Conformer lifetimes of ethyl cyanoformate from exchange-averaged rotational spectra.

PubMed

True, Nancy S

2009-06-25

Ethyl cyanoformate exists as a mixture of two conformers but displays three R-branch a-type band series in its rotational spectrum. Simulations with population fractions 0.37 at 210 K and 0.70 at 297 K undergoing conformer exchange with average conformer lifetimes, , shorter than approximately 40 ps at approximately 210 K and shorter than approximately 37 ps at 297 K reproduce the experimental spectra between 26.5 and 38 GHz, the exchanging species accounting for the third set of bands. The upper-limit 's are 1 order of magnitude longer than RRKM theory predictions and the population fractions are consistent with the total population with energy above 700 cm(-1), approximately twice the conformer interconversion barrier height. Model calculations indicate that extensive K-sublevel mixing in individual molecular eigenstates can produce the large population and the narrow distribution of the rotational-constant sum, B + C, consistent with the observed exchange-averaged band series.

The dynamical behavior of the s-trioxane radical cation-A low-temperature EPR and theoretical study.

PubMed

Naumov, Sergej S; Knolle, Wolfgang; Naumov, Sergej P; Pöppl, Andreas; Janovský, Igor

2014-10-28

The radical cation of s-trioxane, radiolytically generated in a freon (CF3CCl3) matrix, was studied in the 10-140 K temperature region. Reversible changes of the EPR spectra were observed, arising from both ring puckering and ring inversion through the molecular plane. The ESREXN program based on the Liouville density matrix equation, allowing the treatment of dynamical exchange, has been used to analyze the experimental results. Two limiting conformer structures of the s-trioxane radical cation were taken into account, namely "rigid" half-boat and averaged planar ones, differing strongly in their electron distribution. The spectrum due to the "rigid" half-boat conformer can be observed only at very low (<60 K) temperatures, when the exchange of conformers is very slow. Two transition states for interconversion by puckering and ring-inversion were identified, close in activation energy (2.3 and 3.0 kJ/mol calculated). Since the energy difference is very small, both processes set on at a comparable temperature. In the case of nearly complete equilibration (fast exchange) between six energetically equivalent structures at T > 120 K in CF3CCl3, a septet due to six equivalent protons (hfs splitting constant 5.9 mT) is observed, characteristic of the dynamically averaged planar geometry of the radical cation. DFT quantum chemical calculations and spectral simulation including intramolecular dynamical exchange support the interpretation.

Probing RNA Native Conformational Ensembles with Structural Constraints.

PubMed

Fonseca, Rasmus; van den Bedem, Henry; Bernauer, Julie

2016-05-01

Noncoding ribonucleic acids (RNA) play a critical role in a wide variety of cellular processes, ranging from regulating gene expression to post-translational modification and protein synthesis. Their activity is modulated by highly dynamic exchanges between three-dimensional conformational substates, which are difficult to characterize experimentally and computationally. Here, we present an innovative, entirely kinematic computational procedure to efficiently explore the native ensemble of RNA molecules. Our procedure projects degrees of freedom onto a subspace of conformation space defined by distance constraints in the tertiary structure. The dimensionality reduction enables efficient exploration of conformational space. We show that the conformational distributions obtained with our method broadly sample the conformational landscape observed in NMR experiments. Compared to normal mode analysis-based exploration, our procedure diffuses faster through the experimental ensemble while also accessing conformational substates to greater precision. Our results suggest that conformational sampling with a highly reduced but fully atomistic representation of noncoding RNA expresses key features of their dynamic nature.

Coupling of conformational transitions in the N-terminal domain of the 51-kDa FK506-binding protein (FKBP51) near its site of interaction with the steroid receptor proteins

DOE PAGES

LeMaster, David M.; Mustafi, Sourajit M.; Brecher, Matthew; ...

2015-05-07

Interchanging Leu-119 for Pro-119 at the tip of the β 4-β 5 loop in the first FK506 binding domain (FK1) of the FKBP51 and FKBP52 proteins, respectively, has been reported to largely reverse the inhibitory (FKBP51) or stimulatory (FKBP52) effects of these co-chaperones on the transcriptional activity of glucocorticoid and androgen receptor-protein complexes. Previous NMR relaxation studies have identified exchange line broadening, indicative of submillisecond conformational motion, throughout the β 4-β 5 loop in the FK1 domain of FKBP51, which are suppressed by the FKBP52-like L119P substitution. This substitution also attenuates exchange line broadening in the underlying β 2 andmore » β 3a strands that is centered near a bifurcated main chain hydrogen bond interaction between these two strands. The present study demonstrates that these exchange line broadening effects arise from two distinct coupled conformational transitions, and the transition within the β 2 and β 3a strands samples a transient conformation that resembles the crystal structures of the selectively inhibited FK1 domain of FKBP51 recently reported. Although the crystal structures for their series of inhibitors were interpreted as evidence for an induced fit mechanism of association, the presence of a similar conformation being significantly populated in the unliganded FKBP51 domain is more consistent with a conformational selection binding process. As a result, the contrastingly reduced conformational plasticity of the corresponding FK1 domain of FKBP52 is consistent with the current model in which FKBP51 binds to both the apo- and hormone-bound forms of the steroid receptor to modulate its affinity for ligand, whereas FKBP52 binds selectively to the latter state.« less

Structural Dynamics Control Allosteric Activation of Cytohesin Family Arf GTPase Exchange Factors

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

Malaby, Andrew W.; Das, Sanchaita; Chakravarthy, Srinivas

Membrane dynamic processes including vesicle biogenesis depend on Arf guanosine triphosphatase (GTPase) activation by guanine nucleotide exchange factors (GEFs) containing a catalytic Sec7 domain and a membrane-targeting module such as a pleckstrin homology (PH) domain. The catalytic output of cytohesin family Arf GEFs is controlled by autoinhibitory interactions that impede accessibility of the exchange site in the Sec7 domain. These restraints can be relieved through activator Arf-GTP binding to an allosteric site comprising the PH domain and proximal autoinhibitory elements (Sec7-PH linker and C-terminal helix). Small-angle X-ray scattering and negative-stain electron microscopy were used to investigate the structural organization andmore » conformational dynamics of cytohesin-3 (Grp1) in autoinhibited and active states. The results support a model in which hinge dynamics in the autoinhibited state expose the activator site for Arf-GTP binding, while subsequent C-terminal helix unlatching and repositioning unleash conformational entropy in the Sec7-PH linker to drive exposure of the exchange site.« less

Adsorption of virus-like particles on ion exchange surface: Conformational changes at different pH detected by dual polarization interferometry.

PubMed

Yang, Yanli; Mengran Yu; Zhang, Songping; Ma, Guanghui; Su, Zhiguo

2015-08-21

Disassembling of virus-like particles (VLPs) like hepatitis B virus surface antigen (HB-VLPs) during chromatographic process has been identified as a major cause of loss of antigen activity. In this study, dual polarization interferometry (DPI) measurement, together with chromatography experiments, were performed to study the adsorption and conformational change of HB-VLPs on ion exchange surface at three different pHs. Changes in pH values of buffer solution showed only minimal effect on the HB-VLPs assembly and antigen activity, while significantly different degree of HB-VLPs disassembling was observed after ion exchange chromatography (IEC) at different pHs, indicating the conformational change of HB-VLPs caused mainly by its interactions with the adsorbent surface. By creating an ion exchange surface on chip surface, the conformational changes of HB-VLPs during adsorption to the surface were monitored in real time by DPI for the first time. As pH increased from 7.0 to 9.0, strong electrostatic interactions between oppositely charged HB-VLPs and the ion exchange surface make the HB-VLPs spread thinly or even adsorbed in disassembled formation on the surface as revealed by significant decrease in thickness of the adsorbed layer measured by DPI. Such findings were consistent with the results of IEC experiments operated at different pHs, that more disassembled HB-VLPs were detected in the eluted proteins at pH 9.0. At low pH like pH 5.0, however, possible bi-layer adsorption was involved as evidenced by an adsorbed layer thickness higher than average diameter of the HB-VLPs. The "lateral" protein-protein interactions might be unfavorable and would make additional contribution to the disassembling of HB-VLPs besides the primary mechanism related to the protein-surface interactions; therefore, the lowest antigen activity was observed after IEC at pH 5.0. Such real-time information on conformational change of VLPs is helpful for better understanding the real mechanism for the disassembling of VLPs on the solid-liquid interface. Copyright © 2015 Elsevier B.V. All rights reserved.

Equilibrious Strand Exchange Promoted by DNA Conformational Switching

NASA Astrophysics Data System (ADS)

Wu, Zhiguo; Xie, Xiao; Li, Puzhen; Zhao, Jiayi; Huang, Lili; Zhou, Xiang

2013-01-01

Most of DNA strand exchange reactions in vitro are based on toehold strategy which is generally nonequilibrium, and intracellular strand exchange mediated by proteins shows little sequence specificity. Herein, a new strand exchange promoted by equilibrious DNA conformational switching is verified. Duplexes containing c-myc sequence which is potentially converted into G-quadruplex are designed in this strategy. The dynamic equilibrium between duplex and G4-DNA is response to the specific exchange of homologous single-stranded DNA (ssDNA). The SER is enzyme free and sequence specific. No ATP is needed and the displaced ssDNAs are identical to the homologous ssDNAs. The SER products and exchange kenetics are analyzed by PAGE and the RecA mediated SER is performed as the contrast. This SER is a new feature of G4-DNAs and a novel strategy to utilize the dynamic equilibrium of DNA conformations.

Enhanced conformational sampling of carbohydrates by Hamiltonian replica-exchange simulation.

PubMed

Mishra, Sushil Kumar; Kara, Mahmut; Zacharias, Martin; Koca, Jaroslav

2014-01-01

Knowledge of the structure and conformational flexibility of carbohydrates in an aqueous solvent is important to improving our understanding of how carbohydrates function in biological systems. In this study, we extend a variant of the Hamiltonian replica-exchange molecular dynamics (MD) simulation to improve the conformational sampling of saccharides in an explicit solvent. During the simulations, a biasing potential along the glycosidic-dihedral linkage between the saccharide monomer units in an oligomer is applied at various levels along the replica runs to enable effective transitions between various conformations. One reference replica runs under the control of the original force field. The method was tested on disaccharide structures and further validated on biologically relevant blood group B, Lewis X and Lewis A trisaccharides. The biasing potential-based replica-exchange molecular dynamics (BP-REMD) method provided a significantly improved sampling of relevant conformational states compared with standard continuous MD simulations, with modest computational costs. Thus, the proposed BP-REMD approach adds a new dimension to existing carbohydrate conformational sampling approaches by enhancing conformational sampling in the presence of solvent molecules explicitly at relatively low computational cost.

The Area between Exchange Curves as a Measure of Conformational Differences in Hydrogen-Deuterium Exchange Mass Spectrometry Studies

PubMed Central

Mazur, Sharlyn J.; Weber, Daniel P.

2018-01-01

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) provides information about protein conformational mobility under native conditions. The area between exchange curves, Abec, a functional data analysis concept, was adapted to the interpretation of HDX-MS data and provides a useful measure of exchange curve dissimilarity for tests of significance. Importantly, for most globular proteins under native conditions, Abec values provide an estimate of the log ratio of exchange-competent fractions in the two states, and thus are related to differences in the free energy of microdomain unfolding. PMID:28236290

Reliable oligonucleotide conformational ensemble generation in explicit solvent for force field assessment using reservoir replica exchange molecular dynamics simulations

PubMed Central

Henriksen, Niel M.; Roe, Daniel R.; Cheatham, Thomas E.

2013-01-01

Molecular dynamics force field development and assessment requires a reliable means for obtaining a well-converged conformational ensemble of a molecule in both a time-efficient and cost-effective manner. This remains a challenge for RNA because its rugged energy landscape results in slow conformational sampling and accurate results typically require explicit solvent which increases computational cost. To address this, we performed both traditional and modified replica exchange molecular dynamics simulations on a test system (alanine dipeptide) and an RNA tetramer known to populate A-form-like conformations in solution (single-stranded rGACC). A key focus is on providing the means to demonstrate that convergence is obtained, for example by investigating replica RMSD profiles and/or detailed ensemble analysis through clustering. We found that traditional replica exchange simulations still require prohibitive time and resource expenditures, even when using GPU accelerated hardware, and our results are not well converged even at 2 microseconds of simulation time per replica. In contrast, a modified version of replica exchange, reservoir replica exchange in explicit solvent, showed much better convergence and proved to be both a cost-effective and reliable alternative to the traditional approach. We expect this method will be attractive for future research that requires quantitative conformational analysis from explicitly solvated simulations. PMID:23477537

Reliable oligonucleotide conformational ensemble generation in explicit solvent for force field assessment using reservoir replica exchange molecular dynamics simulations.

PubMed

Henriksen, Niel M; Roe, Daniel R; Cheatham, Thomas E

2013-04-18

Molecular dynamics force field development and assessment requires a reliable means for obtaining a well-converged conformational ensemble of a molecule in both a time-efficient and cost-effective manner. This remains a challenge for RNA because its rugged energy landscape results in slow conformational sampling and accurate results typically require explicit solvent which increases computational cost. To address this, we performed both traditional and modified replica exchange molecular dynamics simulations on a test system (alanine dipeptide) and an RNA tetramer known to populate A-form-like conformations in solution (single-stranded rGACC). A key focus is on providing the means to demonstrate that convergence is obtained, for example, by investigating replica RMSD profiles and/or detailed ensemble analysis through clustering. We found that traditional replica exchange simulations still require prohibitive time and resource expenditures, even when using GPU accelerated hardware, and our results are not well converged even at 2 μs of simulation time per replica. In contrast, a modified version of replica exchange, reservoir replica exchange in explicit solvent, showed much better convergence and proved to be both a cost-effective and reliable alternative to the traditional approach. We expect this method will be attractive for future research that requires quantitative conformational analysis from explicitly solvated simulations.

Data Quality and Interoperability Challenges for eHealth Exchange Participants: Observations from the Department of Veterans Affairs' Virtual Lifetime Electronic Record Health Pilot Phase.

PubMed

Botts, Nathan; Bouhaddou, Omar; Bennett, Jamie; Pan, Eric; Byrne, Colene; Mercincavage, Lauren; Olinger, Lois; Hunolt, Elaine; Cullen, Theresa

2014-01-01

Authors studied the United States (U.S.) Department of Veterans Affairs' (VA) Virtual Lifetime Electronic Record (VLER) Health pilot phase relative to two attributes of data quality - the adoption of eHealth Exchange data standards, and clinical content exchanged. The VLER Health pilot was an early effort in testing implementation of eHealth Exchange standards and technology. Testing included evaluation of exchange data from the VLER Health pilot sites partners: VA, U.S. Department of Defense (DoD), and private sector health care organizations. Domains assessed data quality and interoperability as it relates to: 1) conformance with data standards related to the underlying structure of C32 Summary Documents (C32) produced by eHealth Exchange partners; and 2) the types of C32 clinical content exchanged. This analysis identified several standards non-conformance issues in sample C32 files and informed further discourse on the methods needed to effectively monitor Health Information Exchange (HIE) data content and standards conformance.

Mechanism of the Exchange Reaction in HRAS from Multiscale Modeling

PubMed Central

Kapoor, Abhijeet; Travesset, Alex

2014-01-01

HRAS regulates cell growth promoting signaling processes by cycling between active (GTP-bound) and inactive (GDP-bound) states. Understanding the transition mechanism is central for the design of small molecules to inhibit the formation of RAS-driven tumors. Using a multiscale approach involving coarse-grained (CG) simulations, all-atom classical molecular dynamics (CMD; total of 3.02 µs), and steered molecular dynamics (SMD) in combination with Principal Component Analysis (PCA), we identified the structural features that determine the nucleotide (GDP) exchange reaction. We show that weakening the coupling between the SwitchI (residues 25–40) and SwitchII (residues 59–75) accelerates the opening of SwitchI; however, an open conformation of SwitchI is unstable in the absence of guanine nucleotide exchange factors (GEFs) and rises up towards the bound nucleotide to close the nucleotide pocket. Both I21 and Y32, play a crucial role in SwitchI transition. We show that an open SwitchI conformation is not necessary for GDP destabilization but is required for GDP/Mg escape from the HRAS. Further, we present the first simulation study showing displacement of GDP/Mg away from the nucleotide pocket. Both SwitchI and SwitchII, delays the escape of displaced GDP/Mg in the absence of GEF. Based on these results, a model for the mechanism of GEF in accelerating the exchange process is hypothesized. PMID:25272152

Evidence of conformational exchange averaging in the thermal rotational spectrum of ethyl cyanoformate.

PubMed

True, Nancy S

2006-06-15

The Stark modulated low resolution microwave spectrum of ethyl cyanoformate between 21.5 and 24.0 GHz at 210, 300, and 358 K, which shows the J + 1 <-- J = 8 <-- 7 bands of three species, is compared to simulations based on electronic structure calculations at the MP2/6-311++G theory level. Calculations at this theory level reproduce the relative energies of the syn-anti and syn-gauche conformers, obtained in a previous study, and indicate that the barrier to conformer exchange is approximately 360 cm(-1) higher in energy than the syn-anti minimum. Simulated spectra of the eigenstates of the calculated O-ethyl torsional potential function reproduce the relative intensities and shapes of the lower and higher frequency bands which correspond to transitions of the syn-anti and syn-gauche conformers, respectively, but fail to reproduce the intense center band in the experimental spectra. A model incorporating exchange averaging reproduces the intensity of the center band and its temperature dependence. These simulations indicate that a large fraction of the thermal population at all three temperatures undergoes conformational exchange with an average energy specific rate constant, , of approximately 25 GHz. This model can explain anomalies present in rotational spectra of many other compounds composed of mixtures of conformers.

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.

Gas-Phase Hydrogen-Deuterium Exchange Labeling of Select Peptide Ion Conformer Types: a Per-Residue Kinetics Analysis.

PubMed

Khakinejad, Mahdiar; Kondalaji, Samaneh Ghassabi; Tafreshian, Amirmahdi; Valentine, Stephen J

2015-07-01

The per-residue, gas-phase hydrogen deuterium exchange (HDX) kinetics for individual amino acid residues on selected ion conformer types of the model peptide KKDDDDDIIKIIK have been examined using ion mobility spectrometry (IMS) and HDX-tandem mass spectrometry (MS/MS) techniques. The [M + 4H](4+) ions exhibit two major conformer types with collision cross sections of 418 Å(2) and 446 Å(2); the [M + 3H](3+) ions also yield two different conformer types having collision cross sections of 340 Å(2) and 367 Å(2). Kinetics plots of HDX for individual amino acid residues reveal fast- and slow-exchanging hydrogens. The contributions of each amino acid residue to the overall conformer type rate constant have been estimated. For this peptide, N- and C-terminal K residues exhibit the greatest contributions for all ion conformer types. Interior D and I residues show decreased contributions. Several charge state trends are observed. On average, the D residues of the [M + 3H](3+) ions show faster HDX rate contributions compared with [M + 4H](4+) ions. In contrast the interior I8 and I9 residues show increased accessibility to exchange for the more elongated [M + 4H](4+) ion conformer type. The contribution of each residue to the overall uptake rate showed a good correlation with a residue hydrogen accessibility score model calculated using a distance from charge site and initial incorporation site for nominal structures obtained from molecular dynamic simulations (MDS).

