble generation and missing structure completion. PMID:24565175

On the relationship between NMR-derived amide order parameters and protein backbone entropy changes

PubMed Central

Sharp, Kim A.; O’Brien, Evan; Kasinath, Vignesh; Wand, A. Joshua

2015-01-01

Molecular dynamics simulations are used to analyze the relationship between NMR-derived squared generalized order parameters of amide NH groups and backbone entropy. Amide order parameters (O2NH) are largely determined by the secondary structure and average values appear unrelated to the overall flexibility of the protein. However, analysis of the more flexible subset (O2NH < 0.8) shows that these report both on the local flexibility of the protein and on a different component of the conformational entropy than that reported by the side chain methyl axis order parameters, O2axis. A calibration curve for backbone entropy vs. O2NH is developed which accounts for both correlations between amide group motions of different residues, and correlations between backbone and side chain motions. This calibration curve can be used with experimental values of O2NH changes obtained by NMR relaxation measurements to extract backbone entropy changes, e.g. upon ligand binding. In conjunction with our previous calibration for side chain entropy derived from measured O2axis values this provides a prescription for determination of the total protein conformational entropy changes from NMR relaxation measurements. PMID:25739366

On the relationship between NMR-derived amide order parameters and protein backbone entropy changes.

PubMed

Sharp, Kim A; O'Brien, Evan; Kasinath, Vignesh; Wand, A Joshua

2015-05-01

Molecular dynamics simulations are used to analyze the relationship between NMR-derived squared generalized order parameters of amide NH groups and backbone entropy. Amide order parameters (O(2) NH ) are largely determined by the secondary structure and average values appear unrelated to the overall flexibility of the protein. However, analysis of the more flexible subset (O(2) NH  < 0.8) shows that these report both on the local flexibility of the protein and on a different component of the conformational entropy than that reported by the side chain methyl axis order parameters, O(2) axis . A calibration curve for backbone entropy vs. O(2) NH is developed, which accounts for both correlations between amide group motions of different residues, and correlations between backbone and side chain motions. This calibration curve can be used with experimental values of O(2) NH changes obtained by NMR relaxation measurements to extract backbone entropy changes, for example, upon ligand binding. In conjunction with our previous calibration for side chain entropy derived from measured O(2) axis values this provides a prescription for determination of the total protein conformational entropy changes from NMR relaxation measurements. © 2015 Wiley Periodicals, Inc.

The SH2 Domain Regulates c-Abl Kinase Activation by a Cyclin-Like Mechanism and Remodulation of the Hinge Motion

PubMed Central

Dölker, Nicole; Górna, Maria W.; Sutto, Ludovico; Torralba, Antonio S.; Superti-Furga, Giulio; Gervasio, Francesco L.

2014-01-01

Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors. PMID:25299346

The SH2 domain regulates c-Abl kinase activation by a cyclin-like mechanism and remodulation of the hinge motion.

PubMed

Dölker, Nicole; Górna, Maria W; Sutto, Ludovico; Torralba, Antonio S; Superti-Furga, Giulio; Gervasio, Francesco L

2014-10-01

Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors.

Mechanism of allosteric propagation across a β-sheet structure investigated by molecular dynamics simulations.

PubMed

Interlandi, Gianluca; Thomas, Wendy E

2016-07-01

The bacterial adhesin FimH consists of an allosterically regulated mannose-binding lectin domain and a covalently linked inhibitory pilin domain. Under normal conditions, the two domains are bound to each other, and FimH interacts weakly with mannose. However, under tensile force, the domains separate and the lectin domain undergoes conformational changes that strengthen its bond with mannose. Comparison of the crystallographic structures of the low and the high affinity state of the lectin domain reveals conformational changes mainly in the regulatory inter-domain region, the mannose binding site and a large β sheet that connects the two distally located regions. Here, molecular dynamics simulations investigated how conformational changes are propagated within and between different regions of the lectin domain. It was found that the inter-domain region moves towards the high affinity conformation as it becomes more compact and buries exposed hydrophobic surface after separation of the pilin domain. The mannose binding site was more rigid in the high affinity state, which prevented water penetration into the pocket. The large central β sheet demonstrated a soft spring-like twisting. Its twisting motion was moderately correlated to fluctuations in both the regulatory and the binding region, whereas a weak correlation was seen in a direct comparison of these two distal sites. The results suggest a so called "population shift" model whereby binding of the lectin domain to either the pilin domain or mannose locks the β sheet in a rather twisted or flat conformation, stabilizing the low or the high affinity state, respectively. Proteins 2016; 84:990-1008. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Munro, S.L.; Andrews, P.R.; Craik, D.J.

The solution dynamics of a series of clinically potent antidepressants have been investigated by measuring /sup 13/C NMR relaxation parameters. Correlation times and internal motional rates were calculated from spin-lattice relaxation times and nuclear Overhauser effects for the protonated carbons in mianserin, imipramine-like antidepressants, and amitriptyline-like antidepressants. These data were interpreted in terms of overall molecular tumbling, internal rotations, and inherent flexibility of these structures. Of particular interest was the conformational variability of the tricyclic nucleus of the tricyclic antidepressants, where the data indicated a fivefold difference in mobility of the dimethylene bridge of imipramine-like antidepressants relative to amitriptyline-like compounds.more » The implications of such a difference in internal motions is discussed in relation to previous NMR studies and to the reported differences in pharmacological activity of these antidepressants.« less

Retinal isomerization in bacteriorhodopsin captured by a femtosecond x-ray laser.

PubMed

Nogly, Przemyslaw; Weinert, Tobias; James, Daniel; Carbajo, Sergio; Ozerov, Dmitry; Furrer, Antonia; Gashi, Dardan; Borin, Veniamin; Skopintsev, Petr; Jaeger, Kathrin; Nass, Karol; Båth, Petra; Bosman, Robert; Koglin, Jason; Seaberg, Matthew; Lane, Thomas; Kekilli, Demet; Brünle, Steffen; Tanaka, Tomoyuki; Wu, Wenting; Milne, Christopher; White, Thomas; Barty, Anton; Weierstall, Uwe; Panneels, Valerie; Nango, Eriko; Iwata, So; Hunter, Mark; Schapiro, Igor; Schertler, Gebhard; Neutze, Richard; Standfuss, Jörg

2018-06-14

Ultrafast isomerization of retinal is the primary step in photoresponsive biological functions including vision in humans and ion-transport across bacterial membranes. We studied the sub-picosecond structural dynamics of retinal isomerization in the light-driven proton pump bacteriorhodopsin using an x-ray laser. A series of structural snapshots with near-atomic spatial and temporal resolution in the femtosecond regime show how the excited all- trans retinal samples conformational states within the protein binding pocket prior to passing through a twisted geometry and emerging in the 13 -cis conformation. Our findings suggest ultrafast collective motions of aspartic acid residues and functional water molecules in the proximity of the retinal Schiff base as a key ingredient for this stereo-selective and efficient photochemical reaction. Copyright © 2018, American Association for the Advancement of Science.

Ultrafast myoglobin structural dynamics observed with an X-ray free-electron laser

DOE PAGES

Levantino, Matteo; Schirò, Giorgio; Lemke, Henrik Till; ...

2015-04-02

Light absorption can trigger biologically relevant protein conformational changes. The light induced structural rearrangement at the level of a photoexcited chromophore is known to occur in the femtosecond timescale and is expected to propagate through the protein as a quake-like intramolecular motion. Here we report direct experimental evidence of such ‘proteinquake’ observed in myoglobin through femtosecond X-ray solution scattering measurements performed at the Linac Coherent Light Source X-ray free-electron laser. An ultrafast increase of myoglobin radius of gyration occurs within 1 picosecond and is followed by a delayed protein expansion. As the system approaches equilibrium it undergoes damped oscillations withmore » a ~3.6-picosecond time period. Our results unambiguously show how initially localized chemical changes can propagate at the level of the global protein conformation in the picosecond timescale.« less

Using NMR spectroscopy to elucidate the role of molecular motions in enzyme function

PubMed Central

Lisi, George P.; Loria, J. Patrick

2015-01-01

Conformational motions play an essential role in enzyme function, often facilitating the formation of enzyme-substrate complexes and/or product release. Although considerable debate remains regarding the role of molecular motions in the conversion of enzymatic substrates to products, numerous examples have found motions to be crucial for optimization of enzyme scaffolds, effective substrate binding, and product dissociation. Conformational fluctuations are often rate-limiting to enzyme catalysis, primarily through product release, with the chemical reaction occurring much more quickly. As a result, the direct involvement of motions at various stages along the enzyme reaction coordinate remains largely unknown and untested. In the following review, we describe the use of solution NMR techniques designed to probe various timescales of molecular motions and detail examples in which motions play a role in propagating catalytic effects from the active site and directly participate in essential aspects of enzyme function. PMID:26952190

Conformational dynamics of minimal elastin-like polypeptides: the role of proline revealed by molecular dynamics and nuclear magnetic resonance.

PubMed

Glaves, Rachel; Baer, Marcel; Schreiner, Eduard; Stoll, Raphael; Marx, Dominik

2008-12-22

Previous molecular dynamics studies of the elastin-like peptide (ELP) GVG(VPGVG) predict that this ELP undergoes a conformational transition from an open to a more compact closed state upon an increase in temperature. These structural changes occurring in this minimal elastin model at the so-called inverse temperature transition (ITT), which takes place when elastin is heated to temperatures of about 20-40 (omicron)C, are investigated further in this work by means of a combined theoretical and experimental approach. To do this, additional extensive classical molecular dynamics (MD) simulations of the capped octapeptide are carried out, analyzed, and compared to data obtained from homonuclear magnetic resonance (NMR) spectroscopy of the same octapeptide. Moreover, in the previous simulations, the proline residue in the ELP is found to act as a hinge, thereby allowing for the large-amplitude opening and closing conformational motion of the ITT. To explore the role of proline in such elastin repeating units, a point mutant (P5I), which replaces the proline residue with an isoleucine residue, is also investigated using the aforementioned theoretical and experimental techniques. The results show that the site-directed mutation completely alters the properties of this ELP, thus confirming the importance of the highly conserved proline residue in the ITT. Furthermore, a correlation between the two different methods employed is seen. Both methods predict the mutant ELP to be present in an unstructured form and the wild type ELP to have a beta-turn-like structure. Finally, the role of the peptidyl cis to trans isomerization of the proline hinge is assessed in detail.

Consequences of Energetic Frustration on the Ligand-Coupled Folding/Dimerization Dynamics of Allosteric Protein S100A12.

PubMed

Ren, Weitong; Li, Wenfei; Wang, Jun; Zhang, Jian; Wang, Wei

2017-10-26

Allosteric proteins are featured by energetic degeneracy of two (or more) functionally relevant conformations, therefore their energy landscapes are often locally frustrated. How such frustration affects the protein folding/binding dynamics is not well understood. Here, by using molecular simulations we study the consequences of local frustration in the dimerization dynamics of allosteric proteins based on a homodimer protein S100A12. Despite of the structural symmetry of the two EF-hand motifs in the three-dimensional structures, the S100A12 homodimer shows allosteric behaviors and local frustration only in half of its structural elements, i.e., the C-terminal EF-hand. We showed that such spatially asymmetric location of frustration leads to asymmetric dimerization pathways, in which the dimerization is dominantly initiated by the interchain binding of the minimally frustrated N-terminal EF-hands, achieving optimal balance between the requirements of rapid conformational switching and interchain assembling to the energy landscapes. We also showed that the local frustration, as represented by the double-basin topography of the energy landscape, gives rise to multiple cross-linked dimerization pathways, in which the dimerization is coupled with the allosteric motions of the C-terminal EF-hands. Binding of metal ions tends to reshape the energy landscape and modulate the dimerization pathways. In addition, by employing the frustratometer method, we showed that the highly frustrated residue-pairs in the C-terminal EF-hand are partially unfolded during the conformational transitions of the native homodimer, leading to lowing of free energy barrier. Our results revealed tight interplay between the local frustration of the energy landscape and the dimerization dynamics for allosteric proteins.

Understanding nucleotide-regulated FtsZ filament dynamics and the monomer assembly switch with large-scale atomistic simulations.

PubMed

Ramírez-Aportela, Erney; López-Blanco, José Ramón; Andreu, José Manuel; Chacón, Pablo

2014-11-04

Bacterial cytoskeletal protein FtsZ assembles in a head-to-tail manner, forming dynamic filaments that are essential for cell division. Here, we study their dynamics using unbiased atomistic molecular simulations from representative filament crystal structures. In agreement with experimental data, we find different filament curvatures that are supported by a nucleotide-regulated hinge motion between consecutive FtsZ monomers. Whereas GTP-FtsZ filaments bend and twist in a preferred orientation, thereby burying the nucleotide, the differently curved GDP-FtsZ filaments exhibit a heterogeneous distribution of open and closed interfaces between monomers. We identify a coordinated Mg(2+) ion as the key structural element in closing the nucleotide site and stabilizing GTP filaments, whereas the loss of the contacts with loop T7 from the next monomer in GDP filaments leads to open interfaces that are more prone to depolymerization. We monitored the FtsZ monomer assembly switch, which involves opening/closing of the cleft between the C-terminal domain and the H7 helix, and observed the relaxation of isolated and filament minus-end monomers into the closed-cleft inactive conformation. This result validates the proposed switch between the low-affinity monomeric closed-cleft conformation and the active open-cleft FtsZ conformation within filaments. Finally, we observed how the antibiotic PC190723 suppresses the disassembly switch and allosterically induces closure of the intermonomer interfaces, thus stabilizing the filament. Our studies provide detailed structural and dynamic insights into modulation of both the intrinsic curvature of the FtsZ filaments and the molecular switch coupled to the high-affinity end-wise association of FtsZ monomers.

New Insights into Color Confinement, Hadron Dynamics, Spectroscopy, and Jet Hadronization from Light-Front Holography and Superconformal Algebra

NASA Astrophysics Data System (ADS)

Brodsky, S. J.

2017-07-01

A fundamental problem in hadron physics is to obtain a relativistic color-confining, first approximation to QCD which can predict both hadron spectroscopy and the frame-independent light-front (LF) wavefunctions underlying hadron dynamics. The QCD Lagrangian with zero quark mass has no explicit mass scale; the classical theory is conformally invariant. Thus, a fundamental problem is to understand how the mass gap and ratios of masses - such as m ρ/ m p - can arise in chiral QCD. De Alfaro, Fubini, and Furlan have made an important observation that a mass scale can appear in the equations of motion without affecting the conformal invariance of the action if one adds a term to the Hamiltonian proportional to the dilatation operator or the special conformal operator and rescales the time variable. If one applies the same procedure to the light-front Hamiltonian, it leads uniquely to a confinement potential κ 4 ζ 2 for mesons, where ζ 2 is the LF radial variable conjugate to the q\\overline{q} invariant mass squared. The same result, including spin terms, is obtained using light-front holography - the duality between light-front dynamics and AdS5, the space of isometries of the conformal group if one modifies the action of AdS5 by the dilaton {e}^{κ^2}{z}^2 in the fifth dimension z . When one generalizes this procedure using superconformal algebra, the resulting light-front eigensolutions predict unified Regge spectroscopy of meson, baryon, and tetraquarks, including remarkable supersymmetric relations between the masses of mesons and baryons of the same parity. One also predicts observables such as hadron structure functions, transverse momentum distributions, and the distribution amplitudes defined from the hadronic light-front wavefunctions. The mass scale κ underlying confinement and hadron masses can be connected to the parameter {Λ}_{\\overline{MS}} in the QCD running coupling by matching the nonperturbative dynamics to the perturbative QCD regime. The result is an effective coupling α s ( Q 2) defined at all momenta. The matching of the high and low momentum transfer regimes also determines a scale Q0 which sets the interface between perturbative and nonperturbative hadron dynamics.

NMR structure and dynamics of the engineered fluorescein-binding lipocalin FluA reveal rigidification of beta-barrel and variable loops upon enthalpy-driven ligand binding.

PubMed

Mills, Jeffrey L; Liu, Gaohua; Skerra, Arne; Szyperski, Thomas

2009-08-11

The NMR structure of the 21 kDa lipocalin FluA, which was previously obtained by combinatorial design, elucidates a reshaped binding site specific for the dye fluorescein resulting from 21 side chain replacements with respect to the parental lipocalin, the naturally occurring bilin-binding protein (BBP). As expected, FluA exhibits the lipocalin fold of BBP, comprising eight antiparallel beta-strands forming a beta-barrel with an alpha-helix attached to its side. Comparison of the NMR structure of free FluA with the X-ray structures of BBP.biliverdin IX(gamma) and FluA.fluorescein complexes revealed significant conformational changes in the binding pocket, which is formed by four loops at the open end of the beta-barrel as well as adjoining beta-strand segments. An "induced fit" became apparent for the side chain conformations of Arg 88 and Phe 99, which contact the bound fluorescein in the complex and undergo concerted rearrangement upon ligand binding. Moreover, slower internal motional modes of the polypeptide backbone were identified by measuring transverse (15)N backbone spin relaxation times in the rotating frame for free FluA and also for the FluA.fluorescein complex. A reduction in the level of such motions was detected upon complex formation, indicating rigidification of the protein structure and loss of conformational entropy. This hypothesis was confirmed by isothermal titration calorimetry, showing that ligand binding is enthalpy-driven, thus overcompensating for the negative entropy associated with both ligand binding per se and rigidification of the protein. Our investigation of the solution structure and dynamics as well as thermodynamics of lipocalin-ligand interaction not only provides insight into the general mechanism of small molecule accommodation in the deep and narrow cavity of this abundant class of proteins but also supports the future design of corresponding binding proteins with novel specificities, so-called "anticalins".

Molecular dynamics simulations elucidate the mode of protein recognition by Skp1 and the F-box domain in the SCF complex.

PubMed

Chandra Dantu, Sarath; Nathubhai Kachariya, Nitin; Kumar, Ashutosh

2016-01-01

Polyubiquitination of the target protein by a ubiquitin transferring machinery is key to various cellular processes. E3 ligase Skp1-Cul1-F-box (SCF) is one such complex which plays crucial role in substrate recognition and transfer of the ubiquitin molecule. Previous computational studies have focused on S-phase kinase-associated protein 2 (Skp2), cullin, and RING-finger proteins of this complex, but the roles of the adapter protein Skp1 and F-box domain of Skp2 have not been determined. Using sub-microsecond molecular dynamics simulations of full-length Skp1, unbound Skp2, Skp2-Cks1 (Cks1: Cyclin-dependent kinases regulatory subunit 1), Skp1-Skp2, and Skp1-Skp2-Cks1 complexes, we have elucidated the function of Skp1 and the F-box domain of Skp2. We found that the L16 loop of Skp1, which was deleted in previous X-ray crystallography studies, can offer additional stability to the ternary complex via its interactions with the C-terminal tail of Skp2. Moreover, Skp1 helices H6, H7, and H8 display vivid conformational flexibility when not bound to Skp2, suggesting that these helices can recognize and lock the F-box proteins. Furthermore, we observed that the F-box domain could rotate (5°-129°), and that the binding partner determined the degree of conformational flexibility. Finally, Skp1 and Skp2 were found to execute a domain motion in Skp1-Skp2 and Skp1-Skp2-Cks1 complexes that could decrease the distance between ubiquitination site of the substrate and the ubiquitin molecule by 3 nm. Thus, we propose that both the F-box domain of Skp2 and Skp1-Skp2 domain motions displaying preferential conformational control can together facilitate polyubiquitination of a wide variety of substrates. © 2015 Wiley Periodicals, Inc.

A Model for the Flexibility of the Distal Histidine in Dehaloperoxidase-Hemoglobin A Based on X-ray Crystal Structures of the Carbon Monoxide Adduct

PubMed Central

2015-01-01

Dehaloperoxidase hemoglobin A (DHP A) is a multifunctional hemoglobin that appears to have evolved oxidative pathways for the degradation of xenobiotics as a protective function that complements the oxygen transport function. DHP A possesses at least two internal binding sites, one for substrates and one for inhibitors, which include various halogenated phenols and indoles. Herein, we report the X-ray crystallographic structure of the carbonmonoxy complex (DHPCO). Unlike other DHP structures with 6-coordinated heme, the conformation of the distal histidine (H55) in DHPCO is primarily external or solvent exposed, despite the fact that the heme Fe is 6-coordinated. As observed generally in globins, DHP exhibits two distal histidine conformations (one internal and one external). In previous structural studies, we have shown that the distribution of H55 conformations is weighted strongly toward the external position when the DHP heme Fe is 5-coordinated. The large population of the external conformation of the distal histidine observed in DHPCO crystals at pH 6.0 indicates that some structural factor in DHP must account for the difference from other globins, which exhibit a significant external conformation only when pH < 4.5. While the original hypothesis suggested that interaction with a heme-Fe-bound ligand was the determinant of H55 conformation, the current study forces a refinement of that hypothesis. The external or open conformation of H55 is observed to have interactions with two propionate groups in heme, at distances of 3.82 and 2.73 Å, respectively. A relatively weak hydrogen bonding interaction between H55 and CO, combined with strong interactions with heme propionate (position 6), is hypothesized to strengthen the external conformation of H55. Density function theory (DFT) calculations were conducted to test whether there is a weaker hydrogen bond interaction between H55 and heme bonded CO or O2. Molecular dynamics simulations were conducted to examine how the tautomeric forms of H55 affect the dynamic motions of the distal histidine that govern the switching between open and closed conformations. The calculations support the modified hypothesis suggesting a competition between the strength of interactions with heme ligand and the heme propionates as the factors that determine the conformation of the distal histidine. PMID:24670063

Hydrogen exchange mass spectrometry of bacteriorhodopsin reveals light-induced changes in the structural dynamics of a biomolecular machine.