Lanthanide paramagnetic probes for NMR spectroscopic studies of fast molecular conformational dynamics and temperature control. Effective six-site proton exchange in 18-crown-6 by exchange spectroscopy.

PubMed

Babailov, Sergey P

2012-02-06

(1)H and (13)C NMR measurements are reported for the CDCl(3) and CD(2)Cl(2) solutions of [La(18-crown-6)(NO(3))(3)] (I), [Pr(18-crown-6) (NO(3))(3)] (II), [Ce(18-crown-6)(NO(3))(3)] (III), and [Nd(18-crown-6)(NO(3))(3)] (IV) complexes. Temperature dependencies of the (1)H NMR spectra of paramagnetic II-IV have been analyzed using the dynamic NMR (DNMR) methods for six-site exchange. Two types of conformational dynamic processes were identified (the first one is conditioned by interconversion of complex enantiomeric forms and pseudorotation of a macrocycle molecule upon the C(2) symmetry axis; the second one is conditioned by macrocycle molecule inversion). Application of exchange spectroscopy (2D-EXSY) of DNMR for investigation of this dynamic system (II-IV) simplifies the assignment of the NMR signals and represents the first experimental study of multisite exchange. In the present work, the methodology of paramagnetic 4f (Ce, Pr, and Nd) probe applications for the study of free-energy, enthalpy, and entropy changes in chemical exchange processes, as well as the advantages of this method in a comparison with DNMR studies of diamagnetic substances, is discussed. In particular, as a result of paramagnetic chemical shifts in 4f complexes, the range of measurable rate constants expands considerably compared to the analogous range in diamagnetic compounds. Coordination compounds investigated in the paper represent new types of thermometric NMR sensors and lanthanide paramagnetic probes for in situ temperature control in solution.

Enantiodiscrimination of flexible cyclic solutes using NMR spectroscopy in polypeptide chiral mesophases: investigation of cis-decalin and THF.

PubMed

Aroulanda, Christie; Lafon, Olivier; Lesot, Philippe

2009-08-06

The conformational dynamics and orientational behavior of two model cyclic molecules, cis-decalin (cis-dec) and tetrahydrofurane (THF), dissolved in weakly ordering, polypeptidic chiral liquid crystals (CLCs) are theoretically discussed and experimentally investigated using deuterium and carbon-13 NMR spectroscopies. The analysis of enantiomeric and enantiotopic discriminations in these compounds is shown to depend on the rate of conformational exchange regime, slow or fast. The slow exchange regime is illustrated through the case of cis-dec at low temperature (243 K). We show that the deuterium NMR spectra in this regime can be qualitatively and quantitatively interpreted by restricting the conformational pathway of cis-dec to two enantiomeric conformers of C(2)-symmetry. The orientational order parameters of these interconverting enantiomers are calculated by matching the (2)H quadrupolar splittings with calculated conformer structures. The fast exchange regime is investigated through the examples of cis-dec at high temperature (356 K) and THF at room temperature (300 K). The (2)H NMR spectra above the coalescence temperature are analyzed by introducing the concept of "average molecular structure". This fictitious structure allows easily identifying NMR equivalences of solutes dissolved in CLC. However, it cannot be applied to determine consistent orientational order parameters. This study emphasizes that enantiotopic discriminations observed for flexible molecules in the fast exchange regime can be quantitatively interpreted only by considering the orientational order of each conformer.

Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis.

PubMed

Yaffe, Dana; Vergara-Jaque, Ariela; Forrest, Lucy R; Schuldiner, Shimon

2016-11-22

Neurotransporters located in synaptic vesicles are essential for communication between nerve cells in a process mediated by neurotransmitters. Vesicular monoamine transporter (VMAT), a member of the largest superfamily of transporters, mediates transport of monoamines to synaptic vesicles and storage organelles in a process that involves exchange of two H + per substrate. VMAT transport is inhibited by the competitive inhibitor reserpine, a second-line agent to treat hypertension, and by the noncompetitive inhibitor tetrabenazine, presently in use for symptomatic treatment of hyperkinetic disorders. During the transport cycle, VMAT is expected to occupy at least three different conformations: cytoplasm-facing, occluded, and lumen-facing. The lumen- to cytoplasm-facing transition, facilitated by protonation of at least one of the essential membrane-embedded carboxyls, generates a binding site for reserpine. Here we have identified residues in the cytoplasmic gate and show that mutations that disrupt the interactions in this gate also shift the equilibrium toward the cytoplasm-facing conformation, emulating the effect of protonation. These experiments provide significant insight into the role of proton translocation in the conformational dynamics of a mammalian H + -coupled antiporter, and also identify key aspects of the mode of action and binding of two potent inhibitors of VMAT2: reserpine binds the cytoplasm-facing conformation, and tetrabenazine binds the lumen-facing conformation.

Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry*

PubMed Central

Stokasimov, Ema; Rubenstein, Peter A.

2009-01-01

Actin can exist in multiple conformations necessary for normal function. Actin isoforms, although highly conserved in sequence, exhibit different biochemical properties and cellular roles. We used amide proton hydrogen/deuterium (HD) exchange detected by mass spectrometry to analyze conformational differences between Saccharomyces cerevisiae and muscle actins in the G and F forms to gain insight into these differences. We also utilized HD exchange to study interdomain and allosteric communication in yeast-muscle hybrid actins to better understand the conformational dynamics of actin. Areas showing differences in HD exchange between G- and F-actins are areas of intermonomer contacts, consistent with the current filament models. Our results showed greater exchange for yeast G-actin compared with muscle actin in the barbed end pivot region and areas in subdomains 1 and 2 and for F-actin in monomer-monomer contact areas. These results suggest greater flexibility of the yeast actin monomer and filament compared with muscle actin. For hybrid G-actins, the muscle-like and yeastlike parts of the molecule generally showed exchange characteristics resembling their parent actins. A few exceptions were a peptide on top of subdomain 2 and the pivot region between subdomains 1 and 3 with muscle actin-like exchange characteristics although the areas were yeastlike. These results demonstrate that there is cross-talk between subdomains 1 and 2 and the large and small domains. Hybrid F-actin data showing greater exchange compared with both yeast and muscle actins are consistent with mismatched yeast-muscle interfaces resulting in decreased stability of the hybrid filament contacts. PMID:19605362

Difference in dimer conformation between amyloid-β(1-42) and (1-43) proteins: Replica exchange molecular dynamics simulations in water

NASA Astrophysics Data System (ADS)

Yano, Atsushi; Okamoto, Akisumi; Nomura, Kazuya; Higai, Shin'ichi; Kurita, Noriyuki

2014-03-01

We searched stable conformations of amyloid-β (Aβ) dimers composed of Aβ(1-42) or Aβ(1-43) protein in water by replica-exchange molecular dynamics simulations and found that Thr43 of the C-terminal of Aβ(1-43) is hydrogen bonded to Arg5 of the same monomer in the Aβ(1-43) dimer, resulting in its ring-shaped conformation, while Aβ(1-42) has no such hydrogen-bond. This conformation is expected to aggregate more easily into a compact conformation of Aβ fibrils. We also investigated the binding affinity and the specific interactions between Aβ monomers by ab initio fragment molecular orbital calculations to elucidate which Aβ residues contribute to the dimerization.

Mechanism of extracellular ion exchange and binding-site occlusion in the sodium-calcium exchanger

PubMed Central

Lee, ChangKeun; Huang, Yihe; Faraldo-Gómez, José D.; Jiang, Youxing

2016-01-01

Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis. Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3Na+:1Ca2+ exchange stoichiometry, and reveals the conformational changes at the onset of the alternating-access transport mechanism. An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport. PMID:27183196

Mechanism of extracellular ion exchange and binding-site occlusion in a sodium/calcium exchanger

DOE PAGES

Liao, Jun; Marinelli, Fabrizio; Lee, Changkeun; ...

2016-05-16

Na +/Ca 2+ exchangers utilize the Na + electrochemical gradient across the plasma membrane to extrude intracellular Ca 2+, and play a central role in Ca 2+ homeostasis. Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na +, Ca 2+ or Sr 2+ in various occupancies and in an apo state. This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3:1Na +/Ca 2+ exchange stoichiometry, and reveals the conformational changes at the onset ofmore » the alternating-access transport mechanism. An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. Lastly, these calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na +/Ca 2+ antiport.« less

DICOM static and dynamic representation through unified modeling language

NASA Astrophysics Data System (ADS)

Martinez-Martinez, Alfonso; Jimenez-Alaniz, Juan R.; Gonzalez-Marquez, A.; Chavez-Avelar, N.

2004-04-01

The DICOM standard, as all standards, specifies in generic way the management in network and storage media environments of digital medical images and their related information. However, understanding the specifications for particular implementation is not a trivial work. Thus, this work is about understanding and modelling parts of the DICOM standard using Object Oriented methodologies, as part of software development processes. This has offered different static and dynamic views, according with the standard specifications, and the resultant models have been represented through the Unified Modelling Language (UML). The modelled parts are related to network conformance claim: Network Communication Support for Message Exchange, Message Exchange, Information Object Definitions, Service Class Specifications, Data Structures and Encoding, and Data Dictionary. The resultant models have given a better understanding about DICOM parts and have opened the possibility of create a software library to develop DICOM conformable PACS applications.

Exploring methods to expedite the recording of CEST datasets using selective pulse excitation

NASA Astrophysics Data System (ADS)

Yuwen, Tairan; Bouvignies, Guillaume; Kay, Lewis E.

2018-07-01

Chemical Exchange Saturation Transfer (CEST) has emerged as a powerful tool for studies of biomolecular conformational exchange involving the interconversion between a major, visible conformer and one or more minor, invisible states. Applications typically entail recording a large number of 2D datasets, each of which differs in the position of a weak radio frequency field, so as to generate a CEST profile for each nucleus from which the chemical shifts of spins in the invisible state(s) are obtained. Here we compare a number of band-selective CEST schemes for speeding up the process using either DANTE or cosine-modulated excitation approaches. We show that while both are essentially identical for applications such as 15N CEST, in cases where the probed spins are dipolar or scalar coupled to other like spins there can be advantages for the cosine-excitation scheme.

Enhanced conformational sampling using replica exchange with concurrent solute scaling and hamiltonian biasing realized in one dimension.

PubMed

Yang, Mingjun; Huang, Jing; MacKerell, Alexander D

2015-06-09

Replica exchange (REX) is a powerful computational tool for overcoming the quasi-ergodic sampling problem of complex molecular systems. Recently, several multidimensional extensions of this method have been developed to realize exchanges in both temperature and biasing potential space or the use of multiple biasing potentials to improve sampling efficiency. However, increased computational cost due to the multidimensionality of exchanges becomes challenging for use on complex systems under explicit solvent conditions. In this study, we develop a one-dimensional (1D) REX algorithm to concurrently combine the advantages of overall enhanced sampling from Hamiltonian solute scaling and the specific enhancement of collective variables using Hamiltonian biasing potentials. In the present Hamiltonian replica exchange method, termed HREST-BP, Hamiltonian solute scaling is applied to the solute subsystem, and its interactions with the environment to enhance overall conformational transitions and biasing potentials are added along selected collective variables associated with specific conformational transitions, thereby balancing the sampling of different hierarchical degrees of freedom. The two enhanced sampling approaches are implemented concurrently allowing for the use of a small number of replicas (e.g., 6 to 8) in 1D, thus greatly reducing the computational cost in complex system simulations. The present method is applied to conformational sampling of two nitrogen-linked glycans (N-glycans) found on the HIV gp120 envelope protein. Considering the general importance of the conformational sampling problem, HREST-BP represents an efficient procedure for the study of complex saccharides, and, more generally, the method is anticipated to be of general utility for the conformational sampling in a wide range of macromolecular systems.

Local Conformational Stability of HIV-1 gp120 in Unliganded and CD4-Bound States as Defined by Amide Hydrogen/Deuterium Exchange▿ †

PubMed Central

Kong, Leopold; Huang, Chih-chin; Coales, Stephen J.; Molnar, Kathleen S.; Skinner, Jeff; Hamuro, Yoshitomo; Kwong, Peter D.

2010-01-01

The binding reaction of the HIV-1 gp120 envelope glycoprotein to the CD4 receptor involves exceptional changes in enthalpy and entropy. Crystal structures of gp120 in unliganded and various ligand-bound states, meanwhile, reveal an inner domain able to fold into diverse conformations, a structurally invariant outer domain, and, in the CD4-bound state, a bridging sheet minidomain. These studies, however, provide only hints as to the flexibility of each state. Here we use amide hydrogen/deuterium exchange coupled to mass spectrometry to provide quantifications of local conformational stability for HIV-1 gp120 in unliganded and CD4-bound states. On average, unliganded core gp120 displayed >10,000-fold slower exchange of backbone-amide hydrogens than a theoretically unstructured protein of the same composition, with binding by CD4 reducing the rate of gp120 amide exchange a further 10-fold. For the structurally constant CD4, alterations in exchange correlated well with alterations in binding surface (P value = 0.0004). For the structurally variable gp120, however, reductions in flexibility extended outside the binding surface, and regions of expected high structural diversity (inner domain/bridging sheet) displayed roughly 20-fold more rapid exchange in the unliganded state than regions of low diversity (outer domain). Thus, despite an extraordinary reduction in entropy, neither unliganded gp120 nor free CD4 was substantially unstructured, suggesting that most of the diverse conformations that make up the gp120 unliganded state are reasonably ordered. The results provide a framework for understanding how local conformational stability influences entropic change, conformational diversity, and structural rearrangements in the gp120-CD4 binding reaction. PMID:20660185

Swinger RNAs with sharp switches between regular transcription and transcription systematically exchanging ribonucleotides: Case studies.

PubMed

Seligmann, Hervé

2015-09-01

During RNA transcription, DNA nucleotides A,C,G, T are usually matched by ribonucleotides A, C, G and U. However occasionally, this rule does not apply: transcript-DNA homologies are detectable only assuming systematic exchanges between ribonucleotides. Nine symmetric (X ↔ Y, e.g. A ↔ C) and fourteen asymmetric (X ↔ Y ↔ Z, e.g. A ↔ C ↔ G) exchanges exist, called swinger transcriptions. Putatively, polymerases occasionally stabilize in unspecified swinger conformations, possibly similar to transient conformations causing punctual misinsertions. This predicts chimeric transcripts, part regular, part swinger-transformed, reflecting polymerases switching to swinger polymerization conformation(s). Four chimeric Genbank transcripts (three from human mitochondrion and one murine cytosolic) are described here: (a) the 5' and 3' extremities reflect regular polymerization, the intervening sequence exchanges systematically between ribonucleotides (swinger rule G ↔ U, transcript (1), with sharp switches between regular and swinger sequences; (b) the 5' half is 'normal', the 3' half systematically exchanges ribonucleotides (swinger rule C ↔ G, transcript (2), with an intercalated sequence lacking homology; (c) the 3' extremity fits A ↔ G exchanges (10% of transcript length), the 5' half follows regular transcription; the intervening region seems a mix of regular and A ↔ G transcriptions (transcript 3); (d) murine cytosolic transcript 4 switches to A ↔ U + C ↔ G, and is fused with A ↔ U + C ↔ G swinger transformed precursor rRNA. In (c), each concomitant transcript 5' and 3' extremities match opposite genome strands. Transcripts 3 and 4 combine transcript fusions with partial swinger transcriptions. Occasional (usually sharp) switches between regular and swinger transcriptions reveal greater coding potential than detected until now, suggest stable polymerase swinger conformations. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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.

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Role of Detergents in Conformational Exchange of a G Protein-coupled Receptor*

PubMed Central

Chung, Ka Young; Kim, Tae Hun; Manglik, Aashish; Alvares, Rohan; Kobilka, Brian K.; Prosser, R. Scott

2012-01-01

The G protein-coupled β2-adrenoreceptor (β2AR) signals through the heterotrimeric G proteins Gs and Gi and β-arrestin. As such, the energy landscape of β2AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β2AR with a trifluoromethyl probe, 19F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β2AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of β2AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor. PMID:22893704

Role of detergents in conformational exchange of a G protein-coupled receptor.

PubMed

Chung, Ka Young; Kim, Tae Hun; Manglik, Aashish; Alvares, Rohan; Kobilka, Brian K; Prosser, R Scott

2012-10-19

The G protein-coupled β(2)-adrenoreceptor (β(2)AR) signals through the heterotrimeric G proteins G(s) and G(i) and β-arrestin. As such, the energy landscape of β(2)AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β(2)AR with a trifluoromethyl probe, (19)F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β(2)AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of β(2)AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor.

Longitudinal relaxation optimized amide 1H-CEST experiments for studying slow chemical exchange processes in fully protonated proteins.

PubMed

Yuwen, Tairan; Kay, Lewis E

2017-04-01

Chemical Exchange Saturation Transfer (CEST) experiments are increasingly used to study slow timescale exchange processes in biomolecules. Although 15 N- and 13 C-CEST have been the approaches of choice, the development of spin state selective 1 H-CEST pulse sequences that separate the effects of chemical and dipolar exchange [T. Yuwen, A. Sekhar and L. E. Kay, Angew Chem Int Ed Engl 2016 doi: 10.1002/anie.201610759 (Yuwen et al. 2017)] significantly increases the utility of 1 H-based experiments. Pulse schemes have been described previously for studies of highly deuterated proteins. We present here longitudinal-relaxation optimized amide 1 H-CEST experiments for probing chemical exchange in protonated proteins. Applications involving a pair of proteins are presented establishing that accurate 1 H chemical shifts of sparsely populated conformers can be obtained from simple analyses of 1 H-CEST profiles. A discussion of the inherent differences between 15 N-/ 13 C- and 1 H-CEST experiments is presented, leading to an optimal strategy for recording 1 H-CEST experiments.

Observing Holliday junction branch migration one step at a time

NASA Astrophysics Data System (ADS)

Ha, Taekjip

2004-03-01

During genetic recombination, two homologous DNA molecules undergo strand exchange to form a four-way DNA (Holliday) junction and the recognition and processing of this species by branch migration and junction resolving enzymes determine the outcome. We have used single molecule fluorescence techniques to study two intrinsic structural dynamics of the Holliday junction, stacking conformer transitions and spontaneous branch migration. Our studies show that the dynamics of branch migration, resolved with one base pair resolution, is determined by the stability of conformers which in turn depends on the local DNA sequences. Therefore, the energy landscape of Holliday junction branch migation is not uniform, but is rugged.

Enhanced Conformational Sampling of N-Glycans in Solution with Replica State Exchange Metadynamics.