PubMed

Pan, Yan; Brown, Leonid; Konermann, Lars

2011-12-21

Many proteins act as molecular machines that are fuelled by a nonthermal energy source. Examples include transmembrane pumps and stator-rotor complexes. These systems undergo cyclic motions (CMs) that are being driven along a well-defined conformational trajectory. Superimposed on these CMs are thermal fluctuations (TFs) that are coupled to stochastic motions of the solvent. Here we explore whether the TFs of a molecular machine are affected by the occurrence of CMs. Bacteriorhodopsin (BR) is a light-driven proton pump that serves as a model system in this study. The function of BR is based on a photocycle that involves trans/cis isomerization of a retinal chromophore, as well as motions of transmembrane helices. Hydrogen/deuterium exchange (HDX) mass spectrometry was used to monitor the TFs of BR, focusing on the monomeric form of the protein. Comparative HDX studies were conducted under illumination and in the dark. The HDX kinetics of BR are dramatically accelerated in the presence of light. The isotope exchange rates and the number of backbone amides involved in EX2 opening transitions increase roughly 2-fold upon illumination. In contrast, light/dark control experiments on retinal-free protein produced no discernible differences. It can be concluded that the extent of TFs in BR strongly depends on photon-driven CMs. The light-induced differences in HDX behavior are ascribed to protein destabilization. Specifically, the thermodynamic stability of the dark-adapted protein is estimated to be 5.5 kJ mol(-1) under the conditions of our work. This value represents the free energy difference between the folded state F and a significantly unfolded conformer U. Illumination reduces the stability of F by 2.2 kJ mol(-1). Mechanical agitation caused by isomerization of the chromophore is transferred to the surrounding protein scaffold, and subsequently, the energy dissipates into the solvent. Light-induced retinal motions therefore act analogously to an internal heat source that promotes the occurrence of TFs. Overall, our data highlight the potential of HDX methods for probing the structural dynamics of molecular machines under "engine on" and "engine off" conditions. © 2011 American Chemical Society

Understanding bimolecular machines: Theoretical and experimental approaches

NASA Astrophysics Data System (ADS)

Goler, Adam Scott

This dissertation concerns the study of two classes of molecular machines from a physical perspective: enzymes and membrane proteins. Though the functions of these classes of proteins are different, they each represent important test-beds from which new understanding can be developed by the application of different techniques. HIV1 Reverse Transcriptase is an enzyme that performs multiple functions, including reverse transcription of RNA into an RNA/DNA duplex, RNA degradation by the RNaseH domain, and synthesis of dsDNA. These functions allow for the incorporation of the retroviral genes into the host genome. Its catalytic cycle requires repeated large-scale conformational changes fundamental to its mechanism. Motivated by experimental work, these motions were studied theoretically by the application of normal mode analysis. It was observed that the lowest order modes correlate with largest amplitude (low-frequency) motion, which are most likely to be catalytically relevant. Comparisons between normal modes obtained via an elastic network model to those calculated from the essential dynamics of a series of all-atom molecular dynamics simulations show the self-consistency between these calculations. That similar conformational motions are seen between independent theoretical methods reinforces the importance of large-scale subdomain motion for the biochemical action of DNA polymerases in general. Moreover, it was observed that the major subunits of HIV1 Reverse Transcriptase interact quasi-harmonically. The 5HT3A Serotonin receptor and P2X1 receptor, by contrast, are trans-membrane proteins that function as ligand gated ion channels. Such proteins feature a central pore, which allows for the transit of ions necessary for cellular function across a membrane. The pore is opened by the ligation of binding sites on the extracellular portion of different protein subunits. In an attempt to resolve the individual subunits of these membrane proteins beyond the diffraction limit, a super-localization microscope capable of reconstructing super-resolution images was constructed. This novel setup allows for the study of discrete state kinetic mechanisms with spatial resolution good enough to distinguish individual binding sites of these membrane proteins. Further use of this technique may allow for the study of allostery and subunit specific stoichiometry in the presence of agonist or antagonist ligands relevant to pharmacology.

Internal dynamics of F-actin and myosin subfragment-1 studied by quasielastic neutron scattering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsuo, Tatsuhito; Arata, Toshiaki; Oda, Toshiro

2015-04-10

Various biological functions related to cell motility are driven by the interaction between the partner proteins, actin and myosin. To obtain insights into how this interaction occurs, the internal dynamics of F-actin and myosin subfragment-1 (S1) were characterized by the quasielastic neutron scattering measurements on the solution samples of F-actin and S1. Contributions of the internal motions of the proteins to the scattering spectra were separated from those of the global macromolecular diffusion. Analysis of the spectra arising from the internal dynamics showed that the correlation times of the atomic motions were about two times shorter for F-actin than formore » S1, suggesting that F-actin fluctuates more rapidly than S1. It was also shown that the fraction of the immobile atoms is larger for S1 than for F-actin. These results suggest that F-actin actively facilitates the binding of myosin by utilizing the more frequent conformational fluctuations than those of S1. - Highlights: • We studied the internal dynamics of F-actin and myosin S1 by neutron scattering. • The correlation times of the atomic motions were smaller for F-actin than for S1. • The fraction of the immobile atoms was also smaller for F-actin than for S1. • Our results suggest that mobility of atoms in F-actin is higher than that in S1. • We propose that high flexibility of F-actin facilitates the binding of myosin.« less

Dynamics of single-stranded DNA tethered to a solid

NASA Astrophysics Data System (ADS)

Radiom, Milad; Paul, Mark R.; Ducker, William A.

2016-06-01

Tethering is used to deliver specific biological and industrial functions. For example, single-stranded DNA (ssDNA) is tethered to polymerases and long sequences of double-stranded DNA (dsDNA) during replication, and to solids in DNA microarrays. However, tethering ssDNA to a large object limits not only the available ssDNA conformations, but also the range of time-scales over which the mechanical responses of ssDNA are important. In this work we examine the effect of tethering by measurement of the mechanical response of ssDNA that is tethered at each end to two separate atomic force microscope cantilevers in aqueous solution. Thermal motion of the cantilevers drives the ends of the ssDNA chain at frequencies near 2 kHz. The presence of a tethered molecule makes a large difference to the asymmetric cross-correlation of two cantilevers, which enables resolution of the mechanical properties in our experiments. By analysis of the correlated motion of the cantilevers we extract the friction and stiffness of the ssDNA. We find that the measured friction is much larger than the friction that is usually associated with the unencumbered motion of ssDNA. We also find that the measured relaxation time, ∼30 μs, is much greater than prior measurements of the free-molecule relaxation time. We attribute the difference to the loss of conformational possibilities as a result of constraining the ends of the ssDNA.

Do All X-ray Structures of Protein-Ligand Complexes Represent Functional States? EPOR, a Case Study.

PubMed

Corbett, Michael S P; Mark, Alan E; Poger, David

2017-02-28

Based on differences between the x-ray crystal structures of ligand-bound and unbound forms, the activation of the erythropoietin receptor (EPOR) was initially proposed to involve a cross-action scissorlike motion. However, the validity of the motions involved in the scissorlike model has been recently challenged. Here, atomistic molecular dynamics simulations are used to examine the structure of the extracellular domain of the EPOR dimer in the presence and absence of erythropoietin and a series of agonistic or antagonistic mimetic peptides free in solution. The simulations suggest that in the absence of crystal packing effects, the EPOR chains in the different dimers adopt very similar conformations with no clear distinction between the agonist and antagonist-bound complexes. This questions whether the available x-ray crystal structures of EPOR truly represent active or inactive conformations. The study demonstrates the difficulty in using such structures to infer a mechanism of action, especially in the case of membrane receptors where just part of the structure has been considered in addition to potential confounding effects that arise from the comparison of structures in a crystal as opposed to a membrane environment. The work highlights the danger of assigning functional significance to small differences between structures of proteins bound to different ligands in a crystal environment without consideration of the effects of the crystal lattice and thermal motion. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Solution structures and backbone dynamics of Escherichia coli rhodanese PspE in its sulfur-free and persulfide-intermediate forms: implications for the catalytic mechanism of rhodanese.

PubMed

Li, Hongwei; Yang, Fan; Kang, Xue; Xia, Bin; Jin, Changwen

2008-04-15

Rhodanese catalyzes the sulfur-transfer reaction that transfers sulfur from thiosulfate to cyanide by a double-displacement mechanism, in which an active cysteine residue plays a central role. Previous studies indicated that the phage-shock protein E (PspE) from Escherichia coli is a rhodanese composed of a single active domain and is the only accessible rhodanese among the three single-domain rhodaneses in E. coli. To understand the catalytic mechanism of rhodanese at the molecular level, we determined the solution structures of the sulfur-free and persulfide-intermediate forms of PspE by nuclear magnetic resonance (NMR) spectroscopy and identified the active site by NMR titration experiments. To obtain further insights into the catalytic mechanism, we studied backbone dynamics by NMR relaxation experiments. Our results demonstrated that the overall structures in both sulfur-free and persulfide-intermediate forms are highly similar, suggesting that no significant conformational changes occurred during the catalytic reaction. However, the backbone dynamics revealed that the motional properties of PspE in its sulfur-free form are different from the persulfide-intermediate state. The conformational exchanges are largely enhanced in the persulfide-intermediate form of PspE, especially around the active site. The present structural and biochemical studies in combination with backbone dynamics provide further insights in understanding the catalytic mechanism of rhodanese.

Unraveling the structural complexity in a single-stranded RNA tail: implications for efficient ligand binding in the prequeuosine riboswitch

PubMed Central

Eichhorn, Catherine D.; Feng, Jun; Suddala, Krishna C.; Walter, Nils G.; Brooks, Charles L.; Al-Hashimi, Hashim M.

2012-01-01

Single-stranded RNAs (ssRNAs) are ubiquitous RNA elements that serve diverse functional roles. Much of our understanding of ssRNA conformational behavior is limited to structures in which ssRNA directly engages in tertiary interactions or is recognized by proteins. Little is known about the structural and dynamic behavior of free ssRNAs at atomic resolution. Here, we report the collaborative application of nuclear magnetic resonance (NMR) and replica exchange molecular dynamics (REMD) simulations to characterize the 12 nt ssRNA tail derived from the prequeuosine riboswitch. NMR carbon spin relaxation data and residual dipolar coupling measurements reveal a flexible yet stacked core adopting an A-form-like conformation, with the level of order decreasing toward the terminal ends. An A-to-C mutation within the polyadenine tract alters the observed dynamics consistent with the introduction of a dynamic kink. Pre-ordering of the tail may increase the efficacy of ligand binding above that achieved by a random-coil ssRNA. The REMD simulations recapitulate important trends in the NMR data, but suggest more internal motions than inferred from the NMR analysis. Our study unmasks a previously unappreciated level of complexity in ssRNA, which we believe will also serve as an excellent model system for testing and developing computational force fields. PMID:22009676

The detection of conformational disorder by thermal analysis

NASA Astrophysics Data System (ADS)

Wunderlich, B.

Conformational disorder in crystals is found in many molecules that possess a plurality of conformational isomers. Typical examples are linear macromolecules such as polyethylene, polytetrafluoroethylene and trans-1,4-polybutadiene; and small molecules such as paraffins, cycloparaffins, soaps, lipids and many liquid-crystal forming molecules. Conformational motion is often coupled with the cooperative creation of disorder. In this case a heat and entropy of transition is observed by thermal analysis. Levels of transition entropies can be estimated, assuming most of the disorder can be traced to conformational isomerism. In case there is conformational disorder frozen-in at low temperature, thermal analysis can be used to find the glass transition of a condis crystal. An Advanced Thermal Analysis System has been developed, and will be described that permits a detailed interpretation of the thermal analysis traces. It rests with the establishment of high quality heat capacity for the rigid solid state (vibration only) and the mobile liquid state (vibrations and large amplitude cooperative motion).

The detection of conformational disorder by thermal analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wunderlich, B.

1988-01-01

Conformational disorder in crystals is found in many molecules that possess a plurality of conformational isomers. Typical examples are linear macromolecules such as polyethylene, polytetrafluoroethylene and trans-1,4-polybutadiene; and small molecules such as paraffins, cycloparaffins, soaps, lipids and many liquid-crystal forming molecules. Conformational motion is often coupled with the cooperative creation of disorder. In this case a heat and entropy of transition is observed by thermal analysis. Levels of transition entropies can be estimated, assuming most of the disorder can be traced to conformational isomerism. In case there is conformational disorder frozen-in at low temperature, thermal analysis can be used tomore » find the glass transition of a condis crystal. An Advanced Thermal Analysis System has been developed, and will be described that permits a detailed interpretation of the thermal analysis traces. It rests with the establishment of high quality heat capacity for the rigid solid state (vibration only) and the mobile liquid state (vibrations and large amplitude cooperative motion). 36 refs., 3 figs.« less

Brownian dynamics of self-regulated particles with additional degrees of freedom: Symmetry breaking and homochirality

NASA Astrophysics Data System (ADS)

Bhattacharyya, Debankur; Paul, Shibashis; Ghosh, Shyamolina; Ray, Deb Shankar

2018-04-01

We consider the Brownian motion of a collection of particles each with an additional degree of freedom. The degree of freedom of a particle (or, in general, a molecule) can assume distinct values corresponding to certain states or conformations. The time evolution of the additional degree of freedom of a particle is guided by those of its neighbors as well as the temperature of the system. We show that the local averaging over these degrees of freedom results in emergence of a collective order in the dynamics in the form of selection or dominance of one of the isomers leading to a symmetry-broken state. Our statistical model captures the basic features of homochirality, e.g., autocatalysis and chiral inhibition.

Crystal Structure of Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26 at 0.95 Å Resolution: Dynamics of Catalytic Residues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oakley, Aaron J.; Klvana, Martin; Otyepka, Michal

We present the structure of LinB, a 33-kDa haloalkane dehalogenase from Sphingomonas paucimobilis UT26, at 0.95 {angstrom} resolution. The data have allowed us to directly observe the anisotropic motions of the catalytic residues. In particular, the side-chain of the catalytic nucleophile, Asp108, displays a high degree of disorder. It has been modeled in two conformations, one similar to that observed previously (conformation A) and one strained (conformation B) that approached the catalytic base (His272). The strain in conformation B was mainly in the C{sub {alpha}}-C{sub {beta}}-C{sub {gamma}} angle (126{sup o}) that deviated by 13.4{sup o} from the 'ideal' bond anglemore » of 112.6{sup o}. On the basis of these observations, we propose a role for the charge state of the catalytic histidine in determining the geometry of the catalytic residues. We hypothesized that double-protonation of the catalytic base (His272) reduces the distance between the side-chain of this residue and that of the Asp108. The results of molecular dynamics simulations were consistent with the structural data showing that protonation of the His272 side-chain nitrogen atoms does indeed reduce the distance between the side-chains of the residues in question, although the simulations failed to demonstrate the same degree of strain in the Asp108 C{sub {alpha}}-C{sub {beta}}-C{sub {gamma}} angle. Instead, the changes in the molecular dynamics structures were distributed over several bond and dihedral angles. Quantum mechanics calculations on LinB with 1-chloro-2,2-dimethylpropane as a substrate were performed to determine which active site conformations and protonation states were most likely to result in catalysis. It was shown that His272 singly protonated at N{sub {delta}1} and Asp108 in conformation A gave the most exothermic reaction ({Delta}H = -22 kcal/mol). With His272 doubly protonated at N{sub {delta}1} and N{sub {epsilon}2}, the reactions were only slightly exothermic or were endothermic. In all calculations starting with Asp108 in conformation B, the Asp108 C{sub {alpha}}-C{sub {beta}}-C{sub {gamma}} angle changed during the reaction and the Asp108 moved to conformation A. The results presented here indicate that the positions of the catalytic residues and charge state of the catalytic base are important for determining reaction energetics in LinB.« less

Conformation and Dynamics of a Flexible Sheet in Solvent Media by Monte Carlo Simulations

NASA Astrophysics Data System (ADS)

Pandey, Ras; Anderson, Kelly; Heinz, Hendrik; Farmer, Barry

2005-03-01

Flexibility of the clay sheet is limited even in the ex-foliated state in some solvent media. A coarse grained model is used to investigate dynamics and conformation of a flexible sheet to model such a clay platelet in an effective solvent medium on a cubic lattice of size L^3 with lattice constant a. The undeformed sheet is described by a square lattice of size Ls^2, where, each node of the sheet is represented by the unit cube of the cubic lattice and 2a is the minimum distance between the nearest neighbor nodes to incorporate the excluded volume constraints. Additionally, each node interacts with neighboring nodes and solvent (empty) sites within a range ri. Each node execute their stochastic motion with the Metropolis algorithm subject to bond length fluctuation and excluded volume constraints. Mean square displacements of the center node and that of its center of mass are investigated as a function of time step for a set of these parameters. The radius of gyration (Rg) is also examined concurrently to understand its relaxation. Multi-scale segmental dynamics of the sheet is studied by identifying the power-law dependence in various time regimes. Relaxation of Rg and its dependence of temperature are planned to be discussed.

Geometry Dynamics of α-Helices in Different Class I Major Histocompatibility Complexes

PubMed Central

Karch, Rudolf; Schreiner, Wolfgang

2015-01-01

MHC α-helices form the antigen-binding cleft and are of particular interest for immunological reactions. To monitor these helices in molecular dynamics simulations, we applied a parsimonious fragment-fitting method to trace the axes of the α-helices. Each resulting axis was fitted by polynomials in a least-squares sense and the curvature integral was computed. To find the appropriate polynomial degree, the method was tested on two artificially modelled helices, one performing a bending movement and another a hinge movement. We found that second-order polynomials retrieve predefined parameters of helical motion with minimal relative error. From MD simulations we selected those parts of α-helices that were stable and also close to the TCR/MHC interface. We monitored the curvature integral, generated a ruled surface between the two MHC α-helices, and computed interhelical area and surface torsion, as they changed over time. We found that MHC α-helices undergo rapid but small changes in conformation. The curvature integral of helices proved to be a sensitive measure, which was closely related to changes in shape over time as confirmed by RMSD analysis. We speculate that small changes in the conformation of individual MHC α-helices are part of the intrinsic dynamics induced by engagement with the TCR. PMID:26649324

Utilizing a Dynamical Description of IspH to Aid in the Development of Novel Antimicrobial Drugs

PubMed Central

Blachly, Patrick G.; de Oliveira, César A. F.; Williams, Sarah L.; McCammon, J. Andrew

2013-01-01

The nonmevalonate pathway is responsible for isoprenoid production in microbes, including H. pylori, M. tuberculosis and P. falciparum, but is nonexistent in humans, thus providing a desirable route for antibacterial and antimalarial drug discovery. We coordinate a structural study of IspH, a [4Fe-4S] protein responsible for converting HMBPP to IPP and DMAPP in the ultimate step in the nonmevalonate pathway. By performing accelerated molecular dynamics simulations on both substrate-free and HMBPP-bound [Fe4S4]2+ IspH, we elucidate how substrate binding alters the dynamics of the protein. Using principal component analysis, we note that while substrate-free IspH samples various open and closed conformations, the closed conformation observed experimentally for HMBPP-bound IspH is inaccessible in the absence of HMBPP. In contrast, simulations with HMBPP bound are restricted from accessing the open states sampled by the substrate-free simulations. Further investigation of the substrate-free simulations reveals large fluctuations in the HMBPP binding pocket, as well as allosteric pocket openings – both of which are achieved through the hinge motions of the individual domains in IspH. Coupling these findings with solvent mapping and various structural analyses reveals alternative druggable sites that may be exploited in future drug design efforts. PMID:24367248

Novel NMR tools to study structure and dynamics of biomembranes.