PubMed

Galvelis, Raimondas; Re, Suyong; Sugita, Yuji

2017-05-09

Molecular dynamics (MD) simulation of a N-glycan in solution is challenging because of high-energy barriers of the glycosidic linkages, functional group rotational barriers, and numerous intra- and intermolecular hydrogen bonds. In this study, we apply different enhanced conformational sampling approaches, namely, metadynamics (MTD), the replica-exchange MD (REMD), and the recently proposed replica state exchange MTD (RSE-MTD), to a N-glycan in solution and compare the conformational sampling efficiencies of the approaches. MTD helps to cross the high-energy barrier along the ω angle by utilizing a bias potential, but it cannot enhance sampling of the other degrees of freedom. REMD ensures moderate-energy barrier crossings by exchanging temperatures between replicas, while it hardly crosses the barriers along ω. In contrast, RSE-MTD succeeds to cross the high-energy barrier along ω as well as to enhance sampling of the other degrees of freedom. We tested two RSE-MTD schemes: in one scheme, 64 replicas were simulated with the bias potential along ω at different temperatures, while simulations of four replicas were performed with the bias potentials for different CVs at 300 K. In both schemes, one unbiased replica at 300 K was included to compute conformational properties of the glycan. The conformational sampling of the former is better than the other enhanced sampling methods, while the latter shows reasonable performance without spending large computational resources. The latter scheme is likely to be useful when a N-glycan-attached protein is simulated.

Chair interconversion and reactivity of mannuronic acid esters.

PubMed

Rönnols, Jerk; Walvoort, Marthe T C; van der Marel, Gijsbert A; Codée, Jeroen D C; Widmalm, Göran

2013-12-14

Mannopyranosyluronic acids display a very unusual conformation behavior in that they often prefer to adopt a (1)C4 chair conformation. They are endowed with a strikingly high reactivity when used in a glycosylation reaction as a glycosyl donor. To investigate the unusual conformational behavior a series of mannuronic acid ester derivatives, comprising anomeric triflate species and O-methyl glycosides, was examined by dynamic NMR experiments, through lineshape analysis of (1)H and (19)F NMR spectra at various temperatures from -80 °C to 0 °C. Exchange rates between (4)C1 and (1)C4 chair conformations were found to depend on the electronic properties and the size of the C2 substituent (F, N3 or OBn) and the aglycon, with higher exchange rates for the glycosyl triflates and smaller C2 substituents. Low temperature (19)F exchange spectroscopy experiments showed that the covalently bound anomeric triflates did not exchange with free triflate species present in the reaction mixture. To relate the conformational behavior of the intermediate triflates to their reactivity in a glycosylation reaction, their relative reactivity was determined via competition reactions monitored by (1)H NMR spectroscopy at low temperature. The 2-O-benzyl ether compound was found to be most reactive whereas the 2-fluoro compound - the most flexible of the studied compounds - was least reactive. Whereas the ring-flip of the mannuronic acids is important for the enhanced reactivity of the donors, the rate of the ring-flip has little influence on the relative reactivity.

Microcontroller-driven fluid-injection system for atomic force microscopy.

PubMed

Kasas, S; Alonso, L; Jacquet, P; Adamcik, J; Haeberli, C; Dietler, G

2010-01-01

We present a programmable microcontroller-driven injection system for the exchange of imaging medium during atomic force microscopy. Using this low-noise system, high-resolution imaging can be performed during this process of injection without disturbance. This latter circumstance was exemplified by the online imaging of conformational changes in DNA molecules during the injection of anticancer drug into the fluid chamber.

Residue selective 15N CEST and CPMG experiments for studies of millisecond timescale protein dynamics.

PubMed

Niu, Xiaogang; Ding, Jienv; Zhang, Wenbo; Li, Qianwen; Hu, Yunfei; Jin, Changwen

2018-06-01

Proteins are intrinsically dynamic molecules and undergo exchanges among multiple conformations to perform biological functions. The CPMG relaxation dispersion and CEST experiments are two important solution NMR techniques for characterizing the conformational exchange processes on the millisecond timescale. Traditional pseudo 3D 15 N CEST and CPMG experiments have certain limitations in their applications. For example, both experiments have low sensitivity for broadened resonances, and the process of optimizing sample conditions and experimental parameters are often time consuming. To overcome these limitations, we herein present a new set of residue selective 15 N CEST and CPMG pulse sequences by employing the Hartmann-Hahn cross-polarization transfer of magnetization in both 1D and 2D schemes. Combined with frequency labeling in the indirect dimension using only a small number of increments, the pulse sequences in the 2D scheme can be applied on resonances in overlapped regions of the 1 H- 15 N HSQC spectrum. The pulse sequences were further applied on several proteins, demonstrating their advantages over the traditional CEST and CPMG experiments under specific circumstances. Copyright © 2018 Elsevier Inc. All rights reserved.

A dynamic N-capping motif in cytochrome b5: evidence for a pH-controlled conformational switch.

PubMed

Davis, Ronald B; Lecomte, Juliette T J

2006-05-01

Apocytochrome b5 is a marginally stable protein exhibiting under native conditions a slow conformational exchange in its C-terminal region. The affected elements of secondary structure include a 3(10)-helix containing at its N-terminus a histidine Ncap and a subsequent proline. Participation of the neutral histidine side-chain in backbone amide capping lowers the imidazole pKa. To explore the nature of the conformational exchange in the protein and determine whether it is related to cis-trans isomerization of the His-Pro bond, three octapeptides encompassing the helix were synthesized and studied by NMR spectroscopy. One corresponded to the wild-type sequence, the second was the D-histidine epimer, and the third contained an alanine in place of the proline. It was found that the rates of cis-trans interconversion in the proline-containing peptides were slower than the rates of the conformational exchange in the protein. In addition, the wild-type peptide hinted at a predisposition for Ncap formation when in the trans configuration. Analysis of the pH response of the peptides and protein suggested that at pH near neutral, the conformational exchange detected in the protein involved only species with a trans His-Pro bond and could be approximated with a three-state model by which the terminal helix sampled a locally unfolded state. This state, which contained an uncapped histidine with a normal pKa, partitioned into neutral and protonated populations according to pH. The intrinsic conformational bias of the wild-type peptide and the pH-driven equilibria illustrated how a 3(10)-element could serve as a nucleation site for structural rearrangement. 2005 Wiley-Liss, Inc.

Asynchronous Replica Exchange Software for Grid and Heterogeneous Computing.

PubMed

Gallicchio, Emilio; Xia, Junchao; Flynn, William F; Zhang, Baofeng; Samlalsingh, Sade; Mentes, Ahmet; Levy, Ronald M

2015-11-01

Parallel replica exchange sampling is an extended ensemble technique often used to accelerate the exploration of the conformational ensemble of atomistic molecular simulations of chemical systems. Inter-process communication and coordination requirements have historically discouraged the deployment of replica exchange on distributed and heterogeneous resources. Here we describe the architecture of a software (named ASyncRE) for performing asynchronous replica exchange molecular simulations on volunteered computing grids and heterogeneous high performance clusters. The asynchronous replica exchange algorithm on which the software is based avoids centralized synchronization steps and the need for direct communication between remote processes. It allows molecular dynamics threads to progress at different rates and enables parameter exchanges among arbitrary sets of replicas independently from other replicas. ASyncRE is written in Python following a modular design conducive to extensions to various replica exchange schemes and molecular dynamics engines. Applications of the software for the modeling of association equilibria of supramolecular and macromolecular complexes on BOINC campus computational grids and on the CPU/MIC heterogeneous hardware of the XSEDE Stampede supercomputer are illustrated. They show the ability of ASyncRE to utilize large grids of desktop computers running the Windows, MacOS, and/or Linux operating systems as well as collections of high performance heterogeneous hardware devices.

Reactions driving conformational movements (molecular motors) in gels: conformational and structural chemical kinetics.

PubMed

Otero, Toribio F

2017-01-18

In this perspective the empirical kinetics of conducting polymers exchanging anions and solvent during electrochemical reactions to get dense reactive gels is reviewed. The reaction drives conformational movements of the chains (molecular motors), exchange of ions and solvent with the electrolyte and structural (relaxation, swelling, shrinking and compaction) gel changes. Reaction-driven structural changes are identified and quantified from electrochemical responses. The empirical reaction activation energy (E a ), the reaction coefficient (k) and the reaction orders (α and β) change as a function of the conformational energy variation during the reaction. This conformational energy becomes an empirical magnitude. E a , k, α and β include and provide quantitative conformational and structural information. The chemical kinetics becomes structural chemical kinetics (SCK) for reactions driving conformational movements of the reactants. The electrochemically stimulated conformational relaxation model describes empirical results and some results from the literature for biochemical reactions. In parallel the development of an emerging technological world of soft, wet, multifunctional and biomimetic tools and anthropomorphic robots driven by reactions of the constitutive material, as in biological organs, can be now envisaged being theoretically supported by the kinetic model.

Conformational Assessment of Adnectin and Adnectin-Drug Conjugate by Hydrogen/Deuterium Exchange Mass Spectrometry

NASA Astrophysics Data System (ADS)

Huang, Richard Y.-C.; O'Neil, Steven R.; Lipovšek, Daša; Chen, Guodong

2018-05-01

Higher-order structure (HOS) characterization of therapeutic protein-drug conjugates for comprehensive assessment of conjugation-induced protein conformational changes is an important consideration in the biopharmaceutical industry to ensure proper behavior of protein therapeutics. In this study, conformational dynamics of a small therapeutic protein, adnectin 1, together with its drug conjugate were characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS) with different spatial resolutions. Top-down HDX allows detailed assessment of the residue-level deuterium content in the payload conjugation region. HDX-MS dataset revealed the ability of peptide-based payload/linker to retain deuterium in HDX experiments. Combined results from intact, top-down, and bottom-up HDX indicated no significant conformational changes of adnectin 1 upon payload conjugation. [Figure not available: see fulltext.

Monte Carlo replica-exchange based ensemble docking of protein conformations.

PubMed

Zhang, Zhe; Ehmann, Uwe; Zacharias, Martin

2017-05-01

A replica-exchange Monte Carlo (REMC) ensemble docking approach has been developed that allows efficient exploration of protein-protein docking geometries. In addition to Monte Carlo steps in translation and orientation of binding partners, possible conformational changes upon binding are included based on Monte Carlo selection of protein conformations stored as ordered pregenerated conformational ensembles. The conformational ensembles of each binding partner protein were generated by three different approaches starting from the unbound partner protein structure with a range spanning a root mean square deviation of 1-2.5 Å with respect to the unbound structure. Because MC sampling is performed to select appropriate partner conformations on the fly the approach is not limited by the number of conformations in the ensemble compared to ensemble docking of each conformer pair in ensemble cross docking. Although only a fraction of generated conformers was in closer agreement with the bound structure the REMC ensemble docking approach achieved improved docking results compared to REMC docking with only the unbound partner structures or using docking energy minimization methods. The approach has significant potential for further improvement in combination with more realistic structural ensembles and better docking scoring functions. Proteins 2017; 85:924-937. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Regulation of Phenylalanine Hydroxylase: Conformational Changes Upon Phenylalanine Binding Detected by H/D Exchange and Mass Spectrometry†

PubMed Central

Li, Jun; Dangott, Lawrence J.; Fitzpatrick, Paul F.

2010-01-01

Phenylalanine acts as an allosteric activator of the tetrahydropterin-dependent enzyme phenylalanine hydroxylase. Hydrogen/deuterium exchange monitored by mass spectrometry has been used to gain insight into local conformational changes accompanying activation of rat phenylalanine hydroxylase by phenylalanine. Peptides in the regulatory and catalytic domains that lie in the interface between these two domains show large increases in the extent of deuterium incorporation from solvent in the presence of phenylalanine. In contrast, the effects of phenylalanine on the exchange kinetics of a mutant enzyme lacking the regulatory domain are limited to peptides surrounding the binding site for the amino acid substrate. These results support a model in which the N-terminus of the protein acts as an inhibitory peptide, with phenylalanine binding causing a conformational change in the regulatory domain that alters the interaction between the catalytic and regulatory domains. PMID:20307070

Eikonalization of conformal blocks

DOE PAGES

Fitzpatrick, A. Liam; Kaplan, Jared; Walters, Matthew T.; ...

2015-09-03

Classical field configurations such as the Coulomb potential and Schwarzschild solution are built from the t-channel exchange of many light degrees of freedom. We study the CFT analog of this phenomenon, which we term the 'eikonalization' of conformal blocks. We show that when an operator T appears in the OPE Ο(x)Ο(0), then the large spin Fock space states [TT···T] ℓ also appear in this OPE with a computable coefficient. The sum over the exchange of these Fock space states in an correlator build the classical 'T field' in the dual AdS description. In some limits the sum of all Fockmore » space exchanges can be represented as the exponential of a single T exchange in the 4-pt correlator of O. Our results should be useful for systematizing 1/ℓ perturbation theory in general CFTs and simplifying the computation of large spin OPE coefficients. As examples we obtain the leading log ℓ dependence of Fock space conformal block coefficients, and we directly compute the OPE coefficients of the simplest ‘triple-trace’ operators.« less

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Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-20

...-Regulatory Organizations; BATS Y-Exchange, Inc.; Notice of Filing of Proposed Rule Change To Amend BATS Y-Exchange Rules To Conform to the Current Rules of BATS Exchange October 13, 2010. Pursuant to Section 19(b... hereby given that on October 4, 2010, BATS Y-Exchange, Inc. (the ``Exchange'' or ``BYX'') filed with the...

[Hydrogen exchange and proteolytic degradation of ribonuclease A. Similarities and distinctions of the kinetic mechanisms].

PubMed

Abaturov, L V; Nosova, N G

2007-01-01

The studies by IR spectroscopy of the temperature dependence of the H-D exchange rate of the RNase A peptide NH atoms permit one to characterize two types of conformation fluctuations, local and global. A comparison with the temperature dependence of the proteolytic degradation rate of RNase A shows that similar in nature fluctuations allow for the H-D exchange of NH atoms and the splitting of peptide bonds of the native protein. In the low temperature region, both processes occur through local fluctuations, by way of the EX2 mechanism, and in the high temperature region, they occur through global fluctuations with the overall denaturation desorganization of the native structure, by way of the EX1 mechanism. The biphasic dependence of the rate of H-D exchange and proteolytic degradation of RNase A on urea concentration is also explained by the combination of local and global fluctuations.

Structural outline of the detailed mechanism for elongation factor Ts-mediated guanine nucleotide exchange on elongation factor Tu.

PubMed

Thirup, Søren S; Van, Lan Bich; Nielsen, Tine K; Knudsen, Charlotte R

2015-07-01

Translation elongation factor EF-Tu belongs to the superfamily of guanine-nucleotide binding proteins, which play key cellular roles as regulatory switches. All G-proteins require activation via exchange of GDP for GTP to carry out their respective tasks. Often, guanine-nucleotide exchange factors are essential to this process. During translation, EF-Tu:GTP transports aminoacylated tRNA to the ribosome. GTP is hydrolyzed during this process, and subsequent reactivation of EF-Tu is catalyzed by EF-Ts. The reaction path of guanine-nucleotide exchange is structurally poorly defined for EF-Tu and EF-Ts. We have determined the crystal structures of the following reaction intermediates: two structures of EF-Tu:GDP:EF-Ts (2.2 and 1.8Å resolution), EF-Tu:PO4:EF-Ts (1.9Å resolution), EF-Tu:GDPNP:EF-Ts (2.2Å resolution) and EF-Tu:GDPNP:pulvomycin:Mg(2+):EF-Ts (3.5Å resolution). These structures provide snapshots throughout the entire exchange reaction and suggest a mechanism for the release of EF-Tu in its GTP conformation. An inferred sequence of events during the exchange reaction is presented. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Studying ion exchange in solution and at biological membranes by FCS.

PubMed

Widengren, Jerker

2013-01-01

By FCS, a wide range of processes can be studied, covering time ranges from subnanoseconds to seconds. In principle, any process at equilibrium conditions can be measured, which reflects itself by a change in the detected fluorescence intensity. In this review, it is described how FCS and variants thereof can be used to monitor ion exchange, in solution and along biological membranes. Analyzing fluorescence fluctuations of ion-sensitive fluorophores by FCS offers selective advantages over other techniques for measuring local ion concentrations, and, in particular, for studying exchange kinetics of ions on a very local scale. This opens for several areas of application. The FCS approach was used to investigate fundamental aspects of proton exchange at and along biological membranes. The protonation relaxation rate, as measured by FCS for a pH-sensitive dye, can also provide information about local accessibility/interaction of a particular labeling site and conformational states of biomolecules, in a similar fashion as in a fluorescence quenching experiment. The same FCS concept can also be applied to ion exchange studies using other ion-sensitive fluorophores, and by use of dyes sensitive to other ambient conditions the concept can be extended also beyond ion exchange studies. Copyright © 2013 Elsevier Inc. All rights reserved.

NMR based solvent exchange experiments to understand the conformational preference of intrinsically disordered proteins using FG-nucleoporin peptide as a model

PubMed Central

Heisel, Kurt A.; Krishnan, V. V.

2014-01-01

The conformational preference of a peptide with three phenylalanine-glycine (FG) repeats from the intrinsically disordered domain of nucleoporin 159 (nup159) from the yeast nucleopore complex (NPC) is studied. Conformational states of this FG-peptide in dimethyl sulfoxide (DMSO), a non-native solvent are first studied. A solvent exchange scheme is designed and performed to understand how the conformational preferences of the peptide are altered as the solvent shifts from DMSO to water. An ensemble of structures of a 19-residue peptide is determined based on 13Cα, 1Hα, and 1HN chemical shifts and with inter-proton distances. An experimental model is then presented where chemical shifts and amide-proton temperature dependence is probed at changing DMSO to water ratios. These co-solvent experiments provide evidence of a conformational change as the fraction of water increases by the stark change in the behavior of amide protons under varied temperature. This investigation provides a NMR based experimental method in the field of intrinsically disordered proteins to realize conformational transitions from a non-native set of structures (in DMSO) to a native set of disordered conformers (in water). PMID:24037535

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2011-10-06

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HLA-DM mediates epitope selection by a "compare-exchange" mechanism when a potential peptide pool is available.

PubMed

Ferrante, Andrea; Anderson, Matthew W; Klug, Candice S; Gorski, Jack

2008-01-01

HLA-DM (DM) mediates exchange of peptides bound to MHC class II (MHCII) during the epitope selection process. Although DM has been shown to have two activities, peptide release and MHC class II refolding, a clear characterization of the mechanism by which DM facilitates peptide exchange has remained elusive. We have previously demonstrated that peptide binding to and dissociation from MHCII in the absence of DM are cooperative processes, likely related to conformational changes in the peptide-MHCII complex. Here we show that DM promotes peptide release by a non-cooperative process, whereas it enhances cooperative folding of the exchange peptide. Through electron paramagnetic resonance (EPR) and fluorescence polarization (FP) we show that DM releases prebound peptide very poorly in the absence of a candidate peptide for the exchange process. The affinity and concentration of the candidate peptide are also important for the release of the prebound peptide. Increased fluorescence energy transfer between the prebound and exchange peptides in the presence of DM is evidence for a tetramolecular complex which resolves in favor of the peptide that has superior folding properties. This study shows that both the peptide releasing activity on loaded MHCII and the facilitating of MHCII binding by a candidate exchange peptide are integral to DM mediated epitope selection. The exchange process is initiated only in the presence of candidate peptides, avoiding possible release of a prebound peptide and loss of a potential epitope. In a tetramolecular transitional complex, the candidate peptides are checked for their ability to replace the pre-bound peptide with a geometry that allows the rebinding of the original peptide. Thus, DM promotes a "compare-exchange" sorting algorithm on an available peptide pool. Such a "third party"-mediated mechanism may be generally applicable for diverse ligand recognition in other biological systems.

Transition Pathway and Its Free-Energy Profile: A Protocol for Protein Folding Simulations

PubMed Central

Lee, In-Ho; Kim, Seung-Yeon; Lee, Jooyoung

2013-01-01

We propose a protocol that provides a systematic definition of reaction coordinate and related free-energy profile as the function of temperature for the protein-folding simulation. First, using action-derived molecular dynamics (ADMD), we investigate the dynamic folding pathway model of a protein between a fixed extended conformation and a compact conformation. We choose the pathway model to be the reaction coordinate, and the folding and unfolding processes are characterized by the ADMD step index, in contrast to the common a priori reaction coordinate as used in conventional studies. Second, we calculate free-energy profile as the function of temperature, by employing the replica-exchange molecular dynamics (REMD) method. The current method provides efficient exploration of conformational space and proper characterization of protein folding/unfolding dynamics from/to an arbitrary extended conformation. We demonstrate that combination of the two simulation methods, ADMD and REMD, provides understanding on molecular conformational changes in proteins. The protocol is tested on a small protein, penta-peptide of met-enkephalin. For the neuropeptide met-enkephalin system, folded, extended, and intermediate sates are well-defined through the free-energy profile over the reaction coordinate. Results are consistent with those in the literature. PMID:23917881

Efficiency of Adaptive Temperature-Based Replica Exchange for Sampling Large-Scale Protein Conformational Transitions.

PubMed

Zhang, Weihong; Chen, Jianhan

2013-06-11

Temperature-based replica exchange (RE) is now considered a principal technique for enhanced sampling of protein conformations. It is also recognized that existence of sharp cooperative transitions (such as protein folding/unfolding) can lead to temperature exchange bottlenecks and significantly reduce the sampling efficiency. Here, we revisit two adaptive temperature-based RE protocols, namely, exchange equalization (EE) and current maximization (CM), that were previously examined using atomistic simulations (Lee and Olson, J. Chem. Physics2011, 134, 24111). Both protocols aim to overcome exchange bottlenecks by adaptively adjusting the simulation temperatures, either to achieve uniform exchange rates (in EE) or to maximize temperature diffusion (CM). By designing a realistic yet computationally tractable coarse-grained protein model, one can sample many reversible folding/unfolding transitions using conventional constant temperature molecular dynamics (MD), standard REMD, EE-REMD, and CM-REMD. This allows rigorous evaluation of the sampling efficiency, by directly comparing the rates of folding/unfolding transitions and convergence of various thermodynamic properties of interest. The results demonstrate that both EE and CM can indeed enhance temperature diffusion compared to standard RE, by ∼3- and over 10-fold, respectively. Surprisingly, the rates of reversible folding/unfolding transitions are similar in all three RE protocols. The convergence rates of several key thermodynamic properties, including the folding stability and various 1D and 2D free energy surfaces, are also similar. Therefore, the efficiency of RE protocols does not appear to be limited by temperature diffusion, but by the inherent rates of spontaneous large-scale conformational rearrangements. This is particularly true considering that virtually all RE simulations of proteins in practice involve exchange attempt frequencies (∼ps(-1)) that are several orders of magnitude faster than the slowest protein motions (∼μs(-1)). Our results also suggest that the efficiency of RE will not likely be improved by other protocols that aim to accelerate exchange or temperature diffusion. Instead, protocols with some types of guided tempering will likely be necessary to drive faster large-scale conformational transitions.

A Small Molecule Causes a Population Shift in the Conformational Landscape of an Intrinsically Disordered Protein.

PubMed

Ban, David; Iconaru, Luigi I; Ramanathan, Arvind; Zuo, Jian; Kriwacki, Richard W

2017-10-04

Intrinsically disordered proteins (IDPs) have roles in myriad biological processes and numerous human diseases. However, kinetic and amplitude information regarding their ground-state conformational fluctuations has remained elusive. We demonstrate using nuclear magnetic resonance (NMR)-based relaxation dispersion that the D2 domain of p27 Kip1 , a prototypical IDP, samples multiple discrete, rapidly exchanging conformational states. By combining NMR with mutagenesis and small-angle X-ray scattering (SAXS), we show that these states involve aromatic residue clustering through long-range hydrophobic interactions. Theoretical studies have proposed that small molecules bind promiscuously to IDPs, causing expansion of their conformational landscapes. However, on the basis of previous NMR-based screening results, we show here that compound binding only shifts the populations of states that existed within the ground state of apo p27-D2 without changing the barriers between states. Our results provide atomic resolution insight into how a small molecule binds an IDP and emphasize the need to examine motions on the low microsecond time scale when probing these types of interactions.

Conformational analysis of HAMLET, the folding variant of human alpha-lactalbumin associated with apoptosis.

PubMed

Casbarra, Annarita; Birolo, Leila; Infusini, Giuseppe; Dal Piaz, Fabrizio; Svensson, Malin; Pucci, Piero; Svanborg, Catharina; Marino, Gennaro

2004-05-01

A combination of hydrogen/deuterium (H/D) exchange and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the conformation in solution of HAMLET, the folding variant of human alpha-lactalbumin, complexed to oleic acid, that induces apoptosis in tumor and immature cells. Although near- and far-UV CD and fluorescence spectroscopy were not able to discriminate between HAMLET and apo-alpha-lactalbumin, H/D exchange experiments clearly showed that they correspond to two distinct conformational states, with HAMLET incorporating a greater number of deuterium atoms than the apo and holo forms. Complementary proteolysis experiments revealed that HAMLET and apo are both accessible to proteases in the beta-domain but showed substantial differences in accessibility to proteases at specific sites. The overall results indicated that the conformational changes associated with the release of Ca2+ are not sufficient to induce the HAMLET conformation. Metal depletion might represent the first event to produce a partial unfolding in the beta-domain of alpha-lactalbumin, but some more unfolding is needed to generate the active conformation HAMLET, very likely allowing the protein to bind the C18:1 fatty acid moiety. On the basis of these data, a putative binding site of the oleic acid, which stabilizes the HAMLET conformation, is proposed.

Enhanced Conformational Sampling Using Replica Exchange with Collective-Variable Tempering.

PubMed

Gil-Ley, Alejandro; Bussi, Giovanni

2015-03-10

The computational study of conformational transitions in RNA and proteins with atomistic molecular dynamics often requires suitable enhanced sampling techniques. We here introduce a novel method where concurrent metadynamics are integrated in a Hamiltonian replica-exchange scheme. The ladder of replicas is built with different strengths of the bias potential exploiting the tunability of well-tempered metadynamics. Using this method, free-energy barriers of individual collective variables are significantly reduced compared with simple force-field scaling. The introduced methodology is flexible and allows adaptive bias potentials to be self-consistently constructed for a large number of simple collective variables, such as distances and dihedral angles. The method is tested on alanine dipeptide and applied to the difficult problem of conformational sampling in a tetranucleotide.

Probing the conformation of a conserved glutamic acid within the Cl− pathway of a CLC H+/Cl− exchanger

PubMed Central

Vien, Malvin

2017-01-01

The CLC proteins form a broad family of anion-selective transport proteins that includes both channels and exchangers. Despite extensive structural, functional, and computational studies, the transport mechanism of the CLC exchangers remains poorly understood. Several transport models have been proposed but have failed to capture all the key features of these transporters. Multiple CLC crystal structures have suggested that a conserved glutamic acid, Gluex, can adopt three conformations and that the interconversion of its side chain between these states underlies H+/Cl− exchange. One of these states, in which Gluex occupies the central binding site (Scen) while Cl− ions fill the internal and external sites (Sint and Sext), has only been observed in one homologue, the eukaryotic cmCLC. The existence of such a state in other CLCs has not been demonstrated. In this study, we find that during transport, the prototypical prokaryotic CLC exchanger, CLC-ec1, adopts a conformation with functional characteristics that match those predicted for a cmCLC-like state, with Gluex trapped in Scen between two Cl− ions. Transport by CLC-ec1 is reduced when [Cl−] is symmetrically increased on both sides of the membrane and mutations that disrupt the hydrogen bonds stabilizing Gluex in Scen destabilize this trapped state. Furthermore, inhibition of transport by high [Cl−] is abolished in the E148A mutant, in which the Gluex side chain is removed. Collectively, our results suggest that, during the CLC transport cycle, Gluex can occupy Scen as well as the Sext position in which it has been captured crystallographically and that hydrogen bonds with the side chains of residues that coordinate ion binding to Scen play a role in determining the equilibrium between these two conformations. PMID:28246117

Experimentally assessing molecular dynamics sampling of the protein native state conformational distribution

PubMed Central

Hernández, Griselda; Anderson, Janet S.; LeMaster, David M.

2012-01-01

The acute sensitivity to conformation exhibited by amide hydrogen exchange reactivity provides a valuable test for the physical accuracy of model ensembles developed to represent the Boltzmann distribution of the protein native state. A number of molecular dynamics studies of ubiquitin have predicted a well-populated transition in the tight turn immediately preceding the primary site of proteasome-directed polyubiquitylation Lys 48. Amide exchange reactivity analysis demonstrates that this transition is 103-fold rarer than these predictions. More strikingly, for the most populated novel conformational basin predicted from a recent 1 ms MD simulation of bovine pancreatic trypsin inhibitor (at 13% of total), experimental hydrogen exchange data indicates a population below 10−6. The most sophisticated efforts to directly incorporate experimental constraints into the derivation of model protein ensembles have been applied to ubiquitin, as illustrated by three recently deposited studies (PDB codes 2NR2, 2K39 and 2KOX). Utilizing the extensive set of experimental NOE constraints, each of these three ensembles yields a modestly more accurate prediction of the exchange rates for the highly exposed amides than does a standard unconstrained molecular simulation. However, for the less frequently exposed amide hydrogens, the 2NR2 ensemble offers no improvement in rate predictions as compared to the unconstrained MD ensemble. The other two NMR-constrained ensembles performed markedly worse, either underestimating (2KOX) or overestimating (2K39) the extent of conformational diversity. PMID:22425325

Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected ¹³C CPMG relaxation dispersion.

PubMed

Weininger, Ulrich; Respondek, Michal; Akke, Mikael

2012-09-01

Protein dynamics on the millisecond time scale commonly reflect conformational transitions between distinct functional states. NMR relaxation dispersion experiments have provided important insights into biologically relevant dynamics with site-specific resolution, primarily targeting the protein backbone and methyl-bearing side chains. Aromatic side chains represent attractive probes of protein dynamics because they are over-represented in protein binding interfaces, play critical roles in enzyme catalysis, and form an important part of the core. Here we introduce a method to characterize millisecond conformational exchange of aromatic side chains in selectively (13)C labeled proteins by means of longitudinal- and transverse-relaxation optimized CPMG relaxation dispersion. By monitoring (13)C relaxation in a spin-state selective manner, significant sensitivity enhancement can be achieved in terms of both signal intensity and the relative exchange contribution to transverse relaxation. Further signal enhancement results from optimizing the longitudinal relaxation recovery of the covalently attached (1)H spins. We validated the L-TROSY-CPMG experiment by measuring fast folding-unfolding kinetics of the small protein CspB under native conditions. The determined unfolding rate matches perfectly with previous results from stopped-flow kinetics. The CPMG-derived chemical shift differences between the folded and unfolded states are in excellent agreement with those obtained by urea-dependent chemical shift analysis. The present method enables characterization of conformational exchange involving aromatic side chains and should serve as a valuable complement to methods developed for other types of protein side chains.

On-tissue Direct Monitoring of Global Hydrogen/Deuterium Exchange by MALDI Mass Spectrometry: Tissue Deuterium Exchange Mass Spectrometry (TDXMS)*

PubMed Central

Quanico, Jusal; Franck, Julien

2016-01-01

Hydrogen/deuterium exchange mass spectrometric (H/DXMS) methods for protein structural analysis are conventionally performed in solution. We present Tissue Deuterium Exchange Mass Spectrometry (TDXMS), a method to directly monitor deuterium uptake on tissue, as a means to better approximate the deuterium exchange behavior of proteins in their native microenvironment. Using this method, a difference in deuterium uptake behavior was observed when the same proteins were monitored in solution and on tissue. The higher maximum deuterium uptake at equilibrium for all proteins analyzed in solution suggests a more open conformation in the absence of interacting partners normally observed on tissue. We also demonstrate a difference in the deuterium uptake behavior of a few proteins across different morphological regions of the same tissue section. Modifications of the total number of hydrogens exchanged, as well as the kinetics of exchange, were both observed. These results provide information on the implication of protein interactions with partners as well as on the conformational changes related to these interactions, and illustrate the importance of examining protein deuterium exchange behavior in the presence of its specific microenvironment directly at the level of tissues. PMID:27512083

Heteronuclear Adiabatic Relaxation Dispersion (HARD) for quantitative analysis of conformational dynamics in proteins.

PubMed

Traaseth, Nathaniel J; Chao, Fa-An; Masterson, Larry R; Mangia, Silvia; Garwood, Michael; Michaeli, Shalom; Seelig, Burckhard; Veglia, Gianluigi

2012-06-01

NMR relaxation methods probe biomolecular motions over a wide range of timescales. In particular, the rotating frame spin-lock R(1ρ) and Carr-Purcell-Meiboom-Gill (CPMG) R(2) experiments are commonly used to characterize μs to ms dynamics, which play a critical role in enzyme folding and catalysis. In an effort to complement these approaches, we introduced the Heteronuclear Adiabatic Relaxation Dispersion (HARD) method, where dispersion in rotating frame relaxation rate constants (longitudinal R(1ρ) and transverse R(2ρ)) is created by modulating the shape and duration of adiabatic full passage (AFP) pulses. Previously, we showed the ability of the HARD method to detect chemical exchange dynamics in the fast exchange regime (k(ex)∼10(4)-10(5) s(-1)). In this article, we show the sensitivity of the HARD method to slower exchange processes by measuring R(1ρ) and R(2ρ) relaxation rates for two soluble proteins (ubiquitin and 10C RNA ligase). One advantage of the HARD method is its nominal dependence on the applied radio frequency field, which can be leveraged to modulate the dispersion in the relaxation rate constants. In addition, we also include product operator simulations to define the dynamic range of adiabatic R(1ρ) and R(2ρ) that is valid under all exchange regimes. We conclude from both experimental observations and simulations that this method is complementary to CPMG-based and rotating frame spin-lock R(1ρ) experiments to probe conformational exchange dynamics for biomolecules. Finally, this approach is germane to several NMR-active nuclei, where relaxation rates are frequency-offset independent. Copyright © 2012 Elsevier Inc. All rights reserved.

Characterization of Aggregation Propensity of a Human Fc-Fusion Protein Therapeutic by Hydrogen/Deuterium Exchange Mass Spectrometry

NASA Astrophysics Data System (ADS)

Huang, Richard Y.-C.; Iacob, Roxana E.; Krystek, Stanley R.; Jin, Mi; Wei, Hui; Tao, Li; Das, Tapan K.; Tymiak, Adrienne A.; Engen, John R.; Chen, Guodong

2017-05-01

Aggregation of protein therapeutics has long been a concern across different stages of manufacturing processes in the biopharmaceutical industry. It is often indicative of aberrant protein therapeutic higher-order structure. In this study, the aggregation propensity of a human Fc-fusion protein therapeutic was characterized. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was applied to examine the conformational dynamics of dimers collected from a bioreactor. HDX-MS data combined with spatial aggregation propensity calculations revealed a potential aggregation interface in the Fc domain. This study provides a general strategy for the characterization of the aggregation propensity of Fc-fusion proteins at the molecular level.

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.

Enhanced Conformational Sampling Using Replica Exchange with Collective-Variable Tempering

PubMed Central

2015-01-01

The computational study of conformational transitions in RNA and proteins with atomistic molecular dynamics often requires suitable enhanced sampling techniques. We here introduce a novel method where concurrent metadynamics are integrated in a Hamiltonian replica-exchange scheme. The ladder of replicas is built with different strengths of the bias potential exploiting the tunability of well-tempered metadynamics. Using this method, free-energy barriers of individual collective variables are significantly reduced compared with simple force-field scaling. The introduced methodology is flexible and allows adaptive bias potentials to be self-consistently constructed for a large number of simple collective variables, such as distances and dihedral angles. The method is tested on alanine dipeptide and applied to the difficult problem of conformational sampling in a tetranucleotide. PMID:25838811

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

Topping, R.J.; Stone, M.P.; Brush, C.K.

The {sup 1}H NMR spectrum of the tetradeoxynucleotide d(TpCpGpA) was examined as a function of temperature, pH, and concentration. At pH 7 and above the solution conformation for this oligodeoxynucleotide appears to be a mixture of random coil and Watson-Crick duplex. At 25{degree}C, a pH titration of d(TpCpGaA) shown that distinct conformational changes occur as the pH is lowered below 7.0. These conformational changes are reversible upon readjusting the pH to neutrality, indicating the presence of a pH-dependent set of conformational equilibria. At 25{degree}C, the various conformational state in the mixture are in rapid exchange on the NMR time scale.more » Examination of the titration curve shown the presence of distinct conformational states at pH greater than 7, and between pH 4 and pH 5. When the pH titration is repeated at 5{degree}C, the conformational equilibria are in slow exchange on the NMR time scale; distinct signals from each conformational state are observable. The stable conformational state present between pH 4 and pH 5 represents an ordered conformation of d(TpCpGpA) which dissociates to a less ordered structure upon raising the temperature. The ordered conformation differs from the Watson-Crick helix, as is shown from nuclear Overhauser enhancement experiments, as well as chemical shift data. These results indicate that their ordered conformation is similar to the conformation of d(TpCpGpA) observed between pH 4 and pH 5. In the present case it is likely that stabilization of an ordered duplex conformation for d(TpCpGpA) is achieved by protonation of cytosine. A possible model which could explain the data involves formation of Hoogsteen C{sup +}:G base pairs.« less

Comprehensive Peptide Ion Structure Studies Using Ion Mobility Techniques: Part 3. Relating Solution-Phase to Gas-Phase Structures.

PubMed

Kondalaji, Samaneh Ghassabi; Khakinejad, Mahdiar; Valentine, Stephen J

2018-06-01

Molecular dynamics (MD) simulations have been utilized to study peptide ion conformer establishment during the electrospray process. An explicit water model is used for nanodroplets containing a model peptide and hydronium ions. Simulations are conducted at 300 K for two different peptide ion charge configurations and for droplets containing varying numbers of hydronium ions. For all conditions, modeling has been performed until production of the gas-phase ions and the resultant conformers have been compared to proposed gas-phase structures. The latter species were obtained from previous studies in which in silico candidate structures were filtered according to ion mobility and hydrogen-deuterium exchange (HDX) reactivity matches. Results from the present study present three key findings namely (1) the evidence from ion production modeling supports previous structure refinement studies based on mobility and HDX reactivity matching, (2) the modeling of the electrospray process is significantly improved by utilizing initial droplets existing below but close to the calculated Rayleigh limit, and (3) peptide ions in the nanodroplets sample significantly different conformers than those in the bulk solution due to altered physicochemical properties of the solvent. Graphical Abstract ᅟ.

Conformational switching between protein substates studied with 2D IR vibrational echo spectroscopy and molecular dynamics simulations.