PubMed

Gawrisch, Klaus; Eldho, Nadukkudy V; Polozov, Ivan V

2002-06-01

Nuclear magnetic resonance (NMR) studies on biomembranes have benefited greatly from introduction of magic angle spinning (MAS) NMR techniques. Improvements in MAS probe technology, combined with the higher magnetic field strength of modern instruments, enables almost liquid-like resolution of lipid resonances. The cross-relaxation rates measured by nuclear Overhauser enhancement spectroscopy (NOESY) provide new insights into conformation and dynamics of lipids with atomic-scale resolution. The data reflect the tremendous motional disorder in the lipid matrix. Transfer of magnetization by spin diffusion along the proton network of lipids is of secondary relevance, even at a long NOESY mixing time of 300 ms. MAS experiments with re-coupling of anisotropic interactions, like the 13C-(1)H dipolar couplings, benefit from the excellent resolution of 13C shifts that enables assignment of the couplings to specific carbon atoms. The traditional 2H NMR experiments on deuterated lipids have higher sensitivity when conducted on oriented samples at higher magnetic field strength. A very large number of NMR parameters from lipid bilayers is now accessible, providing information about conformation and dynamics for every lipid segment. The NMR methods have the sensitivity and resolution to study lipid-protein interaction, lateral lipid organization, and the location of solvents and drugs in the lipid matrix.

Exploration of Multi-State Conformational Dynamics and Underlying Global Functional Landscape of Maltose Binding Protein

PubMed Central

Wang, Yong; Tang, Chun; Wang, Erkang; Wang, Jin

2012-01-01

An increasing number of biological machines have been revealed to have more than two macroscopic states. Quantifying the underlying multiple-basin functional landscape is essential for understanding their functions. However, the present models seem to be insufficient to describe such multiple-state systems. To meet this challenge, we have developed a coarse grained triple-basin structure-based model with implicit ligand. Based on our model, the constructed functional landscape is sufficiently sampled by the brute-force molecular dynamics simulation. We explored maltose-binding protein (MBP) which undergoes large-scale domain motion between open, apo-closed (partially closed) and holo-closed (fully closed) states responding to ligand binding. We revealed an underlying mechanism whereby major induced fit and minor population shift pathways co-exist by quantitative flux analysis. We found that the hinge regions play an important role in the functional dynamics as well as that increases in its flexibility promote population shifts. This finding provides a theoretical explanation of the mechanistic discrepancies in PBP protein family. We also found a functional “backtracking” behavior that favors conformational change. We further explored the underlying folding landscape in response to ligand binding. Consistent with earlier experimental findings, the presence of ligand increases the cooperativity and stability of MBP. This work provides the first study to explore the folding dynamics and functional dynamics under the same theoretical framework using our triple-basin functional model. PMID:22532792

Efficiently explore the energy landscape of proteins in molecular dynamics simulations by amplifying collective motions

NASA Astrophysics Data System (ADS)

He, Jianbin; Zhang, Zhiyong; Shi, Yunyu; Liu, Haiyan

2003-08-01

We describe a method for efficient sampling of the energy landscape of a protein in atomic molecular dynamics simulations. A simulation is divided into alternatively occurring relaxation phases and excitation phases. In the relaxation phase (conventional simulation), we use a frequently updated reference structure and deviations from this reference structure to mark whether the system has been trapped in a local minimum. In that case, the simulation enters the excitation phase, during which a few slow collective modes of the system are coupled to a higher temperature bath. After the system has escaped from the minimum (also judged by deviations from the reference structure) the simulation reenters the relaxation phase. The collective modes are obtained from a coarse-grained Gaussian elastic network model. The scheme, which we call ACM-AME (amplified collective motion-assisted minimum escaping), is compared with conventional simulations as well as an alternative scheme that elevates the temperature of all degrees of freedom during the excitation phase (amplified overall motion-assisted minimum escaping, or AOM-AME). Comparison is made using simulations on four peptides starting from non-native extended or all helical structures. In terms of sampling low energy conformations and continuously sampling new conformations throughout a simulation, the ACM-AME scheme demonstrates very good performance while the AOM-AME scheme shows little improvement upon conventional simulations. Limited success is achieved in producing structures close to the native structures of the peptides: for an S-peptide analog, the ACM-AME approach is able to reproduce its native helical structure, and starting from an all-helical structure of the villin headpiece subdomain (HP-36) in implicit solvent, two out of three 150 ns ACM-AME runs are able to sample structures with 3-4 Å backbone root-mean-square deviations from the nuclear magnetic resonance structure of the protein.

Geodesic motion around traversable wormholes supported by a massless conformally coupled scalar field

NASA Astrophysics Data System (ADS)

Willenborg, Felix; Grunau, Saskia; Kleihaus, Burkhard; Kunz, Jutta

2018-06-01

We consider a traversable wormhole solution of Einstein's gravity conformally coupled to a massless scalar field, a solution derived by Barcelo and Visser based on the Janis-Newman-Winicour-Wyman spacetime. We study the geodesic motion of timelike and spacelike particles in this spacetime. We solve the equations of motion analytically in terms of the Weierstraß functions and discuss all possible orbit types and their parameter dependence. Interestingly, bound orbits occur for timelike geodesics only in one of the two worlds. Moreover, under no conditions there exist timelike two world bound orbits.

Molecular Dynamics of a Water-Lipid Bilayer Interface

NASA Technical Reports Server (NTRS)

Wilson, Michael A.; Pohorille, Andrew

1994-01-01

We present results of molecular dynamics simulations of a glycerol 1-monooleate bilayer in water. The total length of analyzed trajectories is 5ns. The calculated width of the bilayer agrees well with the experimentally measured value. The interior of the membrane is in a highly disordered fluid state. Atomic density profile, orientational and conformational distribution functions, and order parameters indicate that disorder increases toward the center of the bilayer. Analysis of out-of-plane thermal fluctuations of the bilayer surfaces occurring at the time scale of the present calculations reveals that the distribution of modes agrees with predictions of the capillary wave model. Fluctuations of both bilayer surfaces are uncorrelated, yielding Gaussian distribution of instantaneous widths of the membrane. Fluctuations of the width produce transient thinning defects in the bilayer which occasionally span almost half of the membrane. The leading mechanism of these fluctuations is the orientational and conformational motion of head groups rather than vertical motion of the whole molecules. Water considerably penetrates the head group region of the bilayer but not its hydrocarbon core. The total net excess dipole moment of the interfacial water points toward the aqueous phase, but the water polarization profile is non-monotonic. Both water and head groups significantly contribute to the surface potential across the interface. The calculated sign of the surface potential is in agreement with that from experimental measurements, but the value is markedly overestimated. The structural and electrical properties of the water-bilayer system are discussed in relation to membrane functions, in particular transport of ions and nonelectrolytes across membranes.

Characterizing protein conformations by correlation analysis of coarse-grained contact matrices.

PubMed

Lindsay, Richard J; Siess, Jan; Lohry, David P; McGee, Trevor S; Ritchie, Jordan S; Johnson, Quentin R; Shen, Tongye

2018-01-14

We have developed a method to capture the essential conformational dynamics of folded biopolymers using statistical analysis of coarse-grained segment-segment contacts. Previously, the residue-residue contact analysis of simulation trajectories was successfully applied to the detection of conformational switching motions in biomolecular complexes. However, the application to large protein systems (larger than 1000 amino acid residues) is challenging using the description of residue contacts. Also, the residue-based method cannot be used to compare proteins with different sequences. To expand the scope of the method, we have tested several coarse-graining schemes that group a collection of consecutive residues into a segment. The definition of these segments may be derived from structural and sequence information, while the interaction strength of the coarse-grained segment-segment contacts is a function of the residue-residue contacts. We then perform covariance calculations on these coarse-grained contact matrices. We monitored how well the principal components of the contact matrices is preserved using various rendering functions. The new method was demonstrated to assist the reduction of the degrees of freedom for describing the conformation space, and it potentially allows for the analysis of a system that is approximately tenfold larger compared with the corresponding residue contact-based method. This method can also render a family of similar proteins into the same conformational space, and thus can be used to compare the structures of proteins with different sequences.

Characterizing protein conformations by correlation analysis of coarse-grained contact matrices

NASA Astrophysics Data System (ADS)

Lindsay, Richard J.; Siess, Jan; Lohry, David P.; McGee, Trevor S.; Ritchie, Jordan S.; Johnson, Quentin R.; Shen, Tongye

2018-01-01

We have developed a method to capture the essential conformational dynamics of folded biopolymers using statistical analysis of coarse-grained segment-segment contacts. Previously, the residue-residue contact analysis of simulation trajectories was successfully applied to the detection of conformational switching motions in biomolecular complexes. However, the application to large protein systems (larger than 1000 amino acid residues) is challenging using the description of residue contacts. Also, the residue-based method cannot be used to compare proteins with different sequences. To expand the scope of the method, we have tested several coarse-graining schemes that group a collection of consecutive residues into a segment. The definition of these segments may be derived from structural and sequence information, while the interaction strength of the coarse-grained segment-segment contacts is a function of the residue-residue contacts. We then perform covariance calculations on these coarse-grained contact matrices. We monitored how well the principal components of the contact matrices is preserved using various rendering functions. The new method was demonstrated to assist the reduction of the degrees of freedom for describing the conformation space, and it potentially allows for the analysis of a system that is approximately tenfold larger compared with the corresponding residue contact-based method. This method can also render a family of similar proteins into the same conformational space, and thus can be used to compare the structures of proteins with different sequences.

New insights in the conformation of α 1-acid glycoprotein (orosomucoid). Quenching resolved emission anisotropy studies

NASA Astrophysics Data System (ADS)

Albani, J. R.

1999-09-01

Quenching resolved emission anisotropy method was applied to study the dynamics of the two classes of Trp residues of human α 1-acid glycoprotein (orosomucoid), in the absence and presence of progesterone. In the absence of progesterone, the values of the anisotropies of the surface and buried Trp residues are 0.155 and 0.178, respectively. These values lower than the limiting anisotropy (0.267) indicate that both classes of Trp residues display residual motions. In the presence of progesterone, the values of the anisotropies decrease from 0.155 to 0.146 and from 0.178 to 0.167. Thus, binding of progesterone to orosomucoid increases the internal dynamics of the protein. Also, the fact that in the absence or in the presence of progesterone, the anisotropies of both classes are close, means that the amplitudes of the motions of the two classes are not significantly different. From our data and from the well-known position of the carbohydrate residues on orosomucoid, we suggest the presence of a hydrophobic pocket within the protein and where the 'buried' Trp residues can be found.

Using NMR spectroscopy to elucidate the role of molecular motions in enzyme function.

PubMed

Lisi, George P; Loria, J Patrick

2016-02-01

Conformational motions play an essential role in enzyme function, often facilitating the formation of enzyme-substrate complexes and/or product release. Although considerable debate remains regarding the role of molecular motions in the conversion of enzymatic substrates to products, numerous examples have found motions to be crucial for optimization of enzyme scaffolds, effective substrate binding, and product dissociation. Conformational fluctuations are often rate-limiting to enzyme catalysis, primarily through product release, with the chemical reaction occurring much more quickly. As a result, the direct involvement of motions at various stages along the enzyme reaction coordinate remains largely unknown and untested. In the following review, we describe the use of solution NMR techniques designed to probe various timescales of molecular motions and detail examples in which motions play a role in propagating catalytic effects from the active site and directly participate in essential aspects of enzyme function. Copyright © 2015 Elsevier B.V. All rights reserved.

Driving Calmodulin Protein towards Conformational Shift by Changing Ionization States of Select Residues

NASA Astrophysics Data System (ADS)

Negi, Sunita; Rana Atilgan, Ali; Atilgan, Canan

2012-12-01

Proteins are complex systems made up of many conformational sub-states which are mainly determined by the folded structure. External factors such as solvent type, temperature, pH and ionic strength play a very important role in the conformations sampled by proteins. Here we study the conformational multiplicity of calmodulin (CaM) which is a protein that plays an important role in calcium signaling pathways in the eukaryotic cells. CaM can bind to a variety of other proteins or small organic compounds, and mediates different physiological processes by activating various enzymes. Binding of calcium ions and proteins or small organic molecules to CaM induces large conformational changes that are distinct to each interacting partner. In particular, we discuss the effect of pH variation on the conformations of CaM. By using the pKa values of the charged residues as a basis to assign protonation states, the conformational changes induced in CaM by reducing the pH are studied by molecular dynamics simulations. Our current view suggests that at high pH, barrier crossing to the compact form is prevented by repulsive electrostatic interactions between the two lobes. At reduced pH, not only is barrier crossing facilitated by protonation of residues, but also conformations which are on average more compact are attained. The latter are in accordance with the fluorescence resonance energy transfer experiment results of other workers. The key events leading to the conformational change from the open to the compact conformation are (i) formation of a salt bridge between the N-lobe and the linker, stabilizing their relative motions, (ii) bending of the C-lobe towards the N-lobe, leading to a lowering of the interaction energy between the two-lobes, (iii) formation of a hydrophobic patch between the two lobes, further stabilizing the bent conformation by reducing the entropic cost of the compact form, (iv) sharing of a Ca+2 ion between the two lobes.

Transverse ac-driven and geometric ratchet effects for vortices in conformal crystal pinning arrays

DOE PAGES

Reichhardt, Charles; Reichhardt, Cynthia Jane Olsen

2016-02-11

A conformal pinning array is created by taking a conformal transformation of a uniform hexagonal lattice to create a structure in which the sixfold ordering of the original lattice is preserved but which has a spatial gradient in the pinning site density. With a series of conformal arrays it is possible to create asymmetric substrates, and it was previously shown that when an ac drive is applied parallel to the asymmetry direction, a pronounced ratchet effect occurs with a net dc flow of vortices in the same direction as the ac drive. Here, in this article, we show that whenmore » the ac drive is applied perpendicular to the substrate asymmetry direction, it is possible to realize a transverse ratchet effect where a net dc flow of vortices is generated perpendicular to the ac drive. The conformal transverse ratchet effect is distinct from previous versions of transverse ratchets in that it occurs due to the generation of non-Gaussian transverse vortex velocity fluctuations by the plastic motion of vortices, so that the system behaves as a noise correlation ratchet. The transverse ratchet effect is much more pronounced in the conformal arrays than in random gradient arrays and is absent in square gradient arrays due the different nature of the vortex flow in each geometry. We show that a series of reversals can occur in the transverse ratchet effect due to changes in the vortex flow across the pinning gradient as a function of vortex filling, pinning strength, and ac amplitude. We also consider the case where a dc drive applied perpendicular to the substrate asymmetry direction generates a net flow of vortices perpendicular to the dc drive, producing what is known as a geometric or drift ratchet that again arises due to non-Gaussian dynamically generated fluctuations. The drift ratchet is more efficient than the ac driven ratchet and also exhibits a series of reversals for varied parameters. Lastly, our results should be general to a wide class of systems undergoing nonequilibrium dynamics on conformal substrates, such as colloidal particles on optical traps.« less

Laboratory evolution of protein conformational dynamics.

PubMed

Campbell, Eleanor C; Correy, Galen J; Mabbitt, Peter D; Buckle, Ashley M; Tokuriki, Nobuhiko; Jackson, Colin J

2017-11-08

This review focuses on recent work that has begun to establish specific functional roles for protein conformational dynamics, specifically how the conformational landscapes that proteins can sample can evolve under laboratory based evolutionary selection. We discuss recent technical advances in computational and biophysical chemistry, which have provided us with new ways to dissect evolutionary processes. Finally, we offer some perspectives on the emerging view of conformational dynamics and evolution, and the challenges that we face in rationally engineering conformational dynamics. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hierarchical structure of the energy landscape of proteins revisited by time series analysis. I. Mimicking protein dynamics in different time scales

NASA Astrophysics Data System (ADS)

Alakent, Burak; Camurdan, Mehmet C.; Doruker, Pemra

2005-10-01

Time series models, which are constructed from the projections of the molecular-dynamics (MD) runs on principal components (modes), are used to mimic the dynamics of two proteins: tendamistat and immunity protein of colicin E7 (ImmE7). Four independent MD runs of tendamistat and three independent runs of ImmE7 protein in vacuum are used to investigate the energy landscapes of these proteins. It is found that mean-square displacements of residues along the modes in different time scales can be mimicked by time series models, which are utilized in dividing protein dynamics into different regimes with respect to the dominating motion type. The first two regimes constitute the dominance of intraminimum motions during the first 5ps and the random walk motion in a hierarchically higher-level energy minimum, which comprise the initial time period of the trajectories up to 20-40ps for tendamistat and 80-120ps for ImmE7. These are also the time ranges within which the linear nonstationary time series are completely satisfactory in explaining protein dynamics. Encountering energy barriers enclosing higher-level energy minima constrains the random walk motion of the proteins, and pseudorelaxation processes at different levels of minima are detected in tendamistat, depending on the sampling window size. Correlation (relaxation) times of 30-40ps and 150-200ps are detected for two energy envelopes of successive levels for tendamistat, which gives an overall idea about the hierarchical structure of the energy landscape. However, it should be stressed that correlation times of the modes are highly variable with respect to conformational subspaces and sampling window sizes, indicating the absence of an actual relaxation. The random-walk step sizes and the time length of the second regime are used to illuminate an important difference between the dynamics of the two proteins, which cannot be clarified by the investigation of relaxation times alone: ImmE7 has lower-energy barriers enclosing the higher-level energy minimum, preventing the protein to relax and letting it move in a random-walk fashion for a longer period of time.

Hinge-Like Motions in RNA Kink-Turns: The Role of the Second A-Minor Motif and Nominally Unpaired Bases

PubMed Central

Rázga, Filip; Koča, Jaroslav; Šponer, Jiří; Leontis, Neocles B.

2005-01-01

Kink-turn (K-turn) motifs are asymmetric internal loops found at conserved positions in diverse RNAs, with sharp bends in phosphodiester backbones producing V-shaped structures. Explicit-solvent molecular dynamics simulations were carried out for three K-turns from 23S rRNA, i.e., Kt-38 located at the base of the A-site finger, Kt-42 located at the base of the L7/L12 stalk, and Kt-58 located in domain III, and for the K-turn of human U4 snRNA. The simulations reveal hinge-like K-turn motions on the nanosecond timescale. The first conserved A-minor interaction between the K-turn stems is entirely stable in all simulations. The angle between the helical arms of Kt-38 and Kt-42 is regulated by local variations of the second A-minor (type I) interaction between the stems. Its variability ranges from closed geometries to open ones stabilized by insertion of long-residency waters between adenine and cytosine. The simulated A-minor geometries fully agree with x-ray data. Kt-58 and Kt-U4 exhibit similar elbow-like motions caused by conformational change of the adenosine from the nominally unpaired region. Despite the observed substantial dynamics of K-turns, key tertiary interactions are stable and no sign of unfolding is seen. We suggest that some K-turns are flexible elements mediating large-scale ribosomal motions during the protein synthesis cycle. PMID:15722438

Investigating the dynamic nature of the ABC transporters: ABCB1 and MsbA as examples for the potential synergies of MD theory and EPR applications.

PubMed

Stockner, Thomas; Mullen, Anna; MacMillan, Fraser

2015-10-01

ABC transporters are primary active transporters found in all kingdoms of life. Human multidrug resistance transporter ABCB1, or P-glycoprotein, has an extremely broad substrate spectrum and confers resistance against chemotherapy drug treatment in cancer cells. The bacterial ABC transporter MsbA is a lipid A flippase and a homolog to the human ABCB1 transporter, with which it partially shares its substrate spectrum. Crystal structures of MsbA and ABCB1 have been solved in multiple conformations, providing a glimpse into the possible conformational changes the transporter could be going through during the transport cycle. Crystal structures are inherently static, while a dynamic picture of the transporter in motion is needed for a complete understanding of transporter function. Molecular dynamics (MD) simulations and electron paramagnetic resonance (EPR) spectroscopy can provide structural information on ABC transporters, but the strength of these two methods lies in the potential to characterise the dynamic regime of these transporters. Information from the two methods is quite complementary. MD simulations provide an all atom dynamic picture of the time evolution of the molecular system, though with a narrow time window. EPR spectroscopy can probe structural, environmental and dynamic properties of the transporter in several time regimes, but only through the attachment sites of an exogenous spin label. In this review the synergistic effects that can be achieved by combining the two methods are highlighted, and a brief methodological background is also presented. © 2015 Authors; published by Portland Press Limited.

Celestial ephemerides in an expanding universe

NASA Astrophysics Data System (ADS)

Kopeikin, Sergei M.

2012-09-01

The post-Newtonian theory of motion of celestial bodies and propagation of light was instrumental in conducting the critical experimental tests of general relativity and in building the astronomical ephemerides of celestial bodies in the Solar System with unparalleled precision. The cornerstone of the theory is the postulate that the Solar System is gravitationally isolated from the rest of the Universe and the background spacetime is asymptotically flat. The present article extends this theoretical concept and formulates the principles of celestial dynamics of particles and light moving in the gravitational field of a localized astronomical system embedded to the expanding Friedmann-Lemaître-Robertson-Walker universe. We formulate the precise mathematical concept of the Newtonian limit of Einstein’s field equations in the conformally flat Friedmann-Lemaître-Robertson-Walker spacetime and analyze the geodesic motion of massive particles and light in this limit. We prove that by doing conformal spacetime transformations, one can reduce the equations of motion of particles and light to the classical form of the Newtonian theory. However, the time arguments in the equations of motion of particles and light differ from each other in terms being proportional to the Hubble constant H. This leads to the important conclusion that the equations of light propagation used currently by space navigation centers for fitting range and Doppler-tracking observations of celestial bodies are missing some terms of the cosmological origin that are proportional to the Hubble constant H. We also analyze the effect of the cosmological expansion on motion of electrons in atoms. We prove that the Hubble expansion does not affect the atomic frequencies and hence does not affect the atomic time scale used in the creation of astronomical ephemerides. We derive the cosmological correction to the light travel time equation and argue that its measurement opens an exciting opportunity to determine the local value of the Hubble constant H in the Solar System independently of cosmological observations.

Mechanistic insight into the functional transition of the enzyme guanylate kinase induced by a single mutation

PubMed Central

Zhang, Yuebin; Niu, Huiyan; Li, Yan; Chu, Huiying; Shen, Hujun; Zhang, Dinglin; Li, Guohui

2015-01-01

Dramatic functional changes of enzyme usually require scores of alterations in amino acid sequence. However, in the case of guanylate kinase (GK), the functional novelty is induced by a single (S→P) mutation, leading to the functional transition of the enzyme from a phosphoryl transfer kinase into a phosphorprotein interaction domain. Here, by using molecular dynamic (MD) and metadynamics simulations, we provide a comprehensive description of the conformational transitions of the enzyme after mutating serine to proline. Our results suggest that the serine plays a crucial role in maintaining the closed conformation of wild-type GK and the GMP recognition. On the contrary, the S→P mutant exhibits a stable open conformation and loses the ability of ligand binding, which explains its functional transition from the GK enzyme to the GK domain. Furthermore, the free energy profiles (FEPs) obtained by metadymanics clearly demonstrate that the open-closed conformational transition in WT GK is positive correlated with the process of GMP binding, indicating the GMP-induced closing motion of GK enzyme, which is not observed in the mutant. In addition, the FEPs show that the S→P mutation can also leads to the mis-recognition of GMP, explaining the vanishing of catalytic activity of the mutant. PMID:25672880

Mechanistic insight into the functional transition of the enzyme guanylate kinase induced by a single mutation.

PubMed

Zhang, Yuebin; Niu, Huiyan; Li, Yan; Chu, Huiying; Shen, Hujun; Zhang, Dinglin; Li, Guohui

2015-02-12

Dramatic functional changes of enzyme usually require scores of alterations in amino acid sequence. However, in the case of guanylate kinase (GK), the functional novelty is induced by a single (S→P) mutation, leading to the functional transition of the enzyme from a phosphoryl transfer kinase into a phosphorprotein interaction domain. Here, by using molecular dynamic (MD) and metadynamics simulations, we provide a comprehensive description of the conformational transitions of the enzyme after mutating serine to proline. Our results suggest that the serine plays a crucial role in maintaining the closed conformation of wild-type GK and the GMP recognition. On the contrary, the S→P mutant exhibits a stable open conformation and loses the ability of ligand binding, which explains its functional transition from the GK enzyme to the GK domain. Furthermore, the free energy profiles (FEPs) obtained by metadymanics clearly demonstrate that the open-closed conformational transition in WT GK is positive correlated with the process of GMP binding, indicating the GMP-induced closing motion of GK enzyme, which is not observed in the mutant. In addition, the FEPs show that the S→P mutation can also leads to the mis-recognition of GMP, explaining the vanishing of catalytic activity of the mutant.

Characterizing Conformational Dynamics of Proteins Using Evolutionary Couplings.

PubMed

Feng, Jiangyan; Shukla, Diwakar

2018-01-25

Understanding of protein conformational dynamics is essential for elucidating molecular origins of protein structure-function relationship. Traditionally, reaction coordinates, i.e., some functions of protein atom positions and velocities have been used to interpret the complex dynamics of proteins obtained from experimental and computational approaches such as molecular dynamics simulations. However, it is nontrivial to identify the reaction coordinates a priori even for small proteins. Here, we evaluate the power of evolutionary couplings (ECs) to capture protein dynamics by exploring their use as reaction coordinates, which can efficiently guide the sampling of a conformational free energy landscape. We have analyzed 10 diverse proteins and shown that a few ECs are sufficient to characterize complex conformational dynamics of proteins involved in folding and conformational change processes. With the rapid strides in sequencing technology, we expect that ECs could help identify reaction coordinates a priori and enhance the sampling of the slow dynamical process associated with protein folding and conformational change.

Movie of the structural changes during a catalytic cycle of nucleoside monophosphate kinases.

PubMed

Vonrhein, C; Schlauderer, G J; Schulz, G E

1995-05-15

There are 17 crystal structures of nucleoside monophosphate kinases known. As expected for kinases, they show large conformational changes upon binding of substrates. These are concentrated in two chain segments, or domains, of 30 and 38 residues that are involved in binding of the substrates N1TP and N2MP (nucleoside tri- and monophosphates with bases N1 and N2), respectively. After aligning the 17 structures on the main parts of their polypeptide chains, two domains in various conformational states were revealed. These states were caused by bound substrate (or analogues) and by crystal-packing forces, and ranged between a 'closed' conformation and a less well defined 'open' conformation. The structures were visually sorted yielding an approximately evenly spaced series of domain states that outlines the closing motions when the substrates bind. The packing forces in the crystals are weak, leaving the natural domain trajectories essentially intact. Packing is necessary, however, to produce stable intermediates. The ordered experimental structures were then recorded as still pictures of a movie and animated to represent the motions of the molecule during a catalytic cycle. The motions were smoothed out by adding interpolated structures to the observed ones. The resulting movies are available through the World Wide Web (http:@bio5.chemie.uni-freiburg.de/ak movie.html). Given the proliferating number of homologous proteins known to exist in different conformational states, it is becoming possible to outline the motions of chain segments and combine them into a movie, which can then represent protein action much more effectively than static pictures alone are able to do.

Non-actual motion: phenomenological analysis and linguistic evidence.

PubMed

Blomberg, Johan; Zlatev, Jordan

2015-09-01

Sentences with motion verbs describing static situations have been seen as evidence that language and cognition are geared toward dynamism and change (Talmy in Toward a cognitive semantics, MIT Press, Cambridge, 2000; Langacker in Concept, image, and symbol: the cognitive basis of grammar, Mouton de Gruyter, Berlin and New York, 1990). Different concepts have been used in the literature, e.g., fictive motion, subjective motion and abstract motion to denote this. Based on phenomenological analysis, we reinterpret such concepts as reflecting different motivations for the use of such constructions (Blomberg and Zlatev in Phenom Cogn Sci 13(3):395-418, 2014). To highlight the multifaceted character of the phenomenon, we propose the concept non-actual motion (NAM), which we argue is more compatible with the situated cognition approach than explanations such as "mental simulation" (e.g., Matlock in Studies in linguistic motivation, Mouton de Gruyter, Berlin, 2004). We investigate the expression of NAM by means of a picture-based elicitation task with speakers of Swedish, French and Thai. Pictures represented figures that either afford human motion or not (±afford); crossed with this, the figure extended either across the picture from a third-person perspective (3 pp) or from a first-person perspective (1 pp). All picture types elicited NAM-sentences with the combination [+afford, 1 pp] producing most NAM-sentences in all three languages. NAM-descriptions also conformed to language-specific patterns for the expression of actual motion. We conclude that NAM shows interaction between pre-linguistic motivations and language-specific conventions.

Solution NMR views of dynamical ordering of biomacromolecules.

PubMed

Ikeya, Teppei; Ban, David; Lee, Donghan; Ito, Yutaka; Kato, Koichi; Griesinger, Christian

2018-02-01

To understand the mechanisms related to the 'dynamical ordering' of macromolecules and biological systems, it is crucial to monitor, in detail, molecular interactions and their dynamics across multiple timescales. Solution nuclear magnetic resonance (NMR) spectroscopy is an ideal tool that can investigate biophysical events at the atomic level, in near-physiological buffer solutions, or even inside cells. In the past several decades, progress in solution NMR has significantly contributed to the elucidation of three-dimensional structures, the understanding of conformational motions, and the underlying thermodynamic and kinetic properties of biomacromolecules. This review discusses recent methodological development of NMR, their applications and some of the remaining challenges. Although a major drawback of NMR is its difficulty in studying the dynamical ordering of larger biomolecular systems, current technologies have achieved considerable success in the structural analysis of substantially large proteins and biomolecular complexes over 1MDa and have characterised a wide range of timescales across which biomolecular motion exists. While NMR is well suited to obtain local structure information in detail, it contributes valuable and unique information within hybrid approaches that combine complementary methodologies, including solution scattering and microscopic techniques. For living systems, the dynamic assembly and disassembly of macromolecular complexes is of utmost importance for cellular homeostasis and, if dysregulated, implied in human disease. It is thus instructive for the advancement of the study of the dynamical ordering to discuss the potential possibilities of solution NMR spectroscopy and its applications. This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecular dynamics based enhanced sampling of collective variables with very large time steps.

PubMed

Chen, Pei-Yang; Tuckerman, Mark E

2018-01-14

Enhanced sampling techniques that target a set of collective variables and that use molecular dynamics as the driving engine have seen widespread application in the computational molecular sciences as a means to explore the free-energy landscapes of complex systems. The use of molecular dynamics as the fundamental driver of the sampling requires the introduction of a time step whose magnitude is limited by the fastest motions in a system. While standard multiple time-stepping methods allow larger time steps to be employed for the slower and computationally more expensive forces, the maximum achievable increase in time step is limited by resonance phenomena, which inextricably couple fast and slow motions. Recently, we introduced deterministic and stochastic resonance-free multiple time step algorithms for molecular dynamics that solve this resonance problem and allow ten- to twenty-fold gains in the large time step compared to standard multiple time step algorithms [P. Minary et al., Phys. Rev. Lett. 93, 150201 (2004); B. Leimkuhler et al., Mol. Phys. 111, 3579-3594 (2013)]. These methods are based on the imposition of isokinetic constraints that couple the physical system to Nosé-Hoover chains or Nosé-Hoover Langevin schemes. In this paper, we show how to adapt these methods for collective variable-based enhanced sampling techniques, specifically adiabatic free-energy dynamics/temperature-accelerated molecular dynamics, unified free-energy dynamics, and by extension, metadynamics, thus allowing simulations employing these methods to employ similarly very large time steps. The combination of resonance-free multiple time step integrators with free-energy-based enhanced sampling significantly improves the efficiency of conformational exploration.

Molecular dynamics based enhanced sampling of collective variables with very large time steps

NASA Astrophysics Data System (ADS)

Chen, Pei-Yang; Tuckerman, Mark E.

2018-01-01

Enhanced sampling techniques that target a set of collective variables and that use molecular dynamics as the driving engine have seen widespread application in the computational molecular sciences as a means to explore the free-energy landscapes of complex systems. The use of molecular dynamics as the fundamental driver of the sampling requires the introduction of a time step whose magnitude is limited by the fastest motions in a system. While standard multiple time-stepping methods allow larger time steps to be employed for the slower and computationally more expensive forces, the maximum achievable increase in time step is limited by resonance phenomena, which inextricably couple fast and slow motions. Recently, we introduced deterministic and stochastic resonance-free multiple time step algorithms for molecular dynamics that solve this resonance problem and allow ten- to twenty-fold gains in the large time step compared to standard multiple time step algorithms [P. Minary et al., Phys. Rev. Lett. 93, 150201 (2004); B. Leimkuhler et al., Mol. Phys. 111, 3579-3594 (2013)]. These methods are based on the imposition of isokinetic constraints that couple the physical system to Nosé-Hoover chains or Nosé-Hoover Langevin schemes. In this paper, we show how to adapt these methods for collective variable-based enhanced sampling techniques, specifically adiabatic free-energy dynamics/temperature-accelerated molecular dynamics, unified free-energy dynamics, and by extension, metadynamics, thus allowing simulations employing these methods to employ similarly very large time steps. The combination of resonance-free multiple time step integrators with free-energy-based enhanced sampling significantly improves the efficiency of conformational exploration.

Transition States and transition state analogue interactions with enzymes.

PubMed

Schramm, Vern L

2015-04-21

Enzymatic transition states have lifetimes of a few femtoseconds (fs). Computational analysis of enzyme motions leading to transition state formation suggests that local catalytic site motions on the fs time scale provide the mechanism to locate transition states. An experimental test of protein fs motion and its relation to transition state formation can be provided by isotopically heavy proteins. Heavy enzymes have predictable mass-altered bond vibration states without altered electrostatic properties, according to the Born-Oppenheimer approximation. On-enzyme chemistry is slowed in most heavy proteins, consistent with altered protein bond frequencies slowing the search for the transition state. In other heavy enzymes, structural changes involved in reactant binding and release are also influenced. Slow protein motions associated with substrate binding and catalytic site preorganization are essential to allow the subsequent fs motions to locate the transition state and to facilitate the efficient release of products. In the catalytically competent geometry, local groups move in stochastic atomic motion on the fs time scale, within transition state-accessible conformations created by slower protein motions. The fs time scale for the transition state motions does not permit thermodynamic equilibrium between the transition state and stable enzyme states. Isotopically heavy enzymes provide a diagnostic tool for fast coupled protein motions to transition state formation and mass-dependent conformational changes. The binding of transition state analogue inhibitors is the opposite in catalytic time scale to formation of the transition state but is related by similar geometries of the enzyme-transition state and enzyme-inhibitor interactions. While enzymatic transition states have lifetimes as short as 10(-15) s, transition state analogues can bind tightly to enzymes with release rates greater than 10(3) s. Tight-binding transition state analogues stabilize the rare but evolved enzymatic geometry to form the transition state. Evolution to efficient catalysis optimized this geometry and its stabilization by a transition state mimic results in tight binding. Release rates of transition state analogues are orders of magnitude slower than product release in normal catalytic function. During catalysis, product release is facilitated by altered chemistry. Compared to the weak associations found in Michaelis complexes, transition state analogues involve strong interactions related to those in the transition state. Optimum binding of transition state analogues occurs when the complex retains the system motions intrinsic to transition state formation. Conserved dynamic motion retains the entropic components of inhibitor complexes, improving the thermodynamics of analogue binding.

Two-species-coagulation approach to consensus by group level interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Escudero, Carlos; Macia, Fabricio; Velazquez, Juan J. L.

2010-07-15

We explore the self-organization dynamics of a set of entities by considering the interactions that affect the different subgroups conforming the whole. To this end, we employ the widespread example of coagulation kinetics, and characterize which interaction types lead to consensus formation and which do not, as well as the corresponding different macroscopic patterns. The crucial technical point is extending the usual one species coagulation dynamics to the two species one. This is achieved by means of introducing explicitly solvable kernels which have a clear physical meaning. The corresponding solutions are calculated in the long time limit, in which consensusmore » may or may not be reached. The lack of consensus is characterized by means of scaling limits of the solutions. The possible applications of our results to some topics in which consensus reaching is fundamental, such as collective animal motion and opinion spreading dynamics, are also outlined.« less

Two-species-coagulation approach to consensus by group level interactions

NASA Astrophysics Data System (ADS)

Escudero, Carlos; Macià, Fabricio; Velázquez, Juan J. L.

2010-07-01

We explore the self-organization dynamics of a set of entities by considering the interactions that affect the different subgroups conforming the whole. To this end, we employ the widespread example of coagulation kinetics, and characterize which interaction types lead to consensus formation and which do not, as well as the corresponding different macroscopic patterns. The crucial technical point is extending the usual one species coagulation dynamics to the two species one. This is achieved by means of introducing explicitly solvable kernels which have a clear physical meaning. The corresponding solutions are calculated in the long time limit, in which consensus may or may not be reached. The lack of consensus is characterized by means of scaling limits of the solutions. The possible applications of our results to some topics in which consensus reaching is fundamental, such as collective animal motion and opinion spreading dynamics, are also outlined.

Non-adiabatic dynamics of isolated green fluorescent protein chromophore anion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Li, E-mail: zhaoli282@dicp.ac.cn, E-mail: pwzhou@dicp.ac.cn, E-mail: libinsnet@dicp.ac.cn, E-mail: aihuagao@dicp.ac.cn; Gao, Ai-Hua, E-mail: zhaoli282@dicp.ac.cn, E-mail: pwzhou@dicp.ac.cn, E-mail: libinsnet@dicp.ac.cn, E-mail: aihuagao@dicp.ac.cn; University of the Chinese Academy of Sciences, Beijing 100049

2014-12-21

On-the-fly ab initio molecular dynamics calculations have been performed to investigate the relaxation mechanism of green fluorescent protein chromophore anion under vacuum. The CASSCF surface hopping simulation method based on Zhu-Nakamura theory is applied to present the real-time conformational changes of the target molecule. The static calculations and dynamics simulation results suggest that not only the twisting motion around bridging bonds between imidazolinone and phenoxy groups but the strength mode of C=O and pyramidalization character of bridging atom are major factors on the ultrafast fluorescence quenching process of the isolated chromophore anion. The abovementioned factors bring the molecule to themore » vicinity of conical intersections on its potential energy surface and to finish the internal conversion process. A Hula-like twisting pattern is displayed during the relaxation process and the entire decay process disfavors a photoswitching pattern which corresponds to cis-trans photoisomerization.« less

Mapped Chebyshev Pseudo-Spectral Method for Dynamic Aero-Elastic Problem of Limit Cycle Oscillation

NASA Astrophysics Data System (ADS)

Im, Dong Kyun; Kim, Hyun Soon; Choi, Seongim

2018-05-01

A mapped Chebyshev pseudo-spectral method is developed as one of the Fourier-spectral approaches and solves nonlinear PDE systems for unsteady flows and dynamic aero-elastic problem in a given time interval, where the flows or elastic motions can be periodic, nonperiodic, or periodic with an unknown frequency. The method uses the Chebyshev polynomials of the first kind for the basis function and redistributes the standard Chebyshev-Gauss-Lobatto collocation points more evenly by a conformal mapping function for improved numerical stability. Contributions of the method are several. It can be an order of magnitude more efficient than the conventional finite difference-based, time-accurate computation, depending on the complexity of solutions and the number of collocation points. The method reformulates the dynamic aero-elastic problem in spectral form for coupled analysis of aerodynamics and structures, which can be effective for design optimization of unsteady and dynamic problems. A limit cycle oscillation (LCO) is chosen for the validation and a new method to determine the LCO frequency is introduced based on the minimization of a second derivative of the aero-elastic formulation. Two examples of the limit cycle oscillation are tested: nonlinear, one degree-of-freedom mass-spring-damper system and two degrees-of-freedom oscillating airfoil under pitch and plunge motions. Results show good agreements with those of the conventional time-accurate simulations and wind tunnel experiments.

Modelling duality between bound and resonant meson spectra by means of free quantum motions on the de Sitter space-time dS4

NASA Astrophysics Data System (ADS)

Kirchbach, M.; Compean, C. B.

2016-07-01

The real parts of the complex squared energies defined by the resonance poles of the transfer matrix of the Pöschl-Teller barrier, are shown to equal the squared energies of the levels bound within the trigonometric Scarf well potential. By transforming these potentials into parts of the Laplacians describing free quantum motions on the mutually orthogonal open-time-like hyperbolic-, and closed-space-like spherical geodesics on the conformally invariant de Sitter space-time, dS4, the conformal symmetries of these interactions are revealed. On dS4 the potentials under consideration naturally relate to interactions within colorless two-body systems and to cusped Wilson loops. In effect, with the aid of the dS4 space-time as unifying geometry, a conformal symmetry based bijective correspondence (duality) between bound and resonant meson spectra is established at the quantum mechanics level and related to confinement understood as color charge neutrality. The correspondence allows to link the interpretation of mesons as resonance poles of a scattering matrix with their complementary description as states bound by an instantaneous quark interaction and to introduce a conformal symmetry based classification scheme of mesons. As examples representative of such a duality we organize in good agreement with data 71 of the reported light flavor mesons with masses below ˜ 2350 MeV into four conformal families of particles placed on linear f0, π , η , and a0 resonance trajectories, plotted on the ℓ/ M plane. Upon extending the sec2 χ by a properly constructed conformal color dipole potential, shaped after a tangent function, we predict the masses of 12 "missing" mesons. We furthermore notice that the f0 and π trajectories can be viewed as chiral partners, same as the η and a0 trajectories, an indication that chiral symmetry for mesons is likely to be realized in terms of parity doubled conformal multiplets rather than, as usually assumed, only in terms of parity doubled single SO(3) states. We attribute the striking measured meson degeneracies to conformal symmetry dynamics within color neutral two-body systems, and conclude on the usefulness of the de Sitter space-time, dS4, as a tool for modelling strong interactions, on the one side, and on the relevance of hyperbolic and trigonometric potentials in constituent quark models of hadrons, on the other.