PubMed

Bagchi, Sayan; Thorpe, Dayton G; Thorpe, Ian F; Voth, Gregory A; Fayer, M D

2010-12-30

Myoglobin is an important protein for the study of structure and dynamics. Three conformational substates have been identified for the carbonmonoxy form of myoglobin (MbCO). These are manifested as distinct peaks in the IR absorption spectrum of the CO stretching mode. Ultrafast 2D IR vibrational echo chemical exchange experiments are used to observed switching between two of these substates, A(1) and A(3), on a time scale of <100 ps for two mutants of wild-type Mb. The two mutants are a single mutation of Mb, L29I, and a double mutation, T67R/S92D. Molecular dynamics (MD) simulations are used to model the structural differences between the substates of the two MbCO mutants. The MD simulations are also employed to examine the substate switching in the two mutants as a test of the ability of MD simulations to predict protein dynamics correctly for a system in which there is a well-defined transition over a significant potential barrier between two substates. For one mutant, L29I, the simulations show that translation of the His64 backbone may differentiate the two substates. The simulations accurately reproduce the experimentally observed interconversion time for the L29I mutant. However, MD simulations exploring the same His64 backbone coordinate fail to display substate interconversion for the other mutant, T67R/S92D, thus pointing to the likely complexity of the underlying protein interactions. We anticipate that understanding conformational dynamics in MbCO via ultrafast 2D IR vibrational echo chemical exchange experiments can help to elucidate fast conformational switching processes in other proteins.

Conserved conformational selection mechanism of Hsp70 chaperone-substrate interactions

PubMed Central

Velyvis, Algirdas; Zoltsman, Guy; Rosenzweig, Rina; Bouvignies, Guillaume

2018-01-01

Molecular recognition is integral to biological function and frequently involves preferred binding of a molecule to one of several exchanging ligand conformations in solution. In such a process the bound structure can be selected from the ensemble of interconverting ligands a priori (conformational selection, CS) or may form once the ligand is bound (induced fit, IF). Here we focus on the ubiquitous and conserved Hsp70 chaperone which oversees the integrity of the cellular proteome through its ATP-dependent interaction with client proteins. We directly quantify the flux along CS and IF pathways using solution NMR spectroscopy that exploits a methyl TROSY effect and selective isotope-labeling methodologies. Our measurements establish that both bacterial and human Hsp70 chaperones interact with clients by selecting the unfolded state from a pre-existing array of interconverting structures, suggesting a conserved mode of client recognition among Hsp70s and highlighting the importance of molecular dynamics in this recognition event. PMID:29460778

Anomalous nuclear Overhauser effects in carbon-substituted aziridines: scalar cross-relaxation of the first kind.

PubMed

Kuprov, Ilya; Hodgson, David M; Kloesges, Johannes; Pearson, Christopher I; Odell, Barbara; Claridge, Timothy D W

2015-03-16

Anomalous NOESY cross-peaks that cannot be explained by dipolar cross-relaxation or chemical exchange are described for carbon-substituted aziridines. The origin of these is identified as scalar cross-relaxation of the first kind, as demonstrated by a complete theoretical description of this relaxation process and by computational simulation of the NOESY spectra. It is shown that this process relies on the stochastic modulation of J-coupling by conformational transitions, which in the case of aziridines arise from inversion at the nitrogen center. The observation of scalar cross-relaxation between protons does not appear to have been previously reported for NOESY spectra. Conventional analysis would have assigned the cross-peaks as being indicative of a chemical exchange process occurring between correlated spins, were it not for the fact that the pairs of nuclei displaying them cannot undergo such exchange. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Structural Landscape of the Proline-Rich Domain of Sos1 Nucleotide Exchange Factor

PubMed Central

McDonald, Caleb B.; Bhat, Vikas; Kurouski, Dmitry; Mikles, David C.; Deegan, Brian J.; Seldeen, Kenneth L.; Lednev, Igor K.; Farooq, Amjad

2013-01-01

Despite its key role in mediating a plethora of cellular signaling cascades pertinent to health and disease, little is known about the structural landscape of the proline-rich (PR) domain of Sos1 guanine nucleotide exchange factor. Herein, using a battery of biophysical tools, we provide evidence that the PR domain of Sos1 is structurally disordered and adopts an extended random coil-like conformation in solution. Of particular interest is the observation that while chemical denaturation of PR domain results in the formation of a significant amount of polyproline II (PPII) helices, it has little or negligible effect on its overall size as measured by its hydrodynamic radius. Our data also show that the PR domain displays a highly dynamic conformational basin in agreement with the knowledge that the intrinsically unstructured proteins rapidly interconvert between an ensemble of conformations. Collectively, our study provides new insights into the conformational equilibrium of a key signaling molecule with important consequences on its physiological function. PMID:23528987

Unique Structure and Dynamics of the EphA5 Ligand Binding Domain Mediate Its Binding Specificity as Revealed by X-ray Crystallography, NMR and MD Simulations

PubMed Central

Mitra, Sayantan; Zhu, Wanlong; Qin, Haina; Pasquale, Elena B.; Song, Jianxing

2013-01-01

The 16 EphA and EphB receptors represent the largest family of receptor tyrosine kinases, and their interactions with 9 ephrin-A and ephrin-B ligands initiate bidirectional signals controlling many physiological and pathological processes. Most interactions occur between receptor and ephrins of the same class, and only EphA4 can bind all A and B ephrins. To understand the structural and dynamic principles that enable Eph receptors to utilize the same jellyroll β-sandwich fold to bind ephrins, the VAPB-MSP domain, peptides and small molecules, we have used crystallography, NMR and molecular dynamics (MD) simulations to determine the first structure and dynamics of the EphA5 ligand-binding domain (LBD), which only binds ephrin-A ligands. Unexpectedly, despite being unbound, the high affinity ephrin-binding pocket of EphA5 resembles that of other Eph receptors bound to ephrins, with a helical conformation over the J–K loop and an open pocket. The openness of the pocket is further supported by NMR hydrogen/deuterium exchange data and MD simulations. Additionally, the EphA5 LBD undergoes significant picosecond-nanosecond conformational exchanges over the loops, as revealed by NMR and MD simulations, but lacks global conformational exchanges on the microsecond-millisecond time scale. This is markedly different from the EphA4 LBD, which shares 74% sequence identity and 87% homology. Consequently, the unbound EphA5 LBD appears to comprise an ensemble of open conformations that have only small variations over the loops and appear ready to bind ephrin-A ligands. These findings show how two proteins with high sequence homology and structural similarity are still able to achieve distinctive binding specificities through different dynamics, which may represent a general mechanism whereby the same protein fold can serve for different functions. Our findings also suggest that a promising strategy to design agonists/antagonists with high affinity and selectivity might be to target specific dynamic states of the Eph receptor LBDs. PMID:24086308

Replica-exchange molecular dynamics simulations of cellulose solvated in water and in the ionic liquid 1-butyl-3-methylimidazolium chloride

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

Mostofian, Barmak; Cheng, Xiaolin; Smith, Jeremy C.

2014-09-02

Ionic liquids have become a popular solvent for cellulose pretreatment in biorefineries due to their efficiency in dissolution and their reusability. Understanding the interactions between cations, anions, and cellulose is key to the development of better solvents and the improvement of pretreatment conditions. While previous studies described the interactions between ionic liquids and cellulose fibers, shedding light on the initial stages of the cellulose dissolution process, we study the end state of that process by exploring the structure and dynamics of a single cellulose decamer solvated in 1-butyl-3-methyl-imidazolium chloride (BmimCl) and in water using replica-exchange molecular dynamics. In both solvents,more » global structural features of the cellulose chain are similar. However, analyses of local structural properties show that cellulose explores greater conformational variability in the ionic liquid than in water. For instance, in BmimCl the cellulose intramolecular hydrogen bond O3H'••• O5 is disrupted more often resulting in greater flexibility of the solute. Our results indicate that the cellulose chain is more dynamic in BmimCl than in water, which may play a role in the favorable dissolution of cellulose in the ionic liquid. Here, the calculation of the configurational entropy of the cellulose decamer confirms its higher conformational flexibility in BmimCl than in water at elevated temperatures.« less

Conformation transitions of a single polyelectrolyte chain in a poor solvent: a replica-exchange lattice Monte-Carlo study.

PubMed

Wang, Lang; Wang, Zheng; Jiang, Run; Yin, Yuhua; Li, Baohui

2017-03-15

The thermodynamic behaviors of a strongly charged polyelectrolyte chain in a poor solvent are studied using replica-exchange Monte-Carlo simulations on a lattice model, focusing on the effects of finite chain length and the solvent quality on the chain conformation and conformation transitions. The neutralizing counterions and solvent molecules are considered explicitly. The thermodynamic quantities that vary continuously with temperature over a wide range are computed using the multiple histogram reweighting method. Our results suggest that the strength of the short-range hydrophobic interaction, the chain length, and the temperature of the system, characterized by ε, N, and T, respectively, are important parameters that control the conformations of a charged chain. When ε is moderate, the competition between the electrostatic energy and the short-range hydrophobic interaction leads to rich conformations and conformation transitions for a longer chain with a fixed length. Our results have unambiguously demonstrated the stability of the n-pearl-necklace structures, where n has a maximum value and decreases with decreasing temperature. The maximum n value increases with increasing chain length. Our results have also demonstrated the first-order nature of the conformation transitions between the m-pearl and the (m-1)-pearl necklaces. With the increase of ε, the transition temperature increases and the first-order feature becomes more pronounced. It is deduced that at the thermodynamic limit of infinitely long chain length, the conformational transitions between the m-pearl and the (m-1)-pearl necklaces may remain first order when ε > 0 and m = 2 or 3. Pearl-necklace conformations cannot be observed when either ε is too large or N is too small. To observe a pearl-necklace conformation, the T value needs to be carefully chosen for simulations performed at only a single temperature.

Mapping the Dynamics Landscape of Conformational Transitions in Enzyme: The Adenylate Kinase Case

PubMed Central

Li, Dechang; Liu, Ming S.; Ji, Baohua

2015-01-01

Conformational transition describes the essential dynamics and mechanism of enzymes in pursuing their various functions. The fundamental and practical challenge to researchers is to quantitatively describe the roles of large-scale dynamic transitions for regulating the catalytic processes. In this study, we tackled this challenge by exploring the pathways and free energy landscape of conformational changes in adenylate kinase (AdK), a key ubiquitous enzyme for cellular energy homeostasis. Using explicit long-timescale (up to microseconds) molecular dynamics and bias-exchange metadynamics simulations, we determined at the atomistic level the intermediate conformational states and mapped the transition pathways of AdK in the presence and absence of ligands. There is clearly chronological operation of the functional domains of AdK. Specifically in the ligand-free AdK, there is no significant energy barrier in the free energy landscape separating the open and closed states. Instead there are multiple intermediate conformational states, which facilitate the rapid transitions of AdK. In the ligand-bound AdK, the closed conformation is energetically most favored with a large energy barrier to open it up, and the conformational population prefers to shift to the closed form coupled with transitions. The results suggest a perspective for a hybrid of conformational selection and induced fit operations of ligand binding to AdK. These observations, depicted in the most comprehensive and quantitative way to date, to our knowledge, emphasize the underlying intrinsic dynamics of AdK and reveal the sophisticated conformational transitions of AdK in fulfilling its enzymatic functions. The developed methodology can also apply to other proteins and biomolecular systems. PMID:26244746

“Invisible” Conformers of an Antifungal Disulfide Protein Revealed by Constrained Cold and Heat Unfolding, CEST-NMR Experiments, and Molecular Dynamics Calculations

PubMed Central

Fizil, Ádám; Gáspári, Zoltán; Barna, Terézia; Marx, Florentine; Batta, Gyula

2015-01-01

Transition between conformational states in proteins is being recognized as a possible key factor of function. In support of this, hidden dynamic NMR structures were detected in several cases up to populations of a few percent. Here, we show by two- and three-state analysis of thermal unfolding, that the population of hidden states may weight 20–40 % at 298 K in a disulfide-rich protein. In addition, sensitive 15N-CEST NMR experiments identified a low populated (0.15 %) state that was in slow exchange with the folded PAF protein. Remarkably, other techniques failed to identify the rest of the NMR “dark matter”. Comparison of the temperature dependence of chemical shifts from experiments and molecular dynamics calculations suggests that hidden conformers of PAF differ in the loop and terminal regions and are most similar in the evolutionary conserved core. Our observations point to the existence of a complex conformational landscape with multiple conformational states in dynamic equilibrium, with diverse exchange rates presumably responsible for the completely hidden nature of a considerable fraction. PMID:25676351

Evaluating the Uncertainty in Exchange Parameters Determined from Off-Resonance R1ρ Relaxation Dispersion for Systems in Fast Exchange

PubMed Central

Bothe, Jameson R.; Stein, Zachary W.; Al-Hashimi, Hashim M.

2014-01-01

Spin relaxation in the rotating frame (R1ρ) is a powerful NMR technique for characterizing fast microsecond timescale exchange processes directed toward short-lived excited states in biomolecules. At the limit of fast exchange, only kex = k1 + k−1 and Φıx = pGpE(Δω)2 can be determined from R1ρ data limiting the ability to characterize the structure and energetics of the excited state conformation. Here, we use simulations to examine the uncertainty with which exchange parameters can be determined for two state systems in intermediate-to-fast exchange using off-resonance R1ρ relaxation dispersion. R1ρ data computed by solving the Bloch-McConnell equations reveals small but significant asymmetry with respect to offset (R1ρ(ΔΩ) ≠ R1ρ(−ΔΩ)), which is a hallmark of slow-to-intermediate exchange, even under conditions of fast exchange for free precession chemical exchange line broadening (kex/Δω > 10). A grid search analysis combined with bootstrap and Monte-Carlo based statistical approaches for estimating uncertainty in exchange parameters reveals that both the sign and magnitude of Δω can be determined at a useful level of uncertainty for systems in fast exchange (kex/Δω < 10) but that this depends on the uncertainty in the R1ρ data and requires a thorough examination of the multidimensional variation of χ2 as a function of exchange parameters. Results from simulations are complemented by analysis of experimental R1ρ data measured in three nucleic acid systems with exchange processes occurring on the slow (kex/Δω = 0.2; pE = ~ 0.7%), fast (kex/Δω = ~10–16; pE = ~13%) and very fast (kex = 39,000 s−1) chemical shift timescales. PMID:24819426

Enhanced conformational sampling of nucleic acids by a new Hamiltonian replica exchange molecular dynamics approach.

PubMed

Curuksu, Jeremy; Zacharias, Martin

2009-03-14

Although molecular dynamics (MD) simulations have been applied frequently to study flexible molecules, the sampling of conformational states separated by barriers is limited due to currently possible simulation time scales. Replica-exchange (Rex)MD simulations that allow for exchanges between simulations performed at different temperatures (T-RexMD) can achieve improved conformational sampling. However, in the case of T-RexMD the computational demand grows rapidly with system size. A Hamiltonian RexMD method that specifically enhances coupled dihedral angle transitions has been developed. The method employs added biasing potentials as replica parameters that destabilize available dihedral substates and was applied to study coupled dihedral transitions in nucleic acid molecules. The biasing potentials can be either fixed at the beginning of the simulation or optimized during an equilibration phase. The method was extensively tested and compared to conventional MD simulations and T-RexMD simulations on an adenine dinucleotide system and on a DNA abasic site. The biasing potential RexMD method showed improved sampling of conformational substates compared to conventional MD simulations similar to T-RexMD simulations but at a fraction of the computational demand. It is well suited to study systematically the fine structure and dynamics of large nucleic acids under realistic conditions including explicit solvent and ions and can be easily extended to other types of molecules.

Probing the conformation of a conserved glutamic acid within the Cl- pathway of a CLC H+/Cl- exchanger.

PubMed

Vien, Malvin; Basilio, Daniel; Leisle, Lilia; Accardi, Alessio

2017-04-03

The CLC proteins form a broad family of anion-selective transport proteins that includes both channels and exchangers. Despite extensive structural, functional, and computational studies, the transport mechanism of the CLC exchangers remains poorly understood. Several transport models have been proposed but have failed to capture all the key features of these transporters. Multiple CLC crystal structures have suggested that a conserved glutamic acid, Glu ex , can adopt three conformations and that the interconversion of its side chain between these states underlies H + /Cl - exchange. One of these states, in which Glu ex occupies the central binding site (S cen ) while Cl - ions fill the internal and external sites (S int and S ext ), has only been observed in one homologue, the eukaryotic cmCLC. The existence of such a state in other CLCs has not been demonstrated. In this study, we find that during transport, the prototypical prokaryotic CLC exchanger, CLC-ec1, adopts a conformation with functional characteristics that match those predicted for a cmCLC-like state, with Glu ex trapped in S cen between two Cl - ions. Transport by CLC-ec1 is reduced when [Cl - ] is symmetrically increased on both sides of the membrane and mutations that disrupt the hydrogen bonds stabilizing Glu ex in S cen destabilize this trapped state. Furthermore, inhibition of transport by high [Cl - ] is abolished in the E148A mutant, in which the Glu ex side chain is removed. Collectively, our results suggest that, during the CLC transport cycle, Glu ex can occupy S cen as well as the S ext position in which it has been captured crystallographically and that hydrogen bonds with the side chains of residues that coordinate ion binding to S cen play a role in determining the equilibrium between these two conformations. © 2017 Vien et al.

Comparison of the structures of free and ribosome-bound tRNAPhe by using slow tritium exchange.

PubMed Central

Farber, N; Cantor, C R

1980-01-01

The rate of incorporation of tritium from the solvent into the C-8 position of purines in RNA is markedly sensitive to the microenvironment. This slow tritium exchange reaction has been used to study the structure and interactions of yeast tRNAPhe bound to poly(U)-programed tight-couple 70S ribosomes of Escherichia coli. The tritium incorporation into specific sites of the tRNA was determined by enzymatic digestion and measurement of the specific activity of each of the isolated radioactive fragments. Ribosome binding leads to marked suppression in the exchange rate of a number of fragments. This delineates extensive regions of tRNA-ribosome contact. No change in exchange rates is seen for fragments from the corner of the molecule, indicating that this region of bound tRNA is readily accessible to the solvent. Ribosome binding results in an enhanced exchange rate at the T loop. This appears to be the result of a conformational change that is most likely an unfolding of the T and D loops. Additional tritium exchange reactions suggest this conformational change is induced by ribosomes and not by messenger. PMID:7001473

Manipulating and Visualizing Molecular Interactions in Customized Nanoscale Spaces

NASA Astrophysics Data System (ADS)

Stabile, Francis; Henkin, Gil; Berard, Daniel; Shayegan, Marjan; Leith, Jason; Leslie, Sabrina

We present a dynamically adjustable nanofluidic platform for formatting the conformations of and visualizing the interaction kinetics between biomolecules in solution, offering new time resolution and control of the reaction processes. This platform extends convex lens-induced confinement (CLiC), a technique for imaging molecules under confinement, by introducing a system for in situ modification of the chemical environment; this system uses a deep microchannel to diffusively exchange reagents within the nanoscale imaging region, whose height is fixed by a nanopost array. To illustrate, we visualize and manipulate salt-induced, surfactant-induced, and enzyme-induced reactions between small-molecule reagents and DNA molecules, where the conformations of the DNA molecules are formatted by the imposed nanoscale confinement. By using nanofabricated, nonabsorbing, low-background glass walls to confine biomolecules, our nanofluidic platform facilitates quantitative exploration of physiologically and biotechnologically relevant processes at the nanoscale. This device provides new kinetic information about dynamic chemical processes at the single-molecule level, using advancements in the CLiC design including a microchannel-based diffuser and postarray-based dialysis slit.