Specialized Dynamical Properties of Promiscuous Residues Revealed by Simulated Conformational Ensembles

PubMed Central

2013-01-01

The ability to interact with different partners is one of the most important features in proteins. Proteins that bind a large number of partners (hubs) have been often associated with intrinsic disorder. However, many examples exist of hubs with an ordered structure, and evidence of a general mechanism promoting promiscuity in ordered proteins is still elusive. An intriguing hypothesis is that promiscuous binding sites have specific dynamical properties, distinct from the rest of the interface and pre-existing in the protein isolated state. Here, we present the first comprehensive study of the intrinsic dynamics of promiscuous residues in a large protein data set. Different computational methods, from coarse-grained elastic models to geometry-based sampling methods and to full-atom Molecular Dynamics simulations, were used to generate conformational ensembles for the isolated proteins. The flexibility and dynamic correlations of interface residues with a different degree of binding promiscuity were calculated and compared considering side chain and backbone motions, the latter both on a local and on a global scale. The study revealed that (a) promiscuous residues tend to be more flexible than nonpromiscuous ones, (b) this additional flexibility has a higher degree of organization, and (c) evolutionary conservation and binding promiscuity have opposite effects on intrinsic dynamics. Findings on simulated ensembles were also validated on ensembles of experimental structures extracted from the Protein Data Bank (PDB). Additionally, the low occurrence of single nucleotide polymorphisms observed for promiscuous residues indicated a tendency to preserve binding diversity at these positions. A case study on two ubiquitin-like proteins exemplifies how binding promiscuity in evolutionary related proteins can be modulated by the fine-tuning of the interface dynamics. The interplay between promiscuity and flexibility highlighted here can inspire new directions in protein–protein interaction prediction and design methods. PMID:24250278

How mobile are dye adsorbates and acetonitrile molecules on the surface of TiO2 nanoparticles? A quasi-elastic neutron scattering study

PubMed Central

Vaissier, Valerie; Sakai, Victoria Garcia; Li, Xiaoe; Cabral, João T.; Nelson, Jenny; Barnes, Piers R. F.

2016-01-01

Motions of molecules adsorbed to surfaces may control the rate of charge transport within monolayers in systems such as dye sensitized solar cells. We used quasi-elastic neutron scattering (QENS) to evaluate the possible dynamics of two small dye moieties, isonicotinic acid (INA) and bis-isonicotinic acid (BINA), attached to TiO2 nanoparticles via carboxylate groups. The scattering data indicate that moieties are immobile and do not rotate around the anchoring groups on timescales between around 10 ps and a few ns (corresponding to the instrumental range). This gives an upper limit for the rate at which conformational fluctuations can assist charge transport between anchored molecules. Our observations suggest that if the conformation of larger dye molecules varies with time, it does so on longer timescales and/or in parts of the molecule which are not directly connected to the anchoring group. The QENS measurements also indicate that several layers of acetonitrile solvent molecules are immobilized at the interface with the TiO2 on the measurement time scale, in reasonable agreement with recent classical molecular dynamics results. PMID:27991538

Furfural reduction mechanism of a zinc-dependent alcohol dehydrogenase from Cupriavidus necator JMP134

PubMed Central

Kang, ChulHee; Hayes, Robert; Sanchez, Emiliano J.; Webb, Brian N.; Li, Qunrui; Hooper, Travis; Nissen, Mark S.; Xun, Luying

2012-01-01

Summary FurX is a tetrameric Zn-dependent alcohol dehydrogenase (ADH) from Cupriavidus necator JMP134. The enzyme rapidly reduces furfural with NADH as the reducing power. For the first time among characterized ADHs, the high-resolution structures of all reaction steps were obtained in a time-resolved manner, thereby illustrating the complete catalytic events of NADH-dependent reduction of furfural and the dynamic Zn2+ coordination among Glu66, water, substrate and product. In the fully closed conformation of the NADH complex, the catalytic turnover proved faster than observed for the partially closed conformation due to an effective proton transfer network. The domain motion triggered by NAD(H) association/dissociation appeared to facilitate dynamic interchanges in Zn2+ coordination with substrate and product molecules, ultimately increasing the enzymatic turnover rate. NAD+ dissociation appeared to be a slow process, involving multiple steps in concert with a domain opening and reconfiguration of Glu66. This agrees with the report that the cofactor is not dissociated from FurX during ethanol-dependent reduction of furfural, in which ethanol reduces NAD+ to NADH that is subsequently used for furfural reduction. PMID:22081946

How mobile are dye adsorbates and acetonitrile molecules on the surface of TiO2 nanoparticles? A quasi-elastic neutron scattering study

NASA Astrophysics Data System (ADS)

Vaissier, Valerie; Sakai, Victoria Garcia; Li, Xiaoe; Cabral, João T.; Nelson, Jenny; Barnes, Piers R. F.

2016-12-01

Motions of molecules adsorbed to surfaces may control the rate of charge transport within monolayers in systems such as dye sensitized solar cells. We used quasi-elastic neutron scattering (QENS) to evaluate the possible dynamics of two small dye moieties, isonicotinic acid (INA) and bis-isonicotinic acid (BINA), attached to TiO2 nanoparticles via carboxylate groups. The scattering data indicate that moieties are immobile and do not rotate around the anchoring groups on timescales between around 10 ps and a few ns (corresponding to the instrumental range). This gives an upper limit for the rate at which conformational fluctuations can assist charge transport between anchored molecules. Our observations suggest that if the conformation of larger dye molecules varies with time, it does so on longer timescales and/or in parts of the molecule which are not directly connected to the anchoring group. The QENS measurements also indicate that several layers of acetonitrile solvent molecules are immobilized at the interface with the TiO2 on the measurement time scale, in reasonable agreement with recent classical molecular dynamics results.

Computational investigation of the human SOD1 mutant, Cys146Arg, that directs familial amyotrophic lateral sclerosis.

PubMed

Srinivasan, E; Rajasekaran, R

2017-07-25

The genetic substitution mutation of Cys146Arg in the SOD1 protein is predominantly found in the Japanese population suffering from familial amyotrophic lateral sclerosis (FALS). A complete study of the biophysical aspects of this particular missense mutation through conformational analysis and producing free energy landscapes could provide an insight into the pathogenic mechanism of ALS disease. In this study, we utilized general molecular dynamics simulations along with computational predictions to assess the structural characterization of the protein as well as the conformational preferences of monomeric wild type and mutant SOD1. Our static analysis, accomplished through multiple programs, predicted the deleterious and destabilizing effect of mutant SOD1. Subsequently, comparative molecular dynamic studies performed on the wild type and mutant SOD1 indicated a loss in the protein conformational stability and flexibility. We observed the mutational consequences not only in local but also in long-range variations in the structural properties of the SOD1 protein. Long-range intramolecular protein interactions decrease upon mutation, resulting in less compact structures in the mutant protein rather than in the wild type, suggesting that the mutant structures are less stable than the wild type SOD1. We also presented the free energy landscape to study the collective motion of protein conformations through principal component analysis for the wild type and mutant SOD1. Overall, the study assisted in revealing the cause of the structural destabilization and protein misfolding via structural characterization, secondary structure composition and free energy landscapes. Hence, the computational framework in our study provides a valuable direction for the search for the cure against fatal FALS.

Crankshaft motion in a highly congested bis(triarylmethyl)peroxide.

PubMed

Khuong, Tinh-Alfredo V; Zepeda, Gerardo; Sanrame, Carlos N; Dang, Hung; Bartberger, Michael D; Houk, K N; Garcia-Garibay, Miguel A

2004-11-17

Crankshaft motion has been proposed in the solid state for molecular fragments consisting of three or more rotors linked by single bonds, whereby the two terminal rotors are static and the internal rotors experience circular motion. Bis-[tri-(3,5-di-tert-butyl)phenylmethyl]-peroxide 2 was tested as a model in search of crankshaft motion at the molecular level. In the case of peroxide 2, the bulky trityl groups may be viewed as the external static rotors, while the two peroxide oxygens can undergo the sought after internal rotation. Evidence for this process in the case of peroxide 2 was obtained from conformational dynamics determined by variable-temperature (13)C and (1)H NMR between 190 and 375 K in toluene-d(8). Detailed spectral assignments for the interpretation of two coalescence processes were based on a correlation between NMR spectra obtained in solution at low temperature, in the solid state by (13)C CPMAS NMR, and by GIAO calculations based on a B3LYP/6-31G structure of 2 obtained from its X-ray coordinates as the input. Evidence supporting crankshaft rotation rather than slippage of the trityl groups was obtained from molecular mechanics calculations.

Slow Domain Motions of an Oligomeric Protein from Deep-Sea Hyperthermophile probed by Neutron Spin Echo

NASA Astrophysics Data System (ADS)

Bhowmik, Debsindhu; Shrestha, Utsab; Dhindsa, Gurpreet; Sharp, Melissa; Stingaciu, Laura R.; Chu, Xiang-Qiang; Xiang-Qiang Chu Team

Deep-sea microorganisms have the ability to survive under extreme conditions, such as high pressure and high temperature. In this work, we used the combination of the neutron spin-echo (NSE) and the small angle neutron scattering (SANS) techniques to study the inter-domain motions of the inorganic pyrophosphate (IPPase) enzyme derived from thermostable microorganisms Thermococcus thioreducens. The IPPase has hexameric quaternary structure with molecular mass of approx. 120kDa (each subunit of 20kDa), which is a large oligomeric structure. The understanding of its slow inter-domain motions can be the key to explain how they are able to perform catalytic activity at higher temperature compared to mesophilic enzymes, thus leading to adapt to extreme environment present at the seabed. The NSE can probe these slow motions directly in the time domain up to several tens of nanoseconds at the nanometers length scales, while the corresponding structural change can be explored by the SANS. Our results provide a better picture of the local flexibility and conformational substates unique to these types of proteins, which will help us better understandthe relation between protein dynamics and their biological activities

Structure of Simian Immunodeficiency Virus Envelope Spikes Bound with CD4 and Monoclonal Antibody 36D5.

PubMed

Hu, Guiqing; Liu, Jun; Roux, Kenneth H; Taylor, Kenneth A

2017-08-15

The human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) envelope spike (Env) mediates viral entry into host cells. The V3 loop of the gp120 component of the Env trimer contributes to the coreceptor binding site and is a target for neutralizing antibodies. We used cryo-electron tomography to visualize the binding of CD4 and the V3 loop monoclonal antibody (MAb) 36D5 to gp120 of the SIV Env trimer. Our results show that 36D5 binds gp120 at the base of the V3 loop and suggest that the antibody exerts its neutralization effect by blocking the coreceptor binding site. The antibody does this without altering the dynamics of the spike motion between closed and open states when CD4 is bound. The interaction between 36D5 and SIV gp120 is similar to the interaction between some broadly neutralizing anti-V3 loop antibodies and HIV-1 gp120. Two conformations of gp120 bound with CD4 are revealed, suggesting an intrinsic dynamic nature of the liganded Env trimer. CD4 binding substantially increases the binding of 36D5 to gp120 in the intact Env trimer, consistent with CD4-induced changes in the conformation of gp120 and the antibody binding site. Binding by MAb 36D5 does not substantially alter the proportions of the two CD4-bound conformations. The position of MAb 36D5 at the V3 base changes little between conformations, indicating that the V3 base serves as a pivot point during the transition between these two states. IMPORTANCE Glycoprotein spikes on the surfaces of SIV and HIV are the sole targets available to the immune system for antibody neutralization. Spikes evade the immune system by a combination of a thick layer of polysaccharide on the surface (the glycan shield) and movement between spike domains that masks the epitope conformation. Using SIV virions whose spikes were "decorated" with the primary cellular receptor (CD4) and an antibody (36D5) at part of the coreceptor binding site, we visualized multiple conformations trapped by the rapid freezing step, which were separated using statistical analysis. Our results show that the CD4-induced conformational dynamics of the spike enhances binding of the antibody. Copyright © 2017 American Society for Microbiology.

A surprising role for conformational entropy in protein function

PubMed Central

Wand, A. Joshua; Moorman, Veronica R.; Harpole, Kyle W.

2014-01-01

Formation of high-affinity complexes is critical for the majority of enzymatic reactions involving proteins. The creation of the family of Michaelis and other intermediate complexes during catalysis clearly involves a complicated manifold of interactions that are diverse and complex. Indeed, computing the energetics of interactions between proteins and small molecule ligands using molecular structure alone remains a grand challenge. One of the most difficult contributions to the free energy of protein-ligand complexes to experimentally access is that due to changes in protein conformational entropy. Fortunately, recent advances in solution nuclear magnetic resonance (NMR) relaxation methods have enabled the use of measures-of-motion between conformational states of a protein as a proxy for conformational entropy. This review briefly summarizes the experimental approaches currently employed to characterize fast internal motion in proteins, how this information is used to gain insight into conformational entropy, what has been learned and what the future may hold for this emerging view of protein function. PMID:23478875

The Role of Large-Scale Motions in Catalysis by Dihydrofolate Reductase

PubMed Central

2011-01-01

Dihydrofolate reductase has long been used as a model system to study the coupling of protein motions to enzymatic hydride transfer. By studying environmental effects on hydride transfer in dihydrofolate reductase (DHFR) from the cold-adapted bacterium Moritella profunda (MpDHFR) and comparing the flexibility of this enzyme to that of DHFR from Escherichia coli (EcDHFR), we demonstrate that factors that affect large-scale (i.e., long-range, but not necessarily large amplitude) protein motions have no effect on the kinetic isotope effect on hydride transfer or its temperature dependence, although the rates of the catalyzed reaction are affected. Hydrogen/deuterium exchange studies by NMR-spectroscopy show that MpDHFR is a more flexible enzyme than EcDHFR. NMR experiments with EcDHFR in the presence of cosolvents suggest differences in the conformational ensemble of the enzyme. The fact that enzymes from different environmental niches and with different flexibilities display the same behavior of the kinetic isotope effect on hydride transfer strongly suggests that, while protein motions are important to generate the reaction ready conformation, an optimal conformation with the correct electrostatics and geometry for the reaction to occur, they do not influence the nature of the chemical step itself; large-scale motions do not couple directly to hydride transfer proper in DHFR. PMID:22060818

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wurzburg, Beth A; Jardetzky, Theodore S; Stanford)

The structure of immunoglobulin E (IgE)-Fc 3-4 has been solved in three new crystal forms, providing 13 snapshots of the Fc conformation and revealing a diverse range of open-closed motions among subunit chains and dimers. A more detailed analysis of the open-to-closed motion of IgE-Fc 3-4 was possible with so many structures, and the new structures allow a more thorough examination of the flexibility of IgE-Fc and its implications for receptor binding. The existence of a hydrophobic pocket at the elbow region of the Fc appears to be conformation dependent and suggests a means of regulating the IgE-Fc conformation (andmore » potentially receptor binding) with small molecules.« less

Coupling of Lever Arm Swing and Biased Brownian Motion in Actomyosin

PubMed Central

Nie, Qing-Miao; Togashi, Akio; Sasaki, Takeshi N.; Takano, Mitsunori; Sasai, Masaki; Terada, Tomoki P.

2014-01-01

An important unresolved problem associated with actomyosin motors is the role of Brownian motion in the process of force generation. On the basis of structural observations of myosins and actins, the widely held lever-arm hypothesis has been proposed, in which proteins are assumed to show sequential structural changes among observed and hypothesized structures to exert mechanical force. An alternative hypothesis, the Brownian motion hypothesis, has been supported by single-molecule experiments and emphasizes more on the roles of fluctuating protein movement. In this study, we address the long-standing controversy between the lever-arm hypothesis and the Brownian motion hypothesis through in silico observations of an actomyosin system. We study a system composed of myosin II and actin filament by calculating free-energy landscapes of actin-myosin interactions using the molecular dynamics method and by simulating transitions among dynamically changing free-energy landscapes using the Monte Carlo method. The results obtained by this combined multi-scale calculation show that myosin with inorganic phosphate (Pi) and ADP weakly binds to actin and that after releasing Pi and ADP, myosin moves along the actin filament toward the strong-binding site by exhibiting the biased Brownian motion, a behavior consistent with the observed single-molecular behavior of myosin. Conformational flexibility of loops at the actin-interface of myosin and the N-terminus of actin subunit is necessary for the distinct bias in the Brownian motion. Both the 5.5–11 nm displacement due to the biased Brownian motion and the 3–5 nm displacement due to lever-arm swing contribute to the net displacement of myosin. The calculated results further suggest that the recovery stroke of the lever arm plays an important role in enhancing the displacement of myosin through multiple cycles of ATP hydrolysis, suggesting a unified movement mechanism for various members of the myosin family. PMID:24762409

Coupling of lever arm swing and biased Brownian motion in actomyosin.

PubMed

Nie, Qing-Miao; Togashi, Akio; Sasaki, Takeshi N; Takano, Mitsunori; Sasai, Masaki; Terada, Tomoki P

2014-04-01

An important unresolved problem associated with actomyosin motors is the role of Brownian motion in the process of force generation. On the basis of structural observations of myosins and actins, the widely held lever-arm hypothesis has been proposed, in which proteins are assumed to show sequential structural changes among observed and hypothesized structures to exert mechanical force. An alternative hypothesis, the Brownian motion hypothesis, has been supported by single-molecule experiments and emphasizes more on the roles of fluctuating protein movement. In this study, we address the long-standing controversy between the lever-arm hypothesis and the Brownian motion hypothesis through in silico observations of an actomyosin system. We study a system composed of myosin II and actin filament by calculating free-energy landscapes of actin-myosin interactions using the molecular dynamics method and by simulating transitions among dynamically changing free-energy landscapes using the Monte Carlo method. The results obtained by this combined multi-scale calculation show that myosin with inorganic phosphate (Pi) and ADP weakly binds to actin and that after releasing Pi and ADP, myosin moves along the actin filament toward the strong-binding site by exhibiting the biased Brownian motion, a behavior consistent with the observed single-molecular behavior of myosin. Conformational flexibility of loops at the actin-interface of myosin and the N-terminus of actin subunit is necessary for the distinct bias in the Brownian motion. Both the 5.5-11 nm displacement due to the biased Brownian motion and the 3-5 nm displacement due to lever-arm swing contribute to the net displacement of myosin. The calculated results further suggest that the recovery stroke of the lever arm plays an important role in enhancing the displacement of myosin through multiple cycles of ATP hydrolysis, suggesting a unified movement mechanism for various members of the myosin family.

Can an Endplate-conformed Cervical Cage Provide a Better Biomechanical Environment than a Typical Non-conformed Cage?: A Finite Element Model and Cadaver Study.

PubMed

Zhang, Fan; Xu, Hao-Cheng; Yin, Bo; Xia, Xin-Lei; Ma, Xiao-Sheng; Wang, Hong-Li; Yin, Jun; Shao, Ming-Hao; Lyu, Fei-Zhou; Jiang, Jian-Yuan

2016-08-01

To evaluate the biomechanical characteristics of endplate-conformed cervical cages by finite element method (FEM) analysis and cadaver study. Twelve specimens (C2 -C7 ) and a finite element model (C3 -C7 ) were subjected to biomechanical evaluations. In the cadaver study, specimens were randomly assigned to intact (I), endplate-conformed (C) and non-conformed (N) groups with C4-5 discs as the treated segments. The morphologies of the endplate-conformed cages were individualized according to CT images of group C and the cages fabricated with a 3-D printer. The non-conformed cages were wedge-shaped and similar to commercially available grafts. Axial pre-compression loads of 73.6 N and moment of 1.8 Nm were used to simulate flexion (FLE), extension (EXT), lateral bending (LB) and axial rotation (AR). Range of motion (ROM) at C4-5 of each specimen was recorded and film sensors fixed between the cages and C5 superior endplates were used to detect interface stress. A finite element model was built based on the CT data of a healthy male volunteer. The morphologies of the endplate-conformed and wedge-shaped, non-conformed cervical cages were both simulated by a reverse engineering technique and implanted at the segment of C4-5 in the finite element model for biomechanical evaluation. Force loading and grouping were similar to those applied in the cadaver study. ROM of C4-5 in group I were recorded to validate the finite element model. Additionally, maximum cage-endplate interface stresses, stress distribution contours on adjoining endplates, intra-disc stresses and facet loadings at adjacent segments were measured and compared between groups. In the cadaver study, Group C showed a much lower interface stress in all directions of motion (all P < 0.05) and the ROM of C4-5 was smaller in FLE-EXT (P = 0.001) but larger in AR (P = 0.017). FEM analysis produced similar results: the model implanted with an endplate-conformed cage presented a lower interface stress with a more uniform stress distribution than that implanted with a non-conformed cage. Additionally, intra-disc stress and facet loading at the adjacent segments were obviously increased in both groups C and N, especially those at the supra-jacent segments. However, stress increase was milder in group C than in group N for all directions of motion. Endplate-conformed cages can decrease cage-endplate interface stress in all directions of motion and increase cervical stability in FLE-EXT. Additionally, adjacent segments are possibly protected because intra-disc stress and facet loading are smaller after endplate-conformed cage implantation. However, axial stability was reduced in group C, indicating that endplate-conformed cage should not be used alone and an anterior plate system is still important in anterior cervical discectomy and fusion. © 2016 Chinese Orthopaedic Association and John Wiley & Sons Australia, Ltd.

Modeling the effect of pathogenic mutations on the conformational landscape of protein kinases.

PubMed

Saladino, Giorgio; Gervasio, Francesco Luigi

2016-04-01

Most proteins assume different conformations to perform their cellular functions. This conformational dynamics is physiologically regulated by binding events and post-translational modifications, but can also be affected by pathogenic mutations. Atomistic molecular dynamics simulations complemented by enhanced sampling approaches are increasingly used to probe the effect of mutations on the conformational dynamics and on the underlying conformational free energy landscape of proteins. In this short review we discuss recent successful examples of simulations used to understand the molecular mechanism underlying the deregulation of physiological conformational dynamics due to non-synonymous single point mutations. Our examples are mostly drawn from the protein kinase family. Copyright © 2016 Elsevier Ltd. All rights reserved.

On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein.

PubMed

Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo; Roitberg, Adrian E; Fernandez-Alberti, Sebastian

2015-06-28

The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data.

On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein

NASA Astrophysics Data System (ADS)

Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo; Roitberg, Adrian E.; Fernandez-Alberti, Sebastian

2015-06-01

The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data.

Molecular dynamics and docking simulations as a proof of high flexibility in E. coli FabH and its relevance for accurate inhibitor modeling

NASA Astrophysics Data System (ADS)

Pérez-Castillo, Yunierkis; Froeyen, Matheus; Cabrera-Pérez, Miguel Ángel; Nowé, Ann

2011-04-01

Bacterial β-ketoacyl-acyl carrier protein synthase III (FabH) has become an attractive target for the development of new antibacterial agents which can overcome the increased resistance of these pathogens to antibiotics in clinical use. Despite several efforts have been dedicated to find inhibitors for this enzyme, it is not a straightforward task, mainly due its high flexibility which makes difficult the structure-based design of FabH inhibitors. Here, we present for the first time a Molecular Dynamics (MD) study of the E. colil FabH enzyme to explore its conformational space. We compare the flexibility of this enzyme for the unliganded protein and an enzyme-inhibitor complex and find a correspondence between our modeling results and the experimental evidence previously reported for this enzyme. Furthermore, through a 100 ns MD simulation of the unliganded enzyme we extract useful information related to the concerted motions that take place along the principal components of displacement. We also establish a relation between the presence of water molecules in the oxyanion hole with the conformational stability of structural important loops. Representative conformations of the binding pocket along the whole trajectory of the unliganded protein are selected through cluster analysis and we find that they contain a conformational diversity which is not provided by the X-ray structures of ecFabH. As a proof of this last hypothesis, we use a set of 10 FabH inhibitors and show that they cannot be correctly modeled in any available X-ray structure, while by using our set of conformations extracted from the MD simulations, this task can be accomplish. Finally, we show the ability of short MD simulations for the refinement of the docking binding poses and for MM-PBSA calculations to predict stable protein-inhibitor complexes in this enzyme.

Free-Energy Landscape of Reverse tRNA Translocation through the Ribosome Analyzed by Electron Microscopy Density Maps and Molecular Dynamics Simulations

PubMed Central

Ishida, Hisashi; Matsumoto, Atsushi

2014-01-01

To understand the mechanism of reverse tRNA translocation in the ribosome, all-atom molecular dynamics simulations of the ribosome-tRNAs-mRNA-EFG complex were performed. The complex at the post-translocational state was directed towards the translocational and pre-translocational states by fitting the complex into cryo-EM density maps. Between a series of the fitting simulations, umbrella sampling simulations were performed to obtain the free-energy landscape. Multistep structural changes, such as a ratchet-like motion and rotation of the head of the small subunit were observed. The free-energy landscape showed that there were two main free-energy barriers: one between the post-translocational and intermediate states, and the other between the pre-translocational and intermediate states. The former corresponded to a clockwise rotation, which was coupled to the movement of P-tRNA over the P/E-gate made of G1338, A1339 and A790 in the small subunit. The latter corresponded to an anticlockwise rotation of the head, which was coupled to the location of the two tRNAs in the hybrid state. This indicates that the coupled motion of the head rotation and tRNA translocation plays an important role in opening and closing of the P/E-gate during the ratchet-like movement in the ribosome. Conformational change of EF-G was interpreted to be the result of the combination of the external motion by L12 around an axis passing near the sarcin-ricin loop, and internal hinge-bending motion. These motions contributed to the movement of domain IV of EF-G to maintain its interaction with A/P-tRNA. PMID:24999999

Free-energy landscape of reverse tRNA translocation through the ribosome analyzed by electron microscopy density maps and molecular dynamics simulations.

PubMed

Ishida, Hisashi; Matsumoto, Atsushi

2014-01-01

To understand the mechanism of reverse tRNA translocation in the ribosome, all-atom molecular dynamics simulations of the ribosome-tRNAs-mRNA-EFG complex were performed. The complex at the post-translocational state was directed towards the translocational and pre-translocational states by fitting the complex into cryo-EM density maps. Between a series of the fitting simulations, umbrella sampling simulations were performed to obtain the free-energy landscape. Multistep structural changes, such as a ratchet-like motion and rotation of the head of the small subunit were observed. The free-energy landscape showed that there were two main free-energy barriers: one between the post-translocational and intermediate states, and the other between the pre-translocational and intermediate states. The former corresponded to a clockwise rotation, which was coupled to the movement of P-tRNA over the P/E-gate made of G1338, A1339 and A790 in the small subunit. The latter corresponded to an anticlockwise rotation of the head, which was coupled to the location of the two tRNAs in the hybrid state. This indicates that the coupled motion of the head rotation and tRNA translocation plays an important role in opening and closing of the P/E-gate during the ratchet-like movement in the ribosome. Conformational change of EF-G was interpreted to be the result of the combination of the external motion by L12 around an axis passing near the sarcin-ricin loop, and internal hinge-bending motion. These motions contributed to the movement of domain IV of EF-G to maintain its interaction with A/P-tRNA.

Dynamic Stabilization of Expressed Proteins in Engineered Diatom Biosilica Matrices.

PubMed

Xiong, Yijia; Ford, Nicole R; Hecht, Karen A; Roesijadi, Guritno; Squier, Thomas C

2016-05-18

Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that will enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39-amino-acid targeting sequence (Sil3T8) that directs a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundance of >200 000 proteins per frustule. Using either a fluorescent ligand bound to the scFv or the intrinsic fluorescence of EGFP, we monitored protein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. Like proteins in solution, proteins within isolated frustules undergo isotropic rotational motion, but with 2-fold increases in rotational correlation times that are indicative of weak macromolecular associations within the biosilica. Solvent accessibilities and high-affinity (pM) binding are comparable to those in solution. In contrast to solution conditions, scFv antibodies within the biosilica matrix retain their binding affinity in the presence of chaotropic agents (i.e., 8 M urea). Together, these results argue that dramatic increases in protein conformational stability within the biosilica matrices arise through molecular crowding, acting to retain native protein folds and associated functionality with the potential to allow the utility of engineered proteins under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations.

Autoinhibitory mechanisms of ERG studied by molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Lu, Yan; Salsbury, Freddie R.

2015-01-01

ERG, an ETS-family transcription factor, acts as a regulator of differentiation of early hematopoietic cells. It contains an autoinhibitory domain, which negatively regulates DNA-binding. The mechanism of autoinhibitory is still illusive. To understand the mechanism, we study the dynamical properties of ERG protein by molecular dynamics simulations. These simulations suggest that DNA binding autoinhibition associates with the internal dynamics of ERG. Specifically, we find that (1), The N-C terminal correlation in the inhibited ERG is larger than that in uninhibited ERG that contributes to the autoinhibition of DNA-binding. (2), DNA-binding changes the property of the N-C terminal correlation from being anti-correlated to correlated, that is, changing the relative direction of the correlated motions and (3), For the Ets-domain specifically, the inhibited and uninhibited forms exhibit essentially the same dynamics, but the binding of the DNA decreases the fluctuation of the Ets-domain. We also find from PCA analysis that the three systems, even with quite different dynamics, do have highly similar free energy surfaces, indicating that they share similar conformations.

Nuclear Resonance Vibrational Spectra of Five-Coordinate Imidazole-ligated Iron(II) Porphyrinates

PubMed Central

Hu, Chuanjiang; Barabanschikov, Alexander; Ellison, Mary K.; Zhao, Jiyong; Alp, E. Ercan; Sturhahn, Wolfgang; Zgierski, Marek Z.; Sage, J. Timothy; Scheidt, W. Robert

2012-01-01

Nuclear resonance vibrational spectra have been obtained for six five-coordinate imidazole-ligated iron(II) porphyrinates, [Fe(Por)(L)] (Por = tetraphenylporphyrinate, octaethylporphyrinate, tetratolylporphyrinate or protoporphyrinate IX and L = 2-methylimidazole or 1,2-dimethylimidazole). Measurements have been made on both powder and oriented crystal samples. The spectra are dominated by strong signals around 200–300 cm−1. Although the in-plane and out-of-plane vibrations are seriously overlapped, oriented crystal spectra allow their deconvolution. Thus, oriented crystal experimental data, along with DFT calculations, enable the assignment of key vibrations in the spectra. Molecular dynamics are also discussed. The nature of the Fe–NIm vibrations has been elaborated further than was possible from resonance Raman studies. Our study suggests that the Fe motions are coupled with the porphyrin core and peripheral groups motions. Both peripheral groups and their conformations have significant influence on the vibrational spectra (position and shape). PMID:22243131

High-Dimensional Mutant and Modular Thermodynamic Cycles, Molecular Switching, and Free Energy Transduction

PubMed Central

Carter, Charles W.

2017-01-01

Understanding how distinct parts of proteins produce coordinated behavior has driven and continues to drive advances in protein science and enzymology. However, despite consensus about the conceptual basis for allostery, the idiosyncratic nature of allosteric mechanisms resists general approaches. Computational methods can identify conformational transition states from structural changes, revealing common switching mechanisms that impose multistate behavior. Thermodynamic cycles use factorial perturbations to measure coupling energies between side chains in molecular switches that mediate shear during domain motion. Such cycles have now been complemented by modular cycles that measure energetic coupling between separable domains. For one model system, energetic coupling between domains has been shown to be quantitatively equivalent to that between dynamic side chains. Linkages between domain motion, switching residues, and catalysis make nucleoside triphosphate hydrolysis conditional on domain movement, confirming an essential yet neglected aspect of free energy transduction and suggesting the potential generality of these studies. PMID:28375734

New Insights into Color Confinement, Hadron Dynamics, Spectroscopy, and Jet Hadronization from Light-Front Holography and Superconformal Algebra

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brodsky, S. J.

A fundamental problem in hadron physics is to obtain a relativistic color-confining, first approximation to QCD which can predict both hadron spectroscopy and the frame-independent light-front (LF) wavefunctions underlying hadron dynamics. The QCD Lagrangian with zero quark mass has no explicit mass scale; the classical theory is conformally invariant. Thus, a fundamental problem is to understand how the mass gap and ratios of masses – such as mρ/mp – can arise in chiral QCD. De Alfaro, Fubini, and Furlan have made an important observation that a mass scale can appear in the equations of motion without affecting the conformal invariance of the action if one adds a term to the Hamiltonian proportional to the dilatation operator or the special conformal operator and rescales the time variable. If one applies the same procedure to the light-front Hamiltonian, it leads uniquely to a confinement potential κ 4ζ 2 for mesons, where ζ 2 is the LF radial variable conjugate to themore » $$q\\bar{q}$$ invariant mass squared. The same result, including spin terms, is obtained using light-front holography – the duality between light-front dynamics and AdS 5, the space of isometries of the conformal group if one modifies the action of AdS 5 by the dilaton e $κ^2$ z$^2$ in the fifth dimension z . When one generalizes this procedure using superconformal algebra, the resulting light-front eigensolutions predict unified Regge spectroscopy of meson, baryon, and tetraquarks, including remarkable supersymmetric relations between the masses of mesons and baryons of the same parity. One also predicts observables such as hadron structure functions, transverse momentum distributions, and the distribution amplitudes defined from the hadronic light-front wavefunctions. The mass scale κ underlying confinement and hadron masses can be connected to the parameter Λ $$\\overline{MS}$$ in the QCD running coupling by matching the nonperturbative dynamics to the perturbative QCD regime. The result is an effective coupling α s(Q 2) defined at all momenta. Lastly, the matching of the high and low momentum transfer regimes also determines a scale Q 0 which sets the interface between perturbative and nonperturbative hadron dynamics.« less

New Insights into Color Confinement, Hadron Dynamics, Spectroscopy, and Jet Hadronization from Light-Front Holography and Superconformal Algebra

DOE PAGES

Brodsky, S. J.

2017-07-11

A fundamental problem in hadron physics is to obtain a relativistic color-confining, first approximation to QCD which can predict both hadron spectroscopy and the frame-independent light-front (LF) wavefunctions underlying hadron dynamics. The QCD Lagrangian with zero quark mass has no explicit mass scale; the classical theory is conformally invariant. Thus, a fundamental problem is to understand how the mass gap and ratios of masses – such as mρ/mp – can arise in chiral QCD. De Alfaro, Fubini, and Furlan have made an important observation that a mass scale can appear in the equations of motion without affecting the conformal invariance of the action if one adds a term to the Hamiltonian proportional to the dilatation operator or the special conformal operator and rescales the time variable. If one applies the same procedure to the light-front Hamiltonian, it leads uniquely to a confinement potential κ 4ζ 2 for mesons, where ζ 2 is the LF radial variable conjugate to themore » $$q\\bar{q}$$ invariant mass squared. The same result, including spin terms, is obtained using light-front holography – the duality between light-front dynamics and AdS 5, the space of isometries of the conformal group if one modifies the action of AdS 5 by the dilaton e $κ^2$ z$^2$ in the fifth dimension z . When one generalizes this procedure using superconformal algebra, the resulting light-front eigensolutions predict unified Regge spectroscopy of meson, baryon, and tetraquarks, including remarkable supersymmetric relations between the masses of mesons and baryons of the same parity. One also predicts observables such as hadron structure functions, transverse momentum distributions, and the distribution amplitudes defined from the hadronic light-front wavefunctions. The mass scale κ underlying confinement and hadron masses can be connected to the parameter Λ $$\\overline{MS}$$ in the QCD running coupling by matching the nonperturbative dynamics to the perturbative QCD regime. The result is an effective coupling α s(Q 2) defined at all momenta. Lastly, the matching of the high and low momentum transfer regimes also determines a scale Q 0 which sets the interface between perturbative and nonperturbative hadron dynamics.« less

Energetic and flexibility properties captured by long molecular dynamics simulations of a membrane-embedded pMHCII-TCR complex.

PubMed

Bello, Martiniano; Correa-Basurto, José

2016-04-01

Although crystallographic data have provided important molecular insight into the interactions in the pMHC-TCR complex, the inherent features of this structural approach cause it to only provide a static picture of the interactions. While unbiased molecular dynamics simulations (UMDSs) have provided important information about the dynamic structural behavior of the pMHC-TCR complex, most of them have modeled the pMHC-TCR complex as soluble, when in physiological conditions, this complex is membrane bound; therefore, following this latter UMDS protocol might hamper important dynamic results. In this contribution, we performed three independent 300 ns-long UMDSs of the pMHCII-TCR complex anchored in two opposing membranes to explore the structural and energetic properties of the recognition of pMHCII by the TCR. The conformational ensemble generated through UMDSs was subjected to clustering and Cartesian principal component analyses (cPCA) to explore the dynamical behavior of the pMHCII-TCR association. Furthermore, based on the conformational population sampled through UMDSs, the effective binding free energy, per-residue free energy decomposition, and alanine scanning mutations were explored for the native pMHCII-TCR complex, as well as for 12 mutations (p1-p12MHCII-TCR) introduced in the native peptide. Clustering analyses and cPCA provide insight into the rocking motion of the TCR onto pMHCII, together with the presence of new electrostatic interactions not observed through crystallographic methods. Energetic results provide evidence of the main contributors to the pMHC-TCR complex formation as well as the key residues involved in this molecular recognition process.

Loss of G-A base pairs is insufficient for achieving a large opening of U4 snRNA K-turn motif.

PubMed

Cojocaru, Vlad; Klement, Reinhard; Jovin, Thomas M

2005-01-01

Upon binding to the 15.5K protein, two tandem-sheared G-A base pairs are formed in the internal loop of the kink-turn motif of U4 snRNA (Kt-U4). We have reported that the folding of Kt-U4 is assisted by protein binding. Unstable interactions that contribute to a large opening of the free RNA ('k-e motion') were identified using locally enhanced sampling molecular dynamics simulations, results that agree with experiments. A detailed analysis of the simulations reveals that the k-e motion in Kt-U4 is triggered both by loss of G-A base pairs in the internal loop and backbone flexibility in the stems. Essential dynamics show that the loss of G-A base pairs is correlated along the first mode but anti-correlated along the third mode with the k-e motion. Moreover, when enhanced sampling was confined to the internal loop, the RNA adopted an alternative conformation characterized by a sharper kink, opening of G-A base pairs and modified stacking interactions. Thus, loss of G-A base pairs is insufficient for achieving a large opening of the free RNA. These findings, supported by previously published RNA structure probing experiments, suggest that G-A base pair formation occurs upon protein binding, thereby stabilizing a selective orientation of the stems.

Probing α-relaxation with nuclear magnetic resonance echo decay and relaxation: a study on nitrile butadiene rubber.

PubMed

Sturniolo, Simone; Pieruccini, Marco; Corti, Maurizio; Rigamonti, Attilio

2013-01-01

One dimensional (1)H NMR measurements have been performed to probe slow molecular motions in nitrile butadiene rubber (NBR) around its calorimetric glass transition temperature Tg. The purpose is to show how software aided data analysis can extract meaningful dynamical data from these measurements. Spin-lattice relaxation time, free induction decay (FID) and magic sandwich echo (MSE) measurements have been carried out at different values of the static field, as a function of temperature. It has been evidenced how the efficiency of the MSE signal in reconstructing the original FID exhibits a sudden minimum at a given temperature, with a slight dependence from the measuring frequency. Computer simulations performed with the software SPINEVOLUTION have shown that the minimum in the efficiency reconstruction of the MSE signal corresponds to the average motional frequency taking a value around the inter-proton coupling. The FID signals have been fitted with a truncated form of a newly derived exact correlation function for the transverse magnetization of a dipolar interacting spin pair, which allows one to avoid the restriction of the stationary and Gaussian approximations. A direct estimate of the conformational dynamics on approaching the Tg is obtained, and the results are in agreement with the analysis performed via the MSE reconstruction efficiency. The occurrence of a wide distribution of correlation frequencies for the chains motion, with a Vogel-Fulcher type temperature dependence, is addressed. A route for a fruitful study of the dynamics accompanying the glass transition by a variety of NMR measurements is thus proposed. Copyright © 2013 Elsevier Inc. All rights reserved.

A Wrench in the Works of Human Acetylcholinesterase: Soman Induced Conformational Changes Revealed by Molecular Dynamics Simulations

PubMed Central

Fattebert, Jean-Luc; Emigh, Aiyana

2015-01-01

Irreversible inactivation of human acetylcholinesterase (hAChE) by organophosphorous pesticides (OPs) and chemical weapon agents (CWA) has severe morbidity and mortality consequences. We present data from quantum mechanics/molecular mechanics (QM/MM) and 80 classical molecular dynamics (MD) simulations of the apo and soman-adducted forms of hAChE to investigate the effects on the dynamics and protein structure when the catalytic Serine 203 is phosphonylated. We find that the soman phosphonylation of the active site Ser203 follows a water assisted addition-elimination mechanism with the elimination of the fluoride ion being the highest energy barrier at 6.5 kcal/mole. We observe soman-dependent changes in backbone and sidechain motions compared to the apo form of the protein. These alterations restrict the soman-adducted hAChE to a structural state that is primed for the soman adduct to be cleaved and removed from the active site. The altered motions and resulting structures provide alternative pathways into and out of the hAChE active site. In the soman-adducted protein both side and back door pathways are viable for soman adduct access. Correlation analysis of the apo and soman adducted MD trajectories shows that the correlation of gorge entrance and back door motion is disrupted when hAChE is adducted. This supports the hypothesis that substrate and product can use two different pathways as entry and exit sites in the apo form of the protein. These alternative pathways have important implications for the rational design of medical countermeasures. PMID:25874456

A wrench in the works of human acetylcholinesterase: Soman induced conformational changes revealed by molecular dynamics simulations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bennion, Brian J.; Essiz, Sebnem G.; Lau, Edmond Y.