Free energy surface of an intrinsically disordered protein: comparison between temperature replica exchange molecular dynamics and bias-exchange metadynamics.

PubMed

Zerze, Gül H; Miller, Cayla M; Granata, Daniele; Mittal, Jeetain

2015-06-09

Intrinsically disordered proteins (IDPs), which are expected to be largely unstructured under physiological conditions, make up a large fraction of eukaryotic proteins. Molecular dynamics simulations have been utilized to probe structural characteristics of these proteins, which are not always easily accessible to experiments. However, exploration of the conformational space by brute force molecular dynamics simulations is often limited by short time scales. Present literature provides a number of enhanced sampling methods to explore protein conformational space in molecular simulations more efficiently. In this work, we present a comparison of two enhanced sampling methods: temperature replica exchange molecular dynamics and bias exchange metadynamics. By investigating both the free energy landscape as a function of pertinent order parameters and the per-residue secondary structures of an IDP, namely, human islet amyloid polypeptide, we found that the two methods yield similar results as expected. We also highlight the practical difference between the two methods by describing the path that we followed to obtain both sets of data.

Role of urea on recombinant Apo A-I stability and its utilization in anion exchange chromatography.

PubMed

Angarita, Monica; Arosio, Paolo; Müller-Späth, Thomas; Baur, Daniel; Falkenstein, Roberto; Kuhne, Wolfgang; Morbidelli, Massimo

2014-08-08

Apolipoprotein A-I (Apo A-I) is an important lipid-binding protein involved in the transport and metabolism of cholesterol. High protein purity, in particular with respect to endotoxins is required for therapeutic applications. The use of urea during the purification process of recombinant Apo A-I produced in Escherichia coli has been suggested so as to provide high endotoxin clearance. In this work, we show that urea can be used as a sole modifier during the ion exchange chromatographic purification of Apo A-I and we investigate the molecular mechanism of elution by correlating the effect of urea on self-association, conformation and adsorption equilibrium properties of a modified model Apo A-I. In the absence of urea the protein was found to be present as a population of oligomers represented mainly by trimers, hexamers and nonamers. The addition of urea induced oligomer dissociation and protein structure unfolding. We correlated the changes in protein association and conformation with variations of the adsorption equilibrium of the protein on a strong anion exchanger. It was confirmed that the adsorption isotherms, described by a Langmuir model, were dependent on both protein and urea concentrations. Monomers, observed at low urea concentration (0.5M), were characterized by larger binding affinity and adsorption capacity compared to both protein oligomers (0M) and unfolded monomers (2-8M). The reduction of both the binding strength and maximum adsorption capacity at urea concentrations larger than 0.5M explains the ability of urea of inducing elution of the protein from the ion exchange resin. The dissociation of the protein complexes occurring during the elution could likely be the origin of the effective clearance of endotoxins originally trapped inside the oligomers. Copyright © 2014 Elsevier B.V. All rights reserved.

Asymmetric Preorganization of Inverted Pair Residues in the Sodium-Calcium Exchanger

PubMed Central

Giladi, Moshe; Almagor, Lior; van Dijk, Liat; Hiller, Reuben; Man, Petr; Forest, Eric; Khananshvili, Daniel

2016-01-01

In analogy with many other proteins, Na+/Ca2+ exchangers (NCX) adapt an inverted twofold symmetry of repeated structural elements, while exhibiting a functional asymmetry by stabilizing an outward-facing conformation. Here, structure-based mutant analyses of the Methanococcus jannaschii Na+/Ca2+ exchanger (NCX_Mj) were performed in conjunction with HDX-MS (hydrogen/deuterium exchange mass spectrometry) to identify the structure-dynamic determinants of functional asymmetry. HDX-MS identified hallmark differences in backbone dynamics at ion-coordinating residues of apo-NCX_Mj, whereas Na+or Ca2+ binding to the respective sites induced relatively small, but specific, changes in backbone dynamics. Mutant analysis identified ion-coordinating residues affecting the catalytic capacity (kcat/Km), but not the stability of the outward-facing conformation. In contrast, distinct “noncatalytic” residues (adjacent to the ion-coordinating residues) control the stability of the outward-facing conformation, but not the catalytic capacity. The helix-breaking signature sequences (GTSLPE) on the α1 and α2 repeats (at the ion-binding core) differ in their folding/unfolding dynamics, while providing asymmetric contributions to transport activities. The present data strongly support the idea that asymmetric preorganization of the ligand-free ion-pocket predefines catalytic reorganization of ion-bound residues, where secondary interactions with adjacent residues couple the alternating access. These findings provide a structure-dynamic basis for ion-coupled alternating access in NCX and similar proteins. PMID:26876271

Thymosin-beta(4) changes the conformation and dynamics of actin monomers.

PubMed Central

De La Cruz, E M; Ostap, E M; Brundage, R A; Reddy, K S; Sweeney, H L; Safer, D

2000-01-01

Thymosin-beta(4) (Tbeta(4)) binds actin monomers stoichiometrically and maintains the bulk of the actin monomer pool in metazoan cells. Tbeta(4) binding quenches the fluorescence of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) conjugated to Cys(374) of actin monomers. The K(d) of the actin-Tbeta(4) complex depends on the cation and nucleotide bound to actin but is not affected by the AEDANS probe. The different stabilities are determined primarily by the rates of dissociation. At 25 degrees C, the free energy of Tbeta(4) binding MgATP-actin is primarily enthalpic in origin but entropic for CaATP-actin. Binding is coupled to the dissociation of bound water molecules, which is greater for CaATP-actin than MgATP-actin monomers. Proteolysis of MgATP-actin, but not CaATP-actin, at Gly(46) on subdomain 2 is >12 times faster when Tbeta(4) is bound. The C terminus of Tbeta(4) contacts actin near this cleavage site, at His(40). By tritium exchange, Tbeta(4) slows the exchange rate of approximately eight rapidly exchanging amide protons on actin. We conclude that Tbeta(4) changes the conformation and structural dynamics ("breathing") of actin monomers. The conformational change may reflect the unique ability of Tbeta(4) to sequester actin monomers and inhibit nucleotide exchange. PMID:10777749

Faster protein folding using enhanced conformational sampling of molecular dynamics simulation.

PubMed

Kamberaj, Hiqmet

2018-05-01

In this study, we applied swarm particle-like molecular dynamics (SPMD) approach to enhance conformational sampling of replica exchange simulations. In particular, the approach showed significant improvement in sampling efficiency of conformational phase space when combined with replica exchange method (REM) in computer simulation of peptide/protein folding. First we introduce the augmented dynamical system of equations, and demonstrate the stability of the algorithm. Then, we illustrate the approach by using different fully atomistic and coarse-grained model systems, comparing them with the standard replica exchange method. In addition, we applied SPMD simulation to calculate the time correlation functions of the transitions in a two dimensional surface to demonstrate the enhancement of transition path sampling. Our results showed that folded structure can be obtained in a shorter simulation time using the new method when compared with non-augmented dynamical system. Typically, in less than 0.5 ns using replica exchange runs assuming that native folded structure is known and within simulation time scale of 40 ns in the case of blind structure prediction. Furthermore, the root mean square deviations from the reference structures were less than 2Å. To demonstrate the performance of new method, we also implemented three simulation protocols using CHARMM software. Comparisons are also performed with standard targeted molecular dynamics simulation method. Copyright © 2018 Elsevier Inc. All rights reserved.

Allosteric activation via kinetic control: Potassium accelerates a conformational change in IMP dehydrogenase†

PubMed Central

Riera, Thomas V.; Zheng, Lianqing; Josephine, Helen R.; Min, Donghong; Yang, Wei; Hedstrom, Lizbeth

2011-01-01

Allosteric activators are generally believed to shift the equilibrium distribution of enzyme conformations to favor a catalytically productive structure; the kinetics of conformational exchange is seldom addressed. Several observations suggested that the usual allosteric mechanism might not apply to the activation of IMP dehydrogenase (IMPDH) by monovalent cations. Therefore we investigated the mechanism of K+ activation in IMPDH by delineating the kinetic mechanism in the absence of monovalent cations. Surprisingly, the K+-dependence of kcat derives from the rate of flap closure, which increases by ≥65-fold in the presence of K+. We performed both alchemical free energy simulations and potential of mean force calculations using the orthogonal space random walk strategy to computationally analyze how K+ accelerates this conformational change. The simulations recapitulate the preference of IMPDH for K+, validating the computational models. When K+ is replaced with a dummy ion, the residues of the K+ binding site relax into ordered secondary structure, creating a barrier to conformational exchange. K+ mobilizes these residues by providing alternate interactions for the main chain carbonyls. Potential of mean force calculations indicate that K+ changes the shape of the energy well, shrinking the reaction coordinate by shifting the closed conformation toward the open state. This work suggests that allosteric regulation can be under kinetic as well as thermodynamic control. PMID:21870820

Structural landscape of the proline-rich domain of Sos1 nucleotide exchange factor.

PubMed

McDonald, Caleb B; Bhat, Vikas; Kurouski, Dmitry; Mikles, David C; Deegan, Brian J; Seldeen, Kenneth L; Lednev, Igor K; Farooq, Amjad

2013-01-01

Despite its key role in mediating a plethora of cellular signaling cascades pertinent to health and disease, little is known about the structural landscape of the proline-rich (PR) domain of Sos1 guanine nucleotide exchange factor. Herein, using a battery of biophysical tools, we provide evidence that the PR domain of Sos1 is structurally disordered and adopts an extended random coil-like conformation in solution. Of particular interest is the observation that while chemical denaturation of PR domain results in the formation of a significant amount of polyproline II (PPII) helices, it has little or negligible effect on its overall size as measured by its hydrodynamic radius. Our data also show that the PR domain displays a highly dynamic conformational basin in agreement with the knowledge that the intrinsically unstructured proteins rapidly interconvert between an ensemble of conformations. Collectively, our study provides new insights into the conformational equilibrium of a key signaling molecule with important consequences on its physiological function. Copyright © 2013 Elsevier B.V. All rights reserved.

KRAS G12C Drug Development: Discrimination between Switch II Pocket Configurations Using Hydrogen/Deuterium-Exchange Mass Spectrometry

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

Lu, Jia; Harrison, Rane A.; Li, Lianbo

KRAS G12C, the most common RAS mutation found in non-small-cell lung cancer, has been the subject of multiple recent covalent small-molecule inhibitor campaigns including efforts directed at the guanine nucleotide pocket and separate work focused on an inducible pocket adjacent to the switch motifs. Multiple conformations of switch II have been observed, suggesting that switch II pocket (SIIP) binders may be capable of engaging a range of KRAS conformations. Here we report the use of hydrogen/deuterium-exchange mass spectrometry (HDX MS) to discriminate between conformations of switch II induced by two chemical classes of SIIP binders. We investigated the structural basismore » for differences in HDX MS using X-ray crystallography and discovered a new SIIP configuration in response to binding of a quinazoline chemotype. These results have implications for structure-guided drug design targeting the RAS SIIP.« less

Combining Coarse-Grained Protein Models with Replica-Exchange All-Atom Molecular Dynamics

PubMed Central

Wabik, Jacek; Kmiecik, Sebastian; Gront, Dominik; Kouza, Maksim; Koliński, Andrzej

2013-01-01

We describe a combination of all-atom simulations with CABS, a well-established coarse-grained protein modeling tool, into a single multiscale protocol. The simulation method has been tested on the C-terminal beta hairpin of protein G, a model system of protein folding. After reconstructing atomistic details, conformations derived from the CABS simulation were subjected to replica-exchange molecular dynamics simulations with OPLS-AA and AMBER99sb force fields in explicit solvent. Such a combination accelerates system convergence several times in comparison with all-atom simulations starting from the extended chain conformation, demonstrated by the analysis of melting curves, the number of native-like conformations as a function of time and secondary structure propagation. The results strongly suggest that the proposed multiscale method could be an efficient and accurate tool for high-resolution studies of protein folding dynamics in larger systems. PMID:23665897

Exploring the Dynamics of Propeller Loops in Human Telomeric DNA Quadruplexes Using Atomistic Simulations.

PubMed

Islam, Barira; Stadlbauer, Petr; Gil-Ley, Alejandro; Pérez-Hernández, Guillermo; Haider, Shozeb; Neidle, Stephen; Bussi, Giovanni; Banas, Pavel; Otyepka, Michal; Sponer, Jiri

2017-06-13

We have carried out a series of extended unbiased molecular dynamics (MD) simulations (up to 10 μs long, ∼162 μs in total) complemented by replica-exchange with the collective variable tempering (RECT) approach for several human telomeric DNA G-quadruplex (GQ) topologies with TTA propeller loops. We used different AMBER DNA force-field variants and also processed simulations by Markov State Model (MSM) analysis. The slow conformational transitions in the propeller loops took place on a scale of a few μs, emphasizing the need for long simulations in studies of GQ dynamics. The propeller loops sampled similar ensembles for all GQ topologies and for all force-field dihedral-potential variants. The outcomes of standard and RECT simulations were consistent and captured similar spectrum of loop conformations. However, the most common crystallographic loop conformation was very unstable with all force-field versions. Although the loss of canonical γ-trans state of the first propeller loop nucleotide could be related to the indispensable bsc0 α/γ dihedral potential, even supporting this particular dihedral by a bias was insufficient to populate the experimentally dominant loop conformation. In conclusion, while our simulations were capable of providing a reasonable albeit not converged sampling of the TTA propeller loop conformational space, the force-field description still remained far from satisfactory.

Exploring the Dynamics of Propeller Loops in Human Telomeric DNA Quadruplexes Using Atomistic Simulations

PubMed Central

2017-01-01

We have carried out a series of extended unbiased molecular dynamics (MD) simulations (up to 10 μs long, ∼162 μs in total) complemented by replica-exchange with the collective variable tempering (RECT) approach for several human telomeric DNA G-quadruplex (GQ) topologies with TTA propeller loops. We used different AMBER DNA force-field variants and also processed simulations by Markov State Model (MSM) analysis. The slow conformational transitions in the propeller loops took place on a scale of a few μs, emphasizing the need for long simulations in studies of GQ dynamics. The propeller loops sampled similar ensembles for all GQ topologies and for all force-field dihedral-potential variants. The outcomes of standard and RECT simulations were consistent and captured similar spectrum of loop conformations. However, the most common crystallographic loop conformation was very unstable with all force-field versions. Although the loss of canonical γ-trans state of the first propeller loop nucleotide could be related to the indispensable bsc0 α/γ dihedral potential, even supporting this particular dihedral by a bias was insufficient to populate the experimentally dominant loop conformation. In conclusion, while our simulations were capable of providing a reasonable albeit not converged sampling of the TTA propeller loop conformational space, the force-field description still remained far from satisfactory. PMID:28475322

Hydrogen/deuterium exchange in mass spectrometry.

PubMed

Kostyukevich, Yury; Acter, Thamina; Zherebker, Alexander; Ahmed, Arif; Kim, Sunghwan; Nikolaev, Eugene

2018-03-30

The isotopic exchange approach is in use since the first observation of such reactions in 1933 by Lewis. This approach allows the investigation of the pathways of chemical and biochemical reactions, determination of structure, composition, and conformation of molecules. Mass spectrometry has now become one of the most important analytical tools for the monitoring of the isotopic exchange reactions. Investigation of conformational dynamics of proteins, quantitative measurements, obtaining chemical, and structural information about individual compounds of the complex natural mixtures are mainly based on the use of isotope exchange in combination with high resolution mass spectrometry. The most important reaction is the Hydrogen/Deuterium exchange, which is mainly performed in the solution. Recently we have developed the approach allowing performing of the Hydrogen/Deuterium reaction on-line directly in the ionization source under atmospheric pressure. Such approach simplifies the sample preparation and can accelerate the exchange reaction so that certain hydrogens that are considered as non-labile will also participate in the exchange. The use of in-ionization source H/D exchange in modern mass spectrometry for structural elucidation of molecules serves as the basic theme in this review. We will focus on the mechanisms of the isotopic exchange reactions and on the application of in-ESI, in-APCI, and in-APPI source Hydrogen/Deuterium exchange for the investigation of petroleum, natural organic matter, oligosaccharides, and proteins including protein-protein complexes. The simple scenario for adaptation of H/D exchange reactions into mass spectrometric method is also highlighted along with a couple of examples collected from previous studies. © 2018 Wiley Periodicals, Inc.

Insights into the conformations and dynamics of intrinsically disordered proteins using single-molecule fluorescence.

PubMed

Gomes, Gregory-Neal; Gradinaru, Claudiu C

2017-11-01

Most proteins are not static structures, but many of them are found in a dynamic state, exchanging conformations on various time scales as a key aspect of their biological function. An entire spectrum of structural disorder exists in proteins and obtaining a satisfactory quantitative description of these states remains a challenge. Single-molecule fluorescence spectroscopy techniques are uniquely suited for this task, by measuring conformations without ensemble averaging and kinetics without interference from asynchronous processes. In this paper we review some of the recent successes in applying single-molecule fluorescence to different disordered protein systems, including interactions with their cellular targets and self-aggregation processes. We also discuss the implementation of computational methods and polymer physics models that are essential for inferring global dimension parameters for these proteins from smFRET data. Regarding future directions; 3- or 4-color FRET methods can provide multiple distances within a disordered ensemble simultaneously. In addition, integrating complementary experimental data from smFRET, NMR and SAXS will provide meaningful constraints for molecular simulations and will lead to more accurate structural representations of disordered proteins. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitative determination of conformational, dynamic, and kinetic parameters of a ligand-protein/DNA complex from a complete relaxation and conformational exchange matrix analysis of intermolecular transferred NOESY.

PubMed

Moseley, H N; Lee, W; Arrowsmith, C H; Krishna, N R

1997-05-06

We report a quantitative analysis of the 13C-edited intermolecular transferred NOESY (inter-TrNOESY) spectrum of the trp-repressor/operator complex (trp-rep/op) with [ul-13C/15N]-L-tryptophan corepressor using a computer program implementing complete relaxation and conformational exchange matrix (CORCEMA) methodology [Moseley et al. (1995) J. Magn. Reson. 108B, 243-261]. Using complete mixing time curves of three inter-TrNOESY peaks between the tryptophan and the Trp-rep/op, this self-consistent analysis determined the correlation time of the bound species (tauB = 13.5 ns) and the exchange off-rate (k(off) = 3.6 s(-1)) of the corepressor. In addition, the analysis estimated the correlation time of the free species (tauF approximately 0.15 ns). Also, we demonstrate the sensitivity of these inter-TrNOESY peaks to several factors including the k(off) and orientation of the tryptophan corepressor within the binding site. The analysis indicates that the crystal structure orientation for the corepressor is compatible with the solution NMR data.