Irreversible inactivation of human acetylcholinesterase (hAChE) by organophosphorous pesticides (OPs) and chemical weapon agents (CWA) has severe morbidity and mortality consequences. We present data from quantum mechanics/molecular mechanics (QM/MM) and 80 classical molecular dynamics (MD) simulations of the apo and soman-adducted forms of hAChE to investigate the effects on the dynamics and protein structure when the catalytic Serine 203 is phosphonylated. We find that the soman phosphonylation of the active site Ser203 follows a water assisted addition-elimination mechanism with the elimination of the fluoride ion being the highest energy barrier at 6.5 kcal/mole. We observe soman-dependent changes in backbone andmore » sidechain motions compared to the apo form of the protein. These alterations restrict the soman-adducted hAChE to a structural state that is primed for the soman adduct to be cleaved and removed from the active site. The altered motions and resulting structures provide alternative pathways into and out of the hAChE active site. In the soman-adducted protein both side and back door pathways are viable for soman adduct access. Correlation analysis of the apo and soman adducted MD trajectories shows that the correlation of gorge entrance and back door motion is disrupted when hAChE is adducted. This supports the hypothesis that substrate and product can use two different pathways as entry and exit sites in the apo form of the protein. These alternative pathways have important implications for the rational design of medical countermeasures.« less

A wrench in the works of human acetylcholinesterase: Soman induced conformational changes revealed by molecular dynamics simulations

DOE PAGES

Bennion, Brian J.; Essiz, Sebnem G.; Lau, Edmond Y.; ...

2015-04-13

Irreversible inactivation of human acetylcholinesterase (hAChE) by organophosphorous pesticides (OPs) and chemical weapon agents (CWA) has severe morbidity and mortality consequences. We present data from quantum mechanics/molecular mechanics (QM/MM) and 80 classical molecular dynamics (MD) simulations of the apo and soman-adducted forms of hAChE to investigate the effects on the dynamics and protein structure when the catalytic Serine 203 is phosphonylated. We find that the soman phosphonylation of the active site Ser203 follows a water assisted addition-elimination mechanism with the elimination of the fluoride ion being the highest energy barrier at 6.5 kcal/mole. We observe soman-dependent changes in backbone andmore » sidechain motions compared to the apo form of the protein. These alterations restrict the soman-adducted hAChE to a structural state that is primed for the soman adduct to be cleaved and removed from the active site. The altered motions and resulting structures provide alternative pathways into and out of the hAChE active site. In the soman-adducted protein both side and back door pathways are viable for soman adduct access. Correlation analysis of the apo and soman adducted MD trajectories shows that the correlation of gorge entrance and back door motion is disrupted when hAChE is adducted. This supports the hypothesis that substrate and product can use two different pathways as entry and exit sites in the apo form of the protein. These alternative pathways have important implications for the rational design of medical countermeasures.« less

Conformational Entropy from Slowly Relaxing Local Structure Analysis of 15N-H Relaxation in Proteins: Application to Pheromone Binding to MUP-I in the 283-308 K Temperature Range.

PubMed

Žídek, Lukáš; Meirovitch, Eva

2017-09-21

The slowly relaxing local structure (SRLS) approach is applied to 15 N-H relaxation from the major urinary protein I (MUP-I), and its complex with pheromone 2-sec-butyl-4,5-dihydrothiazol. The objective is to elucidate dynamics, and binding-induced changes in conformational entropy. Experimental data acquired previously in the 283-308 K temperature range are used. The N-H bond is found to reorient globally with correlation time, τ 1,0 , and locally with correlation time, τ 2,0 , where τ 1,0 ≫ τ 2,0 . The local motion is restricted by the potential u = -c 0 2 D 00 2 , where D 00 2 is the Wigner rotation matrix element for L = 2, K = 0, and c 0 2 evaluates the strength of the potential. u yields straightforwardly the order parameter, ⟨D 00 2 ⟩, and the conformational entropy, S k , both given by P eq = exp(-u). The deviation of the local ordering/local diffusion axis from the N-H bond, given by the angle β, is also determined. We find that c 0 2 ≅ 18 ± 4 and τ 2,0 = 0-170 ps for ligand-free MUP-I, whereas c 0 2 ≅ 15 ± 4 and τ 2,0 = 20-270 ps for ligand-bound MUP-I. β is in the 0-10° range. c 0 2 and τ 2,0 decrease, whereas β increases, when the temperature is increased from 283 to 308 K. Thus, SRLS provides physically well-defined structure-related (c 0 2 and ⟨D 00 2 ⟩), motion-related (τ 2,0 ), geometry-related (β), and binding-related (S k ) local parameters, and their temperature-dependences. Intriguingly, upon pheromone binding the conformational entropy of MUP-I decreases at high temperature and increases at low temperature. The very same experimental data were analyzed previously with the model-free (MF) method which yielded "global" (in this context, "relating to the entire 283-308 K range") amplitude (S 2 ) and rate (τ e ) of the local motion, and a phenomenological exchange term (R ex ). S 2 is found to decrease (implying implicitly "global" increase in S k ) upon pheromone binding.

Active Site Conformational Dynamics in Human Uridine Phosphorylase 1

PubMed Central

Roosild, Tarmo P.; Castronovo, Samantha

2010-01-01

Uridine phosphorylase (UPP) is a central enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Human UPP activity has been a focus of cancer research due to its role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil (5-FU) and capecitabine. Additionally, specific molecular inhibitors of this enzyme have been found to raise endogenous uridine concentrations, which can produce a cytoprotective effect on normal tissues exposed to these drugs. Here we report the structure of hUPP1 bound to 5-FU at 2.3 Å resolution. Analysis of this structure reveals new insights as to the conformational motions the enzyme undergoes in the course of substrate binding and catalysis. The dimeric enzyme is capable of a large hinge motion between its two domains, facilitating ligand exchange and explaining observed cooperativity between the two active sites in binding phosphate-bearing substrates. Further, a loop toward the back end of the uracil binding pocket is shown to flexibly adjust to the varying chemistry of different compounds through an “induced-fit” association mechanism that was not observed in earlier hUPP1 structures. The details surrounding these dynamic aspects of hUPP1 structure and function provide unexplored avenues to develop novel inhibitors of this protein with improved specificity and increased affinity. Given the recent emergence of new roles for uridine as a neuron protective compound in ischemia and degenerative diseases, such as Alzheimer's and Parkinson's, inhibitors of hUPP1 with greater efficacy, which are able to boost cellular uridine levels without adverse side-effects, may have a wide range of therapeutic applications. PMID:20856879

Flexibility at a glycosidic linkage revealed by molecular dynamics, stochastic modeling, and (13)C NMR spin relaxation: conformational preferences of α-L-Rhap-α-(1 → 2)-α-L-Rhap-OMe in water and dimethyl sulfoxide solutions.

PubMed

Pendrill, Robert; Engström, Olof; Volpato, Andrea; Zerbetto, Mirco; Polimeno, Antonino; Widmalm, Göran

2016-01-28

The monosaccharide L-rhamnose is common in bacterial polysaccharides and the disaccharide α-L-Rhap-α-(1 → 2)-α-L-Rhap-OMe represents a structural model for a part of Shigella flexneri O-antigen polysaccharides. Utilization of [1'-(13)C]-site-specific labeling in the anomeric position at the glycosidic linkage between the two sugar residues facilitated the determination of transglycosidic NMR (3)JCH and (3)JCC coupling constants. Based on these spin-spin couplings the major state and the conformational distribution could be determined with respect to the ψ torsion angle, which changed between water and dimethyl sulfoxide (DMSO) as solvents, a finding mirrored by molecular dynamics (MD) simulations with explicit solvent molecules. The (13)C NMR spin relaxation parameters T1, T2, and heteronuclear NOE of the probe were measured for the disaccharide in DMSO-d6 at two magnetic field strengths, with standard deviations ≤1%. The combination of MD simulation and a stochastic description based on the diffusive chain model resulted in excellent agreement between calculated and experimentally observed (13)C relaxation parameters, with an average error of <2%. The coupling between the global reorientation of the molecule and the local motion of the spin probe is deemed essential if reproduction of NMR relaxation parameters should succeed, since decoupling of the two modes of motion results in significantly worse agreement. Calculation of (13)C relaxation parameters based on the correlation functions obtained directly from the MD simulation of the solute molecule in DMSO as solvent showed satisfactory agreement with errors on the order of 10% or less.

Crystallographic and Computational Analyses of AUUCU Repeating RNA That Causes Spinocerebellar Ataxia Type 10 (SCA10).

PubMed

Park, HaJeung; González, Àlex L; Yildirim, Ilyas; Tran, Tuan; Lohman, Jeremy R; Fang, Pengfei; Guo, Min; Disney, Matthew D

2015-06-23

Spinocerebellar ataxia type 10 (SCA10) is caused by a pentanucleotide repeat expansion of r(AUUCU) within intron 9 of the ATXN10 pre-mRNA. The RNA causes disease by a gain-of-function mechanism in which it inactivates proteins involved in RNA biogenesis. Spectroscopic studies showed that r(AUUCU) repeats form a hairpin structure; however, there were no high-resolution structural models prior to this work. Herein, we report the first crystal structure of model r(AUUCU) repeats refined to 2.8 Å and analysis of the structure via molecular dynamics simulations. The r(AUUCU) tracts adopt an overall A-form geometry in which 3 × 3 nucleotide (5')UCU(3')/(3')UCU(5') internal loops are closed by AU pairs. Helical parameters of the refined structure as well as the corresponding electron density map on the crystallographic model reflect dynamic features of the internal loop. The computational analyses captured dynamic motion of the loop closing pairs, which can form single-stranded conformations with relatively low energies. Overall, the results presented here suggest the possibility for r(AUUCU) repeats to form metastable A-from structures, which can rearrange into single-stranded conformations and attract proteins such as heterogeneous nuclear ribonucleoprotein K (hnRNP K). The information presented here may aid in the rational design of therapeutics targeting this RNA.

Crystallographic and Computational Analyses of AUUCU Repeating RNA That Causes Spinocerebellar Ataxia Type 10 (SCA10)

PubMed Central

Park, HaJeung; González, Àlex L.; Yildirim, Ilyas; Tran, Tuan; Lohman, Jeremy R.; Fang, Pengfei; Guo, Min; Disney, Matthew D.

2016-01-01

Spinocerebellar ataxia type 10 (SCA10) is caused by a pentanucleotide repeat expansion of r(AUUCU) within intron 9 of the ATXN10 pre-mRNA. The RNA causes disease by a gain-of-function mechanism in which it inactivates proteins involved in RNA biogenesis. Spectroscopic studies showed that r(AUUCU) repeats form a hairpin structure; however, there were no high-resolution structural models prior to this work. Herein, we report the first crystal structure of model r(AUUCU) repeats refined to 2.8 Å and analysis of the structure via molecular dynamics simulations. The r(AUUCU) tracts adopt an overall A-form geometry in which 3 × 3 nucleotide 5′UCU3′/3′UCU5′ internal loops are closed by AU pairs. Helical parameters of the refined structure as well as the corresponding electron density map on the crystallographic model reflect dynamic features of the internal loop. The computational analyses captured dynamic motion of the loop closing pairs, which can form single-stranded conformations with relatively low energies. Overall, the results presented here suggest the possibility for r(AUUCU) repeats to form metastable A-from structures, which can rearrange into single-stranded conformations and attract proteins such as heterogeneous nuclear ribonucleoprotein K (hnRNP K). The information presented here may aid in the rational design of therapeutics targeting this RNA. PMID:26039897

Dynamic Motion and Communication in the Streptococcal C1 Phage Lysin, PlyC

PubMed Central

Reboul, Cyril F.; Cowieson, Nathan P.; Costa, Mauricio G. S.; Kass, Itamar; Jackson, Colin; Perahia, David; Buckle, Ashley M.; McGowan, Sheena

2015-01-01

The growing problem of antibiotic resistance underlies the critical need to develop new treatments to prevent and control resistant bacterial infection. Exogenous application of bacteriophage lysins results in rapid and specific destruction of Gram-positive bacteria and therefore lysins represent novel antibacterial agents. The PlyC phage lysin is the most potent lysin characterized to date and can rapidly lyse Group A, C and E streptococci. Previously, we have determined the X-ray crystal structure of PlyC, revealing a complicated and unique arrangement of nine proteins. The scaffold features a multimeric cell-wall docking assembly bound to two catalytic domains that communicate and work synergistically. However, the crystal structure appeared to be auto-inhibited and raised important questions as to the mechanism underlying its extreme potency. Here we use small angle X-ray scattering (SAXS) and reveal that the conformational ensemble of PlyC in solution is different to that in the crystal structure. We also investigated the flexibility of the enzyme using both normal mode (NM) analysis and molecular dynamics (MD) simulations. Consistent with our SAXS data, MD simulations show rotational dynamics of both catalytic domains, and implicate inter-domain communication in achieving a substrate-ready conformation required for enzyme function. Our studies therefore provide insights into how the domains in the PlyC holoenzyme may act together to achieve its extraordinary potency. PMID:26470022

Early time excited-state structural evolution of pyranine in methanol revealed by femtosecond stimulated Raman spectroscopy.

PubMed

Wang, Yanli; Liu, Weimin; Tang, Longteng; Oscar, Breland; Han, Fangyuan; Fang, Chong

2013-07-25

To understand chemical reactivity of molecules in condensed phase in real time, a structural dynamics technique capable of monitoring molecular conformational motions on their intrinsic time scales, typically on femtoseconds to picoseconds, is needed. We have studied a strong photoacid pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid, HPTS, pK(a)* ≈ 0) in pure methanol and observed that excited-state proton transfer (ESPT) is absent, in sharp contrast with our previous work on HPTS in aqueous solutions wherein ESPT prevails following photoexcitation. Two transient vibrational marker bands at ~1477 (1454) and 1532 (1528) cm(-1) appear in CH3OH (CD3OD), respectively, rising within the instrument response time of ~140 fs and decaying with 390-470 (490-1400) fs and ~200 ps time constants in CH3OH (CD3OD). We attribute the mode onset to small-scale coherent proton motion along the pre-existing H-bonding chain between HPTS and methanol, and the two decay stages to the low-frequency skeletal motion-modulated Franck-Condon relaxation within ~1 ps and subsequent rotational diffusion of H-bonding partners in solution before fluorescence. The early time kinetic isotope effect (KIE) of ~3 upon methanol deuteration argues active proton motions particularly within the first few picoseconds when coherent skeletal motions are underdamped. Pronounced quantum beats are observed for high-frequency modes consisting of strong phenolic COH rocking (1532 cm(-1)) or H-out-of-plane wagging motions (952 cm(-1)) due to anharmonic coupling to coherent low-frequency modes impulsively excited at ca. 96, 120, and 168 cm(-1). The vivid illustration of atomic motions of HPTS in varying H-bonding geometry with neighboring methanol molecules unravels the multidimensional energy relaxation pathways immediately following photoexcitation, and provides compelling evidence that, in lieu of ESPT, the photoacidity of HPTS promptly activates characteristic low-frequency skeletal motions to search phase space mainly concerning the phenolic end and to efficiently dissipate vibrational energy via skeletal deformation and proton shuttling motions within the intermediate, relatively confined excited-state HPTS-methanol complex on a solvent-dependent dynamic potential energy surface.

Theory and Applications of Solid-State NMR Spectroscopy to Biomembrane Structure and Dynamics

NASA Astrophysics Data System (ADS)

Xu, Xiaolin

Solid-state Nuclear Magnetic Resonance (NMR) is one of the premiere biophysical methods that can be applied for addressing the structure and dynamics of biomolecules, including proteins, lipids, and nucleic acids. It illustrates the general problem of determining the average biomolecular structure, including the motional mean-square amplitudes and rates of the fluctuations. Lineshape and relaxtion studies give us a view into the molecular properties under different environments. To help the understanding of NMR theory, both lineshape and relaxation experiments are conducted with hexamethylbezene (HMB). This chemical compound with a simple structure serves as a perfect test molecule. Because of its highly symmetric structure, its motions are not very difficult to understand. The results for HMB set benchmarks for other more complicated systems like membrane proteins. After accumulating a large data set on HMB, we also proceed to develop a completely new method of data analysis, which yields the spectral densities in a body-fixed frame revealing internal motions of the system. Among the possible applications of solid-state NMR spectroscopy, we study the light activation mechanism of visual rhodopsin in lipid membranes. As a prototype of G-protein-coupled receptors, which are a large class of membrane proteins, the cofactor isomerization is triggered by photon absorption, and the local structural change is then propagated to a large-scale conformational change of the protein. Facilitation of the binding of transducin then passes along the visual signal to downstream effector proteins like transducin. To study this process, we introduce 2H labels into the rhodopsin chromophore retinal and the C-terminal peptide of transducin to probe the local structure and dynamics of these two hotspots of the rhodopsin activation process. In addition to the examination of local sites with solid-state 2H NMR spectroscopy, wide angle X-ray scattering (WAXS) provides us the chance of looking at the overall conformational changes through difference scattering profiles. Although the resolution of this method is not as high as NMR spectroscopy, which gives information on atomic scale, the early activation probing is possible because of the short duration of the optical pump and X-ray probe lasers. We can thus visualize the energy dissipation process by observing and comparing the difference scattering profiles at different times after the light activation moments.

Differential Modulation of Functional Dynamics and Allosteric Interactions in the Hsp90-Cochaperone Complexes with p23 and Aha1: A Computational Study

PubMed Central

Blacklock, Kristin; Verkhivker, Gennady M.

2013-01-01

Allosteric interactions of the molecular chaperone Hsp90 with a large cohort of cochaperones and client proteins allow for molecular communication and event coupling in signal transduction networks. The integration of cochaperones into the Hsp90 system is driven by the regulatory mechanisms that modulate the progression of the ATPase cycle and control the recruitment of the Hsp90 clientele. In this work, we report the results of computational modeling of allosteric regulation in the Hsp90 complexes with the cochaperones p23 and Aha1. By integrating protein docking, biophysical simulations, modeling of allosteric communications, protein structure network analysis and the energy landscape theory we have investigated dynamics and stability of the Hsp90-p23 and Hsp90-Aha1 interactions in direct comparison with the extensive body of structural and functional experiments. The results have revealed that functional dynamics and allosteric interactions of Hsp90 can be selectively modulated by these cochaperones via specific targeting of the regulatory hinge regions that could restrict collective motions and stabilize specific chaperone conformations. The protein structure network parameters have quantified the effects of cochaperones on conformational stability of the Hsp90 complexes and identified dynamically stable communities of residues that can contribute to the strengthening of allosteric interactions. According to our results, p23-mediated changes in the Hsp90 interactions may provide “molecular brakes” that could slow down an efficient transmission of the inter-domain allosteric signals, consistent with the functional role of p23 in partially inhibiting the ATPase cycle. Unlike p23, Aha1-mediated acceleration of the Hsp90-ATPase cycle may be achieved via modulation of the equilibrium motions that facilitate allosteric changes favoring a closed dimerized form of Hsp90. The results of our study have shown that Aha1 and p23 can modulate the Hsp90-ATPase activity and direct the chaperone cycle by exerting the precise control over structural stability, global movements and allosteric communications in Hsp90. PMID:23977182

Molecular simulation and docking studies of Gal1p and Gal3p proteins in the presence and absence of ligands ATP and galactose: implication for transcriptional activation of GAL genes

NASA Astrophysics Data System (ADS)

Upadhyay, Sanjay K.; Sasidhar, Yellamraju U.