Hydrogen–Deuterium Exchange and Mass Spectrometry Reveal the pH-Dependent Conformational Changes of Diphtheria Toxin T Domain

PubMed Central

2015-01-01

The translocation (T) domain of diphtheria toxin plays a critical role in moving the catalytic domain across the endosomal membrane. Translocation/insertion is triggered by a decrease in pH in the endosome where conformational changes of T domain occur through several kinetic intermediates to yield a final trans-membrane form. High-resolution structural studies are only applicable to the static T-domain structure at physiological pH, and studies of the T-domain translocation pathway are hindered by the simultaneous presence of multiple conformations. Here, we report the application of hydrogen–deuterium exchange mass spectrometry (HDX-MS) for the study of the pH-dependent conformational changes of the T domain in solution. Effects of pH on intrinsic HDX rates were deconvolved by converting the on-exchange times at low pH into times under our “standard condition” (pH 7.5). pH-Dependent HDX kinetic analysis of T domain clearly reveals the conformational transition from the native state (W-state) to a membrane-competent state (W+-state). The initial transition occurs at pH 6 and includes the destabilization of N-terminal helices accompanied by the separation between N- and C-terminal segments. The structural rearrangements accompanying the formation of the membrane-competent state expose a hydrophobic hairpin (TH8–9) to solvent, prepare it to insert into the membrane. At pH 5.5, the transition is complete, and the protein further unfolds, resulting in the exposure of its C-terminal hydrophobic TH8–9, leading to subsequent aggregation in the absence of membranes. This solution-based study complements high resolution crystal structures and provides a detailed understanding of the pH-dependent structural rearrangement and acid-induced oligomerization of T domain. PMID:25290210

Hydrogen-deuterium exchange and mass spectrometry reveal the pH-dependent conformational changes of diphtheria toxin T domain.

PubMed

Li, Jing; Rodnin, Mykola V; Ladokhin, Alexey S; Gross, Michael L

2014-11-04

The translocation (T) domain of diphtheria toxin plays a critical role in moving the catalytic domain across the endosomal membrane. Translocation/insertion is triggered by a decrease in pH in the endosome where conformational changes of T domain occur through several kinetic intermediates to yield a final trans-membrane form. High-resolution structural studies are only applicable to the static T-domain structure at physiological pH, and studies of the T-domain translocation pathway are hindered by the simultaneous presence of multiple conformations. Here, we report the application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) for the study of the pH-dependent conformational changes of the T domain in solution. Effects of pH on intrinsic HDX rates were deconvolved by converting the on-exchange times at low pH into times under our "standard condition" (pH 7.5). pH-Dependent HDX kinetic analysis of T domain clearly reveals the conformational transition from the native state (W-state) to a membrane-competent state (W(+)-state). The initial transition occurs at pH 6 and includes the destabilization of N-terminal helices accompanied by the separation between N- and C-terminal segments. The structural rearrangements accompanying the formation of the membrane-competent state expose a hydrophobic hairpin (TH8-9) to solvent, prepare it to insert into the membrane. At pH 5.5, the transition is complete, and the protein further unfolds, resulting in the exposure of its C-terminal hydrophobic TH8-9, leading to subsequent aggregation in the absence of membranes. This solution-based study complements high resolution crystal structures and provides a detailed understanding of the pH-dependent structural rearrangement and acid-induced oligomerization of T domain.

How hot? Systematic convergence of the replica exchange method using multiple reservoirs.

PubMed

Ruscio, Jory Z; Fawzi, Nicolas L; Head-Gordon, Teresa

2010-02-01

We have devised a systematic approach to converge a replica exchange molecular dynamics simulation by dividing the full temperature range into a series of higher temperature reservoirs and a finite number of lower temperature subreplicas. A defined highest temperature reservoir of equilibrium conformations is used to help converge a lower but still hot temperature subreplica, which in turn serves as the high-temperature reservoir for the next set of lower temperature subreplicas. The process is continued until an optimal temperature reservoir is reached to converge the simulation at the target temperature. This gradual convergence of subreplicas allows for better and faster convergence at the temperature of interest and all intermediate temperatures for thermodynamic analysis, as well as optimizing the use of multiple processors. We illustrate the overall effectiveness of our multiple reservoir replica exchange strategy by comparing sampling and computational efficiency with respect to replica exchange, as well as comparing methods when converging the structural ensemble of the disordered Abeta(21-30) peptide simulated with explicit water by comparing calculated Rotating Overhauser Effect Spectroscopy intensities to experimentally measured values. Copyright 2009 Wiley Periodicals, Inc.

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding.

PubMed

Goricanec, David; Stehle, Ralf; Egloff, Pascal; Grigoriu, Simina; Plückthun, Andreas; Wagner, Gerhard; Hagn, Franz

2016-06-28

Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein-coupled receptor (GPCR) activation. Agonist-receptor binding causes GDP-to-GTP exchange and dissociation of the Gα subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein α subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Gα subdomain, whereas the apo and GDP-bound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Gα Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein α subunit and the influence of a GPCR in that landscape.

Molecular Dynamics Study of Nitrogen-Pyramidalized Bicyclic β-Proline Oligomers: Length-Dependent Convergence to Organized Structures.

PubMed

Otani, Yuko; Watanabe, Satoshi; Ohwada, Tomohiko; Kitao, Akio

2017-01-12

In this study, the solution structures of the homooligomers of a conformationally constrained bicyclic proline-type β-amino acid were studied by means of molecular dynamics (MD) calculations in explicit methanol and water using the umbrella sampling method. The ratio of trans-amide and cis-amide was estimated by NMR and the rotational barrier of the amide of acetylated bicyclic amino acid monomer was estimated by two-dimensional (2D) exchange spectroscopy (EXSY) or line-shape analysis. A bias potential was introduced with respect to the amide torsion angle ω to enhance conformational exchange including isomerization of amide bonds by lowering the rotation energy barrier. After determination of reweighting parameters to best reproduce the experimental results of the monomer amide, the free energy profile around the amide torsion angle ω was obtained from the MD trajectory by reweighting of the biased probability density. The MD simulation results support the existence of invertomers of nitrogen-pyramidalized amide. Furthermore, extended structures with a high fraction of trans-amide conformation appear to be increasingly stabilized as the oligomer is elongated, both in methanol and in water. Our conformational analysis of natural and non-natural tertiary-amide-based peptide oligomers indicates that these oligomers preferentially adopt a limited number of conformations.

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

PubMed Central

Goricanec, David; Stehle, Ralf; Egloff, Pascal; Grigoriu, Simina; Wagner, Gerhard; Hagn, Franz

2016-01-01

Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein–coupled receptor (GPCR) activation. Agonist–receptor binding causes GDP-to-GTP exchange and dissociation of the Gα subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein α subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Gα subdomain, whereas the apo and GDP-bound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Gα Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein α subunit and the influence of a GPCR in that landscape. PMID:27298341

21 CFR 26.41 - Exchange and endorsement of quality system evaluation reports.

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2014-04-01

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21 CFR 26.41 - Exchange and endorsement of quality system evaluation reports.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 1 2012-04-01 2012-04-01 false Exchange and endorsement of quality system... DEVICE QUALITY SYSTEM AUDIT REPORTS, AND CERTAIN MEDICAL DEVICE PRODUCT EVALUATION REPORTS: UNITED STATES... endorsement of quality system evaluation reports. (a) Listed European Community (EC) conformity assessment...

21 CFR 173.20 - Ion-exchange membranes.

Code of Federal Regulations, 2011 CFR

2011-04-01

... ion-exchange membrane is prepared by subjecting a polyethylene base conforming to § 177.1520 of this chapter to polymerization with styrene until the polystyrene phase of the base is not less than 16 percent nor more than 30 percent by weight. The base is then modified by reaction with chloromethyl methyl...

21 CFR 173.20 - Ion-exchange membranes.

Code of Federal Regulations, 2013 CFR

2013-04-01

... ion-exchange membrane is prepared by subjecting a polyethylene base conforming to § 177.1520 of this chapter to polymerization with styrene until the polystyrene phase of the base is not less than 16 percent nor more than 30 percent by weight. The base is then modified by reaction with chloromethyl methyl...

21 CFR 173.20 - Ion-exchange membranes.

Code of Federal Regulations, 2012 CFR

2012-04-01

... ion-exchange membrane is prepared by subjecting a polyethylene base conforming to § 177.1520 of this chapter to polymerization with styrene until the polystyrene phase of the base is not less than 16 percent nor more than 30 percent by weight. The base is then modified by reaction with chloromethyl methyl...

21 CFR 173.20 - Ion-exchange membranes.

Code of Federal Regulations, 2010 CFR

2010-04-01

... ion-exchange membrane is prepared by subjecting a polyethylene base conforming to § 177.1520 of this chapter to polymerization with styrene until the polystyrene phase of the base is not less than 16 percent nor more than 30 percent by weight. The base is then modified by reaction with chloromethyl methyl...

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... Rule Text To Conform to the Changes Adopted by the Financial Industry Regulatory Authority, Inc. for... public, to adopt new rule text to conform to the changes adopted by the Financial Industry Regulatory... Business Startups Act (the ``JOBS Act'').\\4\\ The text of the proposed rule change is available on the...

NMR Investigation of Chloromethane Complexes of Cryptophane-A and Its Analogue with Butoxy Groups

PubMed Central

2014-01-01

Host–guest complexes between cryptophane-A as host and dichloromethane and chloroform as guests are investigated using 1H and 13C NMR spectroscopy. Moreover, a related cryptophane, with the methoxy groups replaced by butoxy units (cryptophane-But), and its complexes with the same guests were also studied. Variable temperature spectra showed effects of chemical exchange between the free and bound guests, as well as of conformational exchange of the host. The guest exchange was studied quantitatively by exchange spectroscopy or line shape analysis. Extraction of kinetic and thermodynamic parameters led to the characterization of the affinity between guests and hosts. On the other hand, the host exchange was investigated by means of 13C Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion which aims at the determination of the transverse relaxation rate R2, the inverse of the transverse relaxation time T2, as a function of the repetition of the π pulses in a CPMG train. The variation of the measured transverse relaxation rate with the repetition rate νCPMG indicated conformational exchange occurring on the microsecond–millisecond time scale. Structural information was obtained through measurements of cross-relaxation rates, both within the host and between the host and the guest protons. The NMR results were supported by DFT calculations. PMID:24472055

Monomeric ephrinB2 binding induces allosteric changes in Nipah virus G that precede its full activation.

PubMed

Wong, Joyce J W; Young, Tracy A; Zhang, Jiayan; Liu, Shiheng; Leser, George P; Komives, Elizabeth A; Lamb, Robert A; Zhou, Z Hong; Salafsky, Joshua; Jardetzky, Theodore S

2017-10-03

Nipah virus is an emergent paramyxovirus that causes deadly encephalitis and respiratory infections in humans. Two glycoproteins coordinate the infection of host cells, an attachment protein (G), which binds to cell surface receptors, and a fusion (F) protein, which carries out the process of virus-cell membrane fusion. The G protein binds to ephrin B2/3 receptors, inducing G conformational changes that trigger F protein refolding. Using an optical approach based on second harmonic generation, we show that monomeric and dimeric receptors activate distinct conformational changes in G. The monomeric receptor-induced changes are not detected by conformation-sensitive monoclonal antibodies or through electron microscopy analysis of G:ephrinB2 complexes. However, hydrogen/deuterium exchange experiments confirm the second harmonic generation observations and reveal allosteric changes in the G receptor binding and F-activating stalk domains, providing insights into the pathway of receptor-activated virus entry.Nipah virus causes encephalitis in humans. Here the authors use a multidisciplinary approach to study the binding of the viral attachment protein G to its host receptor ephrinB2 and show that monomeric and dimeric receptors activate distinct conformational changes in G and discuss implications for receptor-activated virus entry.

Conformational and functional studies of a cytosolic 90 kDa heat shock protein Hsp90 from sugarcane.

PubMed

da Silva, Viviane C H; Cagliari, Thiago C; Lima, Tatiani B; Gozzo, Fábio C; Ramos, Carlos H I

2013-07-01

Hsp90s are involved in several cellular processes, such as signaling, proteostasis, epigenetics, differentiation and stress defense. Although Hsp90s from different organisms are highly similar, they usually have small variations in conformation and function. Thus, the characterization of different Hsp90s is important to gain insight into the structure-function relationship that makes these chaperones key regulators in protein homeostasis. This work describes the characterization of a cytosolic Hsp90 from sugarcane and its comparison with Hsp90s from other plants. Previous expressed sequence tag (EST) studies in Saccharum spp. (sugarcane) predicted the presence of an mRNA coding for a cytosolic Hsp90. The corresponding cDNA was cloned, and the recombinant protein was purified and its conformation and function characterized. The structural conformation of Hsp90 was assessed by chemical cross-linking and hydrogen/deuterium exchange using mass spectrometry and hydrodynamic assays, which revealed regions accessible to solvent and that Hsp90 is an elongated dimer in solution. The in vivo expression of Hsp90 in sugarcane leaves was confirmed by western blot, and in vitro functional characterization indicated that sugarcane Hsp90 has strong chaperone activity. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Sodium recognition by the Na+/Ca2+ exchanger in the outward-facing conformation

PubMed Central

Marinelli, Fabrizio; Almagor, Lior; Hiller, Reuben; Giladi, Moshe; Khananshvili, Daniel; Faraldo-Gómez, José D.

2014-01-01

Na+/Ca2+ exchangers (NCXs) are ubiquitous membrane transporters with a key role in Ca2+ homeostasis and signaling. NCXs mediate the bidirectional translocation of either Na+ or Ca2+, and thus can catalyze uphill Ca2+ transport driven by a Na+ gradient, or vice versa. In a major breakthrough, a prokaryotic NCX homolog (NCX_Mj) was recently isolated and its crystal structure determined at atomic resolution. The structure revealed an intriguing architecture consisting of two inverted-topology repeats, each comprising five transmembrane helices. These repeats adopt asymmetric conformations, yielding an outward-facing occluded state. The crystal structure also revealed four putative ion-binding sites, but the occupancy and specificity thereof could not be conclusively established. Here, we use molecular-dynamics simulations and free-energy calculations to identify the ion configuration that best corresponds to the crystallographic data and that is also thermodynamically optimal. In this most probable configuration, three Na+ ions occupy the so-called Sext, SCa, and Sint sites, whereas the Smid site is occupied by one water molecule and one H+, which protonates an adjacent aspartate side chain (D240). Experimental measurements of Na+/Ca2+ and Ca2+/Ca2+ exchange by wild-type and mutagenized NCX_Mj confirm that transport of both Na+ and Ca2+ requires protonation of D240, and that this side chain does not coordinate either ion at Smid. These results imply that the ion exchange stoichiometry of NCX_Mj is 3:1 and that translocation of Na+ across the membrane is electrogenic, whereas transport of Ca2+ is not. Altogether, these findings provide the basis for further experimental and computational studies of the conformational mechanism of this exchanger. PMID:25468964

Sodium recognition by the Na+/Ca2+ exchanger in the outward-facing conformation.

PubMed

Marinelli, Fabrizio; Almagor, Lior; Hiller, Reuben; Giladi, Moshe; Khananshvili, Daniel; Faraldo-Gómez, José D

2014-12-16

Na(+)/Ca(2+) exchangers (NCXs) are ubiquitous membrane transporters with a key role in Ca(2+) homeostasis and signaling. NCXs mediate the bidirectional translocation of either Na(+) or Ca(2+), and thus can catalyze uphill Ca(2+) transport driven by a Na(+) gradient, or vice versa. In a major breakthrough, a prokaryotic NCX homolog (NCX_Mj) was recently isolated and its crystal structure determined at atomic resolution. The structure revealed an intriguing architecture consisting of two inverted-topology repeats, each comprising five transmembrane helices. These repeats adopt asymmetric conformations, yielding an outward-facing occluded state. The crystal structure also revealed four putative ion-binding sites, but the occupancy and specificity thereof could not be conclusively established. Here, we use molecular-dynamics simulations and free-energy calculations to identify the ion configuration that best corresponds to the crystallographic data and that is also thermodynamically optimal. In this most probable configuration, three Na(+) ions occupy the so-called Sext, SCa, and Sint sites, whereas the Smid site is occupied by one water molecule and one H(+), which protonates an adjacent aspartate side chain (D240). Experimental measurements of Na(+)/Ca(2+) and Ca(2+)/Ca(2+) exchange by wild-type and mutagenized NCX_Mj confirm that transport of both Na(+) and Ca(2+) requires protonation of D240, and that this side chain does not coordinate either ion at Smid. These results imply that the ion exchange stoichiometry of NCX_Mj is 3:1 and that translocation of Na(+) across the membrane is electrogenic, whereas transport of Ca(2+) is not. Altogether, these findings provide the basis for further experimental and computational studies of the conformational mechanism of this exchanger.

Teacher Exchange and Rotation Is Not Equivalent to Partner Assistance

ERIC Educational Resources Information Center

Guilin, Yuan

2018-01-01

Given that education quality has long lagged behind in China's rural schools, one-way "partner assistance" no longer conforms to the new situation of integrated urban-rural governance and the equalization of public services. Only two-way "exchange and rotation" with full participation can truly support schools and teachers in…

How amide hydrogens exchange in native proteins.

PubMed

Persson, Filip; Halle, Bertil

2015-08-18

Amide hydrogen exchange (HX) is widely used in protein biophysics even though our ignorance about the HX mechanism makes data interpretation imprecise. Notably, the open exchange-competent conformational state has not been identified. Based on analysis of an ultralong molecular dynamics trajectory of the protein BPTI, we propose that the open (O) states for amides that exchange by subglobal fluctuations are locally distorted conformations with two water molecules directly coordinated to the N-H group. The HX protection factors computed from the relative O-state populations agree well with experiment. The O states of different amides show little or no temporal correlation, even if adjacent residues unfold cooperatively. The mean residence time of the O state is ∼100 ps for all examined amides, so the large variation in measured HX rate must be attributed to the opening frequency. A few amides gain solvent access via tunnels or pores penetrated by water chains including native internal water molecules, but most amides access solvent by more local structural distortions. In either case, we argue that an overcoordinated N-H group is necessary for efficient proton transfer by Grotthuss-type structural diffusion.

How amide hydrogens exchange in native proteins

PubMed Central

Persson, Filip; Halle, Bertil

2015-01-01

Amide hydrogen exchange (HX) is widely used in protein biophysics even though our ignorance about the HX mechanism makes data interpretation imprecise. Notably, the open exchange-competent conformational state has not been identified. Based on analysis of an ultralong molecular dynamics trajectory of the protein BPTI, we propose that the open (O) states for amides that exchange by subglobal fluctuations are locally distorted conformations with two water molecules directly coordinated to the N–H group. The HX protection factors computed from the relative O-state populations agree well with experiment. The O states of different amides show little or no temporal correlation, even if adjacent residues unfold cooperatively. The mean residence time of the O state is ∼100 ps for all examined amides, so the large variation in measured HX rate must be attributed to the opening frequency. A few amides gain solvent access via tunnels or pores penetrated by water chains including native internal water molecules, but most amides access solvent by more local structural distortions. In either case, we argue that an overcoordinated N–H group is necessary for efficient proton transfer by Grotthuss-type structural diffusion. PMID:26195754

76 FR 27691 - Self-Regulatory Organizations; NYSE Arca, Inc.; Notice of Filing of Proposed Rule Change Amending...

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Light activation of the LOV protein vivid generates a rapidly exchanging dimer.