2012-07-01

The Gal4p mediated transcriptional activation of GAL genes requires the interaction between Gal3p bound with ATP and galactose and Gal80p. Though numerous studies suggest that galactose and ATP activate Gal3p/Gal1p interaction with Gal80p, neither the mechanism of activation nor the interacting surface that binds to Gal80p is well understood. In this study we investigated the dynamics of Gal3p and Gal1p in the presence and absence of ligands ATP and galactose to understand the role played by dynamics in the function of these proteins through molecular dynamics simulation and protein-protein docking studies. We performed simulations totaling to 510 ns on both Gal1p and Gal3p proteins in the presence and absence of ligands ATP and galactose. We find that, while binding of ligands ATP and galactose to Gal3p/Gal1p do not affect the global conformation of proteins, some local conformational changes around upper-lip helix including insertion domain are observed. We observed that only in the presence of ATP and galactose, Gal3p displays opening and closing motion between the two domains. And because of this motion, a binding interface, which is largely hydrophobic, opens up on the surface of Gal3p and this surface can bind to Gal80p. From our simulation studies we infer probable docking sites for Gal80p on Gal3p/Gal1p, which were further ascertained by the docking of Gal80p on to ligand bound Gal1p and Gal3p proteins, and the residues at the interface between Gal3p and Gal80p are identified. Our results correlate quite well with the existing body of literature on functional and dynamical aspects of Gal1p and Gal3p proteins.

Molecular simulation and docking studies of Gal1p and Gal3p proteins in the presence and absence of ligands ATP and galactose: implication for transcriptional activation of GAL genes.

PubMed

Upadhyay, Sanjay K; Sasidhar, Yellamraju U

2012-07-01

The Gal4p mediated transcriptional activation of GAL genes requires the interaction between Gal3p bound with ATP and galactose and Gal80p. Though numerous studies suggest that galactose and ATP activate Gal3p/Gal1p interaction with Gal80p, neither the mechanism of activation nor the interacting surface that binds to Gal80p is well understood. In this study we investigated the dynamics of Gal3p and Gal1p in the presence and absence of ligands ATP and galactose to understand the role played by dynamics in the function of these proteins through molecular dynamics simulation and protein-protein docking studies. We performed simulations totaling to 510 ns on both Gal1p and Gal3p proteins in the presence and absence of ligands ATP and galactose. We find that, while binding of ligands ATP and galactose to Gal3p/Gal1p do not affect the global conformation of proteins, some local conformational changes around upper-lip helix including insertion domain are observed. We observed that only in the presence of ATP and galactose, Gal3p displays opening and closing motion between the two domains. And because of this motion, a binding interface, which is largely hydrophobic, opens up on the surface of Gal3p and this surface can bind to Gal80p. From our simulation studies we infer probable docking sites for Gal80p on Gal3p/Gal1p, which were further ascertained by the docking of Gal80p on to ligand bound Gal1p and Gal3p proteins, and the residues at the interface between Gal3p and Gal80p are identified. Our results correlate quite well with the existing body of literature on functional and dynamical aspects of Gal1p and Gal3p proteins.

Impact of mutation on proton transfer reactions in ketosteroid isomerase: insights from molecular dynamics simulations.

PubMed

Chakravorty, Dhruva K; Hammes-Schiffer, Sharon

2010-06-02

The two proton transfer reactions catalyzed by ketosteroid isomerase (KSI) involve a dienolate intermediate stabilized by hydrogen bonds with Tyr14 and Asp99. Molecular dynamics simulations based on an empirical valence bond model are used to examine the impact of mutating these residues on the hydrogen-bonding patterns, conformational changes, and van der Waals and electrostatic interactions during the proton transfer reactions. While the rate constants for the two proton transfer steps are similar for wild-type (WT) KSI, the simulations suggest that the rate constant for the first proton transfer step is smaller in the mutants due to the significantly higher free energy of the dienolate intermediate relative to the reactant. The calculated rate constants for the mutants D99L, Y14F, and Y14F/D99L relative to WT KSI are qualitatively consistent with the kinetic experiments indicating a significant reduction in the catalytic rates along the series of mutants. In the simulations, WT KSI retained two hydrogen-bonding interactions between the substrate and the active site, while the mutants typically retained only one hydrogen-bonding interaction. A new hydrogen-bonding interaction between the substrate and Tyr55 was observed in the double mutant, leading to the prediction that mutation of Tyr55 will have a greater impact on the proton transfer rate constants for the double mutant than for WT KSI. The electrostatic stabilization of the dienolate intermediate relative to the reactant was greater for WT KSI than for the mutants, providing a qualitative explanation for the significantly reduced rates of the mutants. The active site exhibited restricted motion during the proton transfer reactions, but small conformational changes occurred to facilitate the proton transfer reactions by strengthening the hydrogen-bonding interactions and by bringing the proton donor and acceptor closer to each other with the proper orientation for proton transfer. Thus, these calculations suggest that KSI forms a preorganized active site but that the structure of this preorganized active site is altered upon mutation. Moreover, small conformational changes due to stochastic thermal motions are required within this preorganized active site to facilitate the proton transfer reactions.

Asymmetric processing of a substrate protein in sequential allosteric cycles of AAA+ nanomachines

NASA Astrophysics Data System (ADS)

Kravats, Andrea N.; Tonddast-Navaei, Sam; Bucher, Ryan J.; Stan, George

2013-09-01

Essential protein quality control includes mechanisms of substrate protein (SP) unfolding and translocation performed by powerful ring-shaped AAA+ (ATPases associated with various cellular activities) nanomachines. These SP remodeling actions are effected by mechanical forces imparted by AAA+ loops that protrude into the central channel. Sequential intra-ring allosteric motions, which underlie repetitive SP-loop interactions, have been proposed to comprise clockwise (CW), counterclockwise (CCW), or random (R) conformational transitions of individual AAA+ subunits. To probe the effect of these allosteric mechanisms on unfoldase and translocase functions, we perform Langevin dynamics simulations of a coarse-grained model of an all-alpha SP processed by the single-ring ClpY ATPase or by the double-ring p97 ATPase. We find that, in all three allosteric mechanisms, the SP undergoes conformational transitions along a common set of pathways, which reveals that the active work provided by the ClpY machine involves single loop-SP interactions. Nevertheless, the rates and yields of SP unfolding and translocation are controlled by mechanism-dependent loop-SP binding events, as illustrated by faster timescales of SP processing in CW allostery compared with CCW and R allostery. The distinct efficacy of allosteric mechanisms is due to the asymmetric collaboration of adjacent subunits, which involves CW-biased structural motions of AAA+ loops and results in CW-compatible torque applied onto the SP. Additional simulations of mutant ClpY rings, which render a subset of subunits catalytically-defective or reduce their SP binding affinity, reveal that subunit-based conformational transitions play the major role in SP remodeling. Based on these results we predict that the minimally functional AAA+ ring includes three active subunits, only two of which are adjacent.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo

The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use inmore » combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data.« less

Molecular dynamics of conformational substates for a simplified protein model

NASA Astrophysics Data System (ADS)

Grubmüller, Helmut; Tavan, Paul

1994-09-01

Extended molecular dynamics simulations covering a total of 0.232 μs have been carried out on a simplified protein model. Despite its simplified structure, that model exhibits properties similar to those of more realistic protein models. In particular, the model was found to undergo transitions between conformational substates at a time scale of several hundred picoseconds. The computed trajectories turned out to be sufficiently long as to permit a statistical analysis of that conformational dynamics. To check whether effective descriptions neglecting memory effects can reproduce the observed conformational dynamics, two stochastic models were studied. A one-dimensional Langevin effective potential model derived by elimination of subpicosecond dynamical processes could not describe the observed conformational transition rates. In contrast, a simple Markov model describing the transitions between but neglecting dynamical processes within conformational substates reproduced the observed distribution of first passage times. These findings suggest, that protein dynamics generally does not exhibit memory effects at time scales above a few hundred picoseconds, but confirms the existence of memory effects at a picosecond time scale.

Dielectric Properties of Poly(ethylene oxide) from Molecular Dynamics Simulations

NASA Technical Reports Server (NTRS)

Smith, Grant D.

1994-01-01

The order, conformations and dynamics of poly(oxyethylene) (POE) melts have been investigated through molecular dynamics simulations. The potential energy functions were determined from detailed ab initio electronic structure calculations of the conformational energies of the model molecules 1,2-dimethoxyethane (DME) and diethylether. The x-ray structure factor for POE from simulation will be compared to experiment. In terms of conformation, simulations reveal that chains are extended in the melt relative to isolated chains due to the presence of strong intermolecular O...H interactions, which occur at the expense of intramolecular O...H interactions. Conformational dynamics about the C-C bond were found to be significantly faster than in polymethylene, while conformational dynamics about the C-O bond even faster than the C-C dynamics. The faster local dynamics in POE relative to polymethylene is consistent with C-13 NMR spin-lattice relaxation experiments. Conformational transitions showed significant second-neighbor correlation, as was found for polymethylene. This correlation of transitions with C-C neighbors was found to be reduced relative to C-O neighbors. Dielectric relaxation from simulation will also be compared with experiment.

Single-Molecule Microscopy and Force Spectroscopy of Membrane Proteins

NASA Astrophysics Data System (ADS)

Engel, Andreas; Janovjak, Harald; Fotiadis, Dimtrios; Kedrov, Alexej; Cisneros, David; Müller, Daniel J.

Single-molecule atomic force microscopy (AFM) provides novel ways to characterize the structure-function relationship of native membrane proteins. High-resolution AFM topographs allow observing the structure of single proteins at sub-nanometer resolution as well as their conformational changes, oligomeric state, molecular dynamics and assembly. We will review these feasibilities illustrating examples of membrane proteins in native and reconstituted membranes. Classification of individual topographs of single proteins allows understanding the principles of motions of their extrinsic domains, to learn about their local structural flexibilities and to find the entropy minima of certain conformations. Combined with the visualization of functionally related conformational changes these insights allow understanding why certain flexibilities are required for the protein to function and how structurally flexible regions allow certain conformational changes. Complementary to AFM imaging, single-molecule force spectroscopy (SMFS) experiments detect molecular interactions established within and between membrane proteins. The sensitivity of this method makes it possible to measure interactions that stabilize secondary structures such as transmembrane α-helices, polypeptide loops and segments within. Changes in temperature or protein-protein assembly do not change the locations of stable structural segments, but influence their stability established by collective molecular interactions. Such changes alter the probability of proteins to choose a certain unfolding pathway. Recent examples have elucidated unfolding and refolding pathways of membrane proteins as well as their energy landscapes.

Straight-line climbing flight aerodynamics of a fruit bat

NASA Astrophysics Data System (ADS)

Viswanath, K.; Nagendra, K.; Cotter, J.; Frauenthal, M.; Tafti, D. K.

2014-02-01

From flight data obtained on a fruit bat, Cynopterus brachyotis, a kinematic model for straight-line flapping motion is extracted and analyzed in a computational fluid dynamics (CFD) framework to gain insight into the complexity of bat flight. The intricate functional mechanics and architecture of the bat wings set it apart from other vertebrate flight. The extracted kinematic model is simulated for a range of Reynolds numbers, to observe the effect these phenomena have on the unsteady transient mechanisms of the flow produced by the flapping wings. The Strouhal number calculated from the data is high indicating that the oscillatory motion dominates the flow physics. From the obtained data, the bat exhibits fine control of its mechanics by actively varying wing camber, wing area, torsional rotation of the wing, forward and backward translational sweep of the wing, and wing conformation to dictate the fluid dynamics. As is common in flapping flight, the primary force generation is through the attached unsteady vortices on the wing surface. The bat through varying the wing camber and the wing area modulates this force output. The power requirement for the kinematics is analyzed and correlated with the aerodynamic performance.

Understanding the core of RNA interference: The dynamic aspects of Argonaute-mediated processes.

PubMed

Zhu, Lizhe; Jiang, Hanlun; Sheong, Fu Kit; Cui, Xuefeng; Wang, Yanli; Gao, Xin; Huang, Xuhui

2017-09-01

At the core of RNA interference, the Argonaute proteins (Ago) load and utilize small guide nucleic acids to silence mRNAs or cleave foreign nucleic acids in a sequence specific manner. In recent years, based on extensive structural studies of Ago and its interaction with the nucleic acids, considerable progress has been made to reveal the dynamic aspects of various Ago-mediated processes. Here we review these novel insights into the guide-strand loading, duplex unwinding, and effects of seed mismatch, with a focus on two representative Agos, the human Ago 2 (hAgo2) and the bacterial Thermus thermophilus Ago (TtAgo). In particular, comprehensive molecular simulation studies revealed that although sharing similar overall structures, the two Agos have vastly different conformational landscapes and guide-strand loading mechanisms because of the distinct rigidity of their L1-PAZ hinge. Given the central role of the PAZ motions in regulating the exposure of the nucleic acid binding channel, these findings exemplify the importance of protein motions in distinguishing the overlapping, yet distinct, mechanisms of Ago-mediated processes in different organisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dynamics and unfolding pathway of chimeric azurin variants: insights from molecular dynamics simulation.

PubMed

Evoli, Stefania; Guzzi, Rita; Rizzuti, Bruno

2013-10-01

The spectroscopic, thermal, and functional properties of blue copper proteins can be modulated by mutations in the metal binding loop. Molecular dynamics simulation was used to compare the conformational properties of azurin and two chimeric variants, which were obtained by inserting into the azurin scaffold the copper binding loop of amicyanin and plastocyanin, respectively. Simulations at room temperature show that the proteins retain their overall structure and exhibit concerted motions among specific inner regions, as revealed by principal component analysis. Molecular dynamics at high temperature indicates that the first events in the unfolding pathway are structurally similar in the three proteins and unfolding starts from the region of the α-helix that is far from the metal binding loop. The results provide details of the denaturation process that are consistent with experimental data and in close agreement with other computational approaches, suggesting a distinct mechanism of unfolding of azurin and its chimeric variants. Moreover, differences observed in the dynamics of specific regions in the three proteins correlate with their thermal behavior, contributing to the determination of the basic factors that influence the stability.

Dynamics of Surface Reorganization of Poly(methyl methacrylate) in Contact with Water

NASA Astrophysics Data System (ADS)

Horinouchi, Ayanobu; Atarashi, Hironori; Fujii, Yoshihisa; Tanaka, Keiji

2013-03-01

New tools for tailor-made diagnostics, such as DNA arrays and tips for micro-total-analysis systems, are generally made from polymers. In these applications, the polymer surface is in contact with a water phase. However, despite the importance of detailed knowledge of the fundamental interactions of polymer interfaces with liquids, such studies are very limited. As an initial benchmark for designing and constructing specialized biomedical surfaces containing polymer, aggregation states and dynamics of chains at the water interface should be systematically examined. We here apply time-resolved contact angle measurement to study the dynamics of the surface reorganization of poly(methyl methacrylate) (PMMA) in contact with water. By doing the measurements at various temperatures, it is possible to discuss the surface dynamics of PMMA based on the apparent activation energy. Also, sum-frequency generation spectroscopy revealed that the surface reorganization involves the conformational changes in the main chain part as well as the side chains. Hence, the dynamics observed here may reflect the segmental motion at the outermost region of the PMMA film, in which water plays as a plasticizer.

Rotation of Guanine Amino Groups in G-Quadruplexes: A Probe for Local Structure and Ligand Binding.

PubMed

Adrian, Michael; Winnerdy, Fernaldo Richtia; Heddi, Brahim; Phan, Anh Tuân

2017-08-22

Nucleic acids are dynamic molecules whose functions may depend on their conformational fluctuations and local motions. In particular, amino groups are dynamic components of nucleic acids that participate in the formation of various secondary structures such as G-quadruplexes. Here, we present a cost-efficient NMR method to quantify the rotational dynamics of guanine amino groups in G-quadruplex nucleic acids. An isolated spectrum of amino protons from a specific tetrad-bound guanine can be extracted from the nuclear Overhauser effect spectroscopy spectrum based on the close proximity between the intra-residue imino and amino protons. We apply the method in different structural contexts of G-quadruplexes and their complexes. Our results highlight the role of stacking and hydrogen-bond interactions in restraining amino-group rotation. The measurement of the rotation rate of individual amino groups could give insight into the dynamic processes occurring at specific locations within G-quadruplex nucleic acids, providing valuable probes for local structure, dynamics, and ligand binding. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Structure-activity relationships of pyrethroid insecticides. Part 2. The use of molecular dynamics for conformation searching and average parameter calculation

NASA Astrophysics Data System (ADS)

Hudson, Brian D.; George, Ashley R.; Ford, Martyn G.; Livingstone, David J.

1992-04-01

Molecular dynamics simulations have been performed on a number of conformationally flexible pyrethroid insecticides. The results indicate that molecular dynamics is a suitable tool for conformational searching of small molecules given suitable simulation parameters. The structures derived from the simulations are compared with the static conformation used in a previous study. Various physicochemical parameters have been calculated for a set of conformations selected from the simulations using multivariate analysis. The averaged values of the parameters over the selected set (and the factors derived from them) are compared with the single conformation values used in the previous study.

Reversible ratchet effects for vortices in conformal pinning arrays

DOE PAGES

Reichhardt, Charles; Ray, Dipanjan; Reichhardt, Cynthia Jane Olson

2015-05-04

A conformal transformation of a uniform triangular pinning array produces a structure called a conformal crystal which preserves the sixfold ordering of the original lattice but contains a gradient in the pinning density. Here we use numerical simulations to show that vortices in type-II superconductors driven with an ac drive over gradient pinning arrays produce the most pronounced ratchet effect over a wide range of parameters for a conformal array, while square gradient or random gradient arrays with equivalent pinning densities give reduced ratchet effects. In the conformal array, the larger spacing of the pinning sites in the direction transversemore » to the ac drive permits easy funneling of interstitial vortices for one driving direction, producing the enhanced ratchet effect. In the square array, the transverse spacing between pinning sites is uniform, giving no asymmetry in the funneling of the vortices as the driving direction switches, while in the random array, there are numerous easy-flow channels present for either direction of drive. We find multiple ratchet reversals in the conformal arrays as a function of vortex density and ac amplitude, and correlate the features with a reversal in the vortex ordering, which is greater for motion in the ratchet direction. In conclusion, the enhanced conformal pinning ratchet effect can also be realized for colloidal particles moving over a conformal array, indicating the general usefulness of conformal structures for controlling the motion of particles.« less

Maintaining and Enhancing Diversity of Sampled Protein Conformations in Robotics-Inspired Methods.

PubMed

Abella, Jayvee R; Moll, Mark; Kavraki, Lydia E

2018-01-01

The ability to efficiently sample structurally diverse protein conformations allows one to gain a high-level view of a protein's energy landscape. Algorithms from robot motion planning have been used for conformational sampling, and several of these algorithms promote diversity by keeping track of "coverage" in conformational space based on the local sampling density. However, large proteins present special challenges. In particular, larger systems require running many concurrent instances of these algorithms, but these algorithms can quickly become memory intensive because they typically keep previously sampled conformations in memory to maintain coverage estimates. In addition, robotics-inspired algorithms depend on defining useful perturbation strategies for exploring the conformational space, which is a difficult task for large proteins because such systems are typically more constrained and exhibit complex motions. In this article, we introduce two methodologies for maintaining and enhancing diversity in robotics-inspired conformational sampling. The first method addresses algorithms based on coverage estimates and leverages the use of a low-dimensional projection to define a global coverage grid that maintains coverage across concurrent runs of sampling. The second method is an automatic definition of a perturbation strategy through readily available flexibility information derived from B-factors, secondary structure, and rigidity analysis. Our results show a significant increase in the diversity of the conformations sampled for proteins consisting of up to 500 residues when applied to a specific robotics-inspired algorithm for conformational sampling. The methodologies presented in this article may be vital components for the scalability of robotics-inspired approaches.

Extensive domain motion and electron transfer in the human electron transferring flavoprotein.medium chain Acyl-CoA dehydrogenase complex.

PubMed

Toogood, Helen S; van Thiel, Adam; Basran, Jaswir; Sutcliffe, Mike J; Scrutton, Nigel S; Leys, David

2004-07-30

The crystal structure of the human electron transferring flavoprotein (ETF).medium chain acyl-CoA dehydrogenase (MCAD) complex reveals a dual mode of protein-protein interaction, imparting both specificity and promiscuity in the interaction of ETF with a range of structurally distinct primary dehydrogenases. ETF partitions the functions of partner binding and electron transfer between (i) the recognition loop, which acts as a static anchor at the ETF.MCAD interface, and (ii) the highly mobile redox active FAD domain. Together, these enable the FAD domain of ETF to sample a range of conformations, some compatible with fast interprotein electron transfer. Disorders in amino acid or fatty acid catabolism can be attributed to mutations at the protein-protein interface. Crucially, complex formation triggers mobility of the FAD domain, an induced disorder that contrasts with general models of protein-protein interaction by induced fit mechanisms. The subsequent interfacial motion in the MCAD.ETF complex is the basis for the interaction of ETF with structurally diverse protein partners. Solution studies using ETF and MCAD with mutations at the protein-protein interface support this dynamic model and indicate ionic interactions between MCAD Glu(212) and ETF Arg alpha(249) are likely to transiently stabilize productive conformations of the FAD domain leading to enhanced electron transfer rates between both partners.