PubMed

Zoltowski, Brian D; Crane, Brian R

2008-07-08

The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (light, oxygen, voltage) protein, couples light-induced cysteinyl adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal beta-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap-PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of N. crassa and indicate that LOV-LOV homo- or heterodimerization may be a mechanism for regulating light-activated gene expression.

Equilibrium Molecular Thermodynamics from Kirkwood Sampling

PubMed Central

2015-01-01

We present two methods for barrierless equilibrium sampling of molecular systems based on the recently proposed Kirkwood method (J. Chem. Phys.2009, 130, 134102). Kirkwood sampling employs low-order correlations among internal coordinates of a molecule for random (or non-Markovian) sampling of the high dimensional conformational space. This is a geometrical sampling method independent of the potential energy surface. The first method is a variant of biased Monte Carlo, where Kirkwood sampling is used for generating trial Monte Carlo moves. Using this method, equilibrium distributions corresponding to different temperatures and potential energy functions can be generated from a given set of low-order correlations. Since Kirkwood samples are generated independently, this method is ideally suited for massively parallel distributed computing. The second approach is a variant of reservoir replica exchange, where Kirkwood sampling is used to construct a reservoir of conformations, which exchanges conformations with the replicas performing equilibrium sampling corresponding to different thermodynamic states. Coupling with the Kirkwood reservoir enhances sampling by facilitating global jumps in the conformational space. The efficiency of both methods depends on the overlap of the Kirkwood distribution with the target equilibrium distribution. We present proof-of-concept results for a model nine-atom linear molecule and alanine dipeptide. PMID:25915525

Molecular Simulation Uncovers the Conformational Space of the λ Cro Dimer in Solution

PubMed Central

Ahlstrom, Logan S.; Miyashita, Osamu

2011-01-01

The significant variation among solved structures of the λ Cro dimer suggests its flexibility. However, contacts in the crystal lattice could have stabilized a conformation which is unrepresentative of its dominant solution form. Here we report on the conformational space of the Cro dimer in solution using replica exchange molecular dynamics in explicit solvent. The simulated ensemble shows remarkable correlation with available x-ray structures. Network analysis and a free energy surface reveal the predominance of closed and semi-open dimers, with a modest barrier separating these two states. The fully open conformation lies higher in free energy, indicating that it requires stabilization by DNA or crystal contacts. Most NMR models are found to be unstable conformations in solution. Intersubunit salt bridging between Arg4 and Glu53 during simulation stabilizes closed conformations. Because a semi-open state is among the low-energy conformations sampled in simulation, we propose that Cro-DNA binding may not entail a large conformational change relative to the dominant dimer forms in solution. PMID:22098751

Effects of urea induced protein conformational changes on ion exchange chromatographic behavior.

PubMed

Hou, Ying; Hansen, Thomas B; Staby, Arne; Cramer, Steven M

2010-11-19

Urea is widely employed to facilitate protein separations in ion exchange chromatography at various scales. In this work, five model proteins were used to examine the chromatographic effects of protein conformational changes induced by urea in ion exchange chromatography. Linear gradient experiments were carried out at various urea concentrations and the protein secondary and tertiary structures were evaluated by far UV CD and fluorescence measurements, respectively. The results indicated that chromatographic retention times were well correlated with structural changes and that they were more sensitive to tertiary structural change. Steric Mass Action (SMA) isotherm parameters were also examined and the results indicated that urea induced protein conformational changes could affect both the characteristic charge and equilibrium constants in these systems. Dynamic light scattering analysis of changes in protein size due to urea-induced unfolding indicated that the size of the protein was not correlated with SMA parameter changes. These results indicate that while urea-induced structural changes can have a marked effect on protein chromatographic behavior in IEX, this behavior can be quite complicated and protein specific. These differences in protein behavior may provide insight into how these partially unfolded proteins are interacting with the resin material. Copyright © 2010 Elsevier B.V. All rights reserved.

"Invisible" conformers of an antifungal disulfide protein revealed by constrained cold and heat unfolding, CEST-NMR experiments, and molecular dynamics calculations.

PubMed

Fizil, Ádám; Gáspári, Zoltán; Barna, Terézia; Marx, Florentine; Batta, Gyula

2015-03-23

Transition between conformational states in proteins is being recognized as a possible key factor of function. In support of this, hidden dynamic NMR structures were detected in several cases up to populations of a few percent. Here, we show by two- and three-state analysis of thermal unfolding, that the population of hidden states may weight 20-40 % at 298 K in a disulfide-rich protein. In addition, sensitive (15) N-CEST NMR experiments identified a low populated (0.15 %) state that was in slow exchange with the folded PAF protein. Remarkably, other techniques failed to identify the rest of the NMR "dark matter". Comparison of the temperature dependence of chemical shifts from experiments and molecular dynamics calculations suggests that hidden conformers of PAF differ in the loop and terminal regions and are most similar in the evolutionary conserved core. Our observations point to the existence of a complex conformational landscape with multiple conformational states in dynamic equilibrium, with diverse exchange rates presumably responsible for the completely hidden nature of a considerable fraction. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Effect of sequence and stereochemistry reversal on p53 peptide mimicry.

PubMed

Atzori, Alessio; Baker, Audrey E; Chiu, Mark; Bryce, Richard A; Bonnet, Pascal

2013-01-01

Peptidomimetics effective in modulating protein-protein interactions and resistant to proteolysis have potential in therapeutic applications. An appealing yet underperforming peptidomimetic strategy is to employ D-amino acids and reversed sequences to mimic a lead peptide conformation, either separately or as the combined retro-inverso peptide. In this work, we examine the conformations of inverse, reverse and retro-inverso peptides of p53(15-29) using implicit solvent molecular dynamics simulation and circular dichroism spectroscopy. In order to obtain converged ensembles for the peptides, we find enhanced sampling is required via the replica exchange molecular dynamics method. From these replica exchange simulations, the D-peptide analogues of p53(15-29) result in a predominantly left-handed helical conformation. When the parent sequence is reversed sequence as either the L-peptide and D-peptide, these peptides display a greater helical propensity, feature reflected by NMR and CD studies in TFE/water solvent. The simulations also indicate that, while approximately similar orientations of the side-chains are possible by the peptide analogues, their ability to mimic the parent peptide is severely compromised by backbone orientation (for D-amino acids) and side-chain orientation (for reversed sequences). A retro-inverso peptide is disadvantaged as a mimic in both aspects, and further chemical modification is required to enable this concept to be used fruitfully in peptidomimetic design. The replica exchange molecular simulation approach adopted here, with its ability to provide detailed conformational insights into modified peptides, has potential as a tool to guide structure-based design of new improved peptidomimetics.

Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry*

PubMed Central

Jensen, Pernille Foged; Larraillet, Vincent; Schlothauer, Tilman; Kettenberger, Hubert; Hilger, Maximiliane; Rand, Kasper D.

2015-01-01

The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest in understanding and modulating the IgG–FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG1 and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation to map sites perturbed by binding on both partners of the IgG–FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found that several regions in the IgG Fab region also showed reduced deuterium uptake. Our findings indicate the presence of hitherto unknown FcRn interaction sites in the Fab region or a possible conformational link between the IgG Fc and Fab regions upon FcRn binding. Further, we investigated the role of IgG glycosylation in the conformational response of the IgG–FcRn interaction. Removal of antibody glycans increased the flexibility of the FcRn binding site in the Fc region. Consequently, FcRn binding did not induce a similar conformational stabilization of deglycosylated IgG as observed for the wild-type glycosylated IgG. Our results provide new molecular insight into the IgG–FcRn interaction and illustrate the capability of hydrogen/deuterium exchange mass spectrometry to advance structural proteomics by providing detailed information on the conformation and dynamics of large protein complexes in solution. PMID:25378534

G-quadruplex dynamics.

PubMed

Harkness, Robert W; Mittermaier, Anthony K

2017-11-01

G-quadruplexes (GQs) are four-stranded nucleic acid secondary structures formed by guanosine (G)-rich DNA and RNA sequences. It is becoming increasingly clear that cellular processes including gene expression and mRNA translation are regulated by GQs. GQ structures have been extensively characterized, however little attention to date has been paid to their conformational dynamics, despite the fact that many biological GQ sequences populate multiple structures of similar free energies, leading to an ensemble of exchanging conformations. The impact of these dynamics on biological function is currently not well understood. Recently, structural dynamics have been demonstrated to entropically stabilize GQ ensembles, potentially modulating gene expression. Transient, low-populated states in GQ ensembles may additionally regulate nucleic acid interactions and function. This review will underscore the interplay of GQ dynamics and biological function, focusing on several dynamic processes for biological GQs and the characterization of GQ dynamics by nuclear magnetic resonance (NMR) spectroscopy in conjunction with other biophysical techniques. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

Nullspace Sampling with Holonomic Constraints Reveals Molecular Mechanisms of Protein Gαs.

PubMed

Pachov, Dimitar V; van den Bedem, Henry

2015-07-01

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.

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

From integrability to conformal symmetry: Bosonic superconformal Toda theories

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

Bo-Yu Hou; Liu Chao

In this paper the authors study the conformal integrable models obtained from conformal reductions of WZNW theory associated with second order constraints. These models are called bosonic superconformal Toda models due to their conformal spectra and their resemblance to the usual Toda theories. From the reduction procedure they get the equations of motion and the linearized Lax equations in a generic Z gradation of the underlying Lie algebra. Then, in the special case of principal gradation, they derive the classical r matrix, fundamental Poisson relation, exchange algebra of chiral operators and find out the classical vertex operators. The result showsmore » that their model is very similar to the ordinary Toda theories in that one can obtain various conformal properties of the model from its integrability.« less

A heteronuclear zero quantum coherence Nz-exchange experiment that resolves resonance overlap and its application to measure the rates of heme binding to the IsdC protein.

PubMed

Robson, Scott A; Peterson, Robert; Bouchard, Louis-S; Villareal, Valerie A; Clubb, Robert T

2010-07-21

Chemical exchange phenomena in NMR spectra can be quantitatively interpreted to measure the rates of ligand binding, as well as conformational and chemical rearrangements. In macromolecules, processes that occur slowly on the chemical shift time scale are frequently studied using 2D heteronuclear ZZ or N(z)-exchange spectroscopy. However, to successfully apply this method, peaks arising from each exchanging species must have unique chemical shifts in both dimensions, a condition that is often not satisfied in protein-ligand binding equilibria for (15)N nuclei. To overcome the problem of (15)N chemical shift degeneracy we developed a heteronuclear zero-quantum (and double-quantum) coherence N(z)-exchange experiment that resolves (15)N chemical shift degeneracy in the indirect dimension. We demonstrate the utility of this new experiment by measuring the heme binding kinetics of the IsdC protein from Staphylococcus aureus. Because of peak overlap, we could not reliably analyze binding kinetics using conventional methods. However, our new experiment resulted in six well-resolved systems that yielded interpretable data. We measured a relatively slow k(off) rate of heme from IsdC (<10 s(-1)), which we interpret as necessary so heme loaded IsdC has time to encounter downstream binding partners to which it passes the heme. The utility of using this new exchange experiment can be easily expanded to (13)C nuclei. We expect our heteronuclear zero-quantum coherence N(z)-exchange experiment will expand the usefulness of exchange spectroscopy to slow chemical exchange events that involve ligand binding.

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

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Protein structural dynamics at the gas/water interface examined by hydrogen exchange mass spectrometry.

PubMed

Xiao, Yiming; Konermann, Lars

2015-08-01

Gas/water interfaces (such as air bubbles or foam) are detrimental to the stability of proteins, often causing aggregation. This represents a potential problem for industrial processes, for example, the production and handling of protein drugs. Proteins possess surfactant-like properties, resulting in a high affinity for gas/water interfaces. The tendency of previously buried nonpolar residues to maximize contact with the gas phase can cause significant structural distortion. Most earlier studies in this area employed spectroscopic tools that could only provide limited information. Here we use hydrogen/deuterium exchange (HDX) mass spectrometry (MS) for probing the conformational dynamics of the model protein myoglobin (Mb) in the presence of N(2) bubbles. HDX/MS relies on the principle that unfolded and/or highly dynamic regions undergo faster deuteration than tightly folded segments. In bubble-free solution Mb displays EX2 behavior, reflecting the occurrence of short-lived excursions to partially unfolded conformers. A dramatically different behavior is seen in the presence of N(2) bubbles; EX2 dynamics still take place, but in addition the protein shows EX1 behavior. The latter results from interconversion of the native state with conformers that are globally unfolded and long-lived. These unfolded species likely correspond to Mb that is adsorbed to the surface of gas bubbles. N(2) sparging also induces aggregation. To explain the observed behavior we propose a simple model, that is, "semi-unfolded" ↔ "native" ↔ "globally unfolded" → "aggregated". This model quantitatively reproduces the experimentally observed kinetics. To the best of our knowledge, the current study marks the first exploration of surface denaturation phenomena by HDX/MS. © 2015 The Protein Society.

Fluid to fluid contact heat exchanger

NASA Technical Reports Server (NTRS)

Clark, W. E.

1986-01-01

Heat transfer and pressure drop test results for a fluid to fluid contact heat exchanger are reported. The heat exchanger, fabricated and tested to demonstrate one method of transferring heat between structures in space, had a total contact area of 0.18 sq m. It utilized contact surfaces which were flexible and conformed to the mating contact surfaces upon pressurization of the fluid circulating within the heat exchanger. During proof-of-concept performance tests, the heat exchanger was operated in a typical earth environment. It demonstrated a contact conductance of 3.8 kW/sq m C at contact pressures in the 15 to 70 kPa range.

Dynamic NMR study of trans-cyclodecene

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

Pawar, D.M.; Noe, E.A.

1996-12-18

The slow-exchange {sup 13}C spectrum of trans-cyclodecene at -154.9{degree}C shows eight peaks of the olefinic carbons, and these are interpreted in terms of five conformations. Three of the conformations are of C{sub 1} symmetry, and two are of C{sub 2} symmetry. Further evidence for the number of conformations and their symmetries came from a proton NMR spectrum of the olefinic hydrogens taken at -154.9{degree}C with decoupling the allylic hydrogens. Populations ranged from 3.0% to 37.6% with the least-populated conformation having a free energy of 0.59 kcal/mol, relative to the most stable conformer. The conformations studied by Saunders and Jimenez-Vazquez usingmore » Allinger`s MM3 force field are described, and the calculated strain energies and populations are discussed. Energies for six conformations were also obtained from ab initio calculations at the HF/6-311G{sup *} level. 22 refs., 4 figs., 3 tabs.« less

Light cone thermodynamics

NASA Astrophysics Data System (ADS)

De Lorenzo, Tommaso; Perez, Alejandro

2018-02-01

We show that null surfaces defined by the outgoing and infalling wave fronts emanating from and arriving at a sphere in Minkowski spacetime have thermodynamical properties that are in strict formal correspondence with those of black hole horizons in curved spacetimes. Such null surfaces, made of pieces of light cones, are bifurcate conformal Killing horizons for suitable conformally stationary observers. They can be extremal and nonextremal depending on the radius of the shining sphere. Such conformal Killing horizons have a constant light cone (conformal) temperature, given by the standard expression in terms of the generalization of surface gravity for conformal Killing horizons. Exchanges of conformally invariant energy across the horizon are described by a first law where entropy changes are given by 1 /(4 ℓp2) of the changes of a geometric quantity with the meaning of horizon area in a suitable conformal frame. These conformal horizons satisfy the zeroth to the third laws of thermodynamics in an appropriate way. In the extremal case they become light cones associated with a single event; these have vanishing temperature as well as vanishing entropy.

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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.

Light activation of the LOV protein Vivid generates a rapidly exchanging dimer†‡

PubMed Central

Zoltowski, Brian D.; Crane, Brian R.

2009-01-01

The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (Light, Oxygen, Voltage) protein, couples light-induced cysteinyl-adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal β-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap-to-PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of Neurospora crassa, and indicate that LOV:LOV homo or hetero dimerization may be a mechanism for regulating light-activated gene expression. PMID:18553928

Substrate Binding Drives Active-Site Closing of Human Blood Group B Galactosyltransferase as Revealed by Hot-Spot Labeling and NMR Spectroscopy Experiments.

PubMed

Weissbach, Sophie; Flügge, Friedemann; Peters, Thomas

2018-05-04

Crystallography has shown that human blood group A (GTA) and B (GTB) glycosyltransferases undergo transitions between "open", "semiclosed", and "closed" conformations upon substrate binding. However, the timescales of the corresponding conformational reorientations are unknown. Crystal structures show that the Trp and Met residues are located at "conformational hot spots" of the enzymes. Therefore, we utilized 15 N side-chain labeling of Trp residues and 13 C-methyl labeling of Met residues to study substrate-induced conformational transitions of GTB. Chemical-shift perturbations (CSPs) of Met and Trp residues in direct contact with substrate ligands reflect binding kinetics, whereas the CSPs of Met and Trp residues at remote sites reflect conformational changes of the enzyme upon substrate binding. Acceptor binding is fast on the chemical-shift timescale with rather small CSPs in the range of less than approximately 20 Hz. Donor binding matches the intermediate exchange regime to yield an estimate for exchange rate constants of approximately 200-300 Hz. Donor or acceptor binding to GTB saturated with acceptor or donor substrate, respectively, is slow (<10 Hz), as are coupled protein motions, reflecting mutual allosteric control of donor and acceptor binding. Remote CSPs suggest that substrate binding drives the enzyme into the closed state required for catalysis. These findings should contribute to better understanding of the mechanism of glycosyl transfer of GTA and GTB. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biofouling on polymeric heat exchanger surfaces with E. coli and native biofilms.

PubMed

Pohl, S; Madzgalla, M; Manz, W; Bart, H J

2015-01-01

The biofouling affinity of different polymeric surfaces (polypropylene, polysulfone, polyethylene terephthalate, and polyether ether ketone) in comparison to stainless steel (SS) was studied for the model bacterium Escherichia coli K12 DSM 498 and native biofilms originating from Rhine water. The biofilm mass deposited on the polymer surfaces was minimized by several magnitudes compared to SS. The cell count and the accumulated biomass of E. coli on the polymer surfaces showed an opposing linear trend. The promising low biofilm formation on the polymers is attributed to the combination of inherent surface properties (roughness, surface energy and hydrophobicity) when compared to SS. The fouling characteristics of E. coli biofilms show good conformity with the more complex native biofilms investigated. The results can be utilized for the development of new polymer heat exchangers when using untreated river water as coolant or for other processes needing antifouling materials.

Effect of tension and curvature on the chemical potential of lipids in lipid aggregates.

PubMed

Grafmüller, Andrea; Lipowsky, Reinhard; Knecht, Volker

2013-01-21

Understanding the factors that influence the free energy of lipids in bilayer membranes is an essential step toward understanding exchange processes of lipids between membranes. In general, both lipid composition and membrane geometry can affect lipid exchange rates between bilayer membranes. Here, the free energy change ΔG(des) for the desorption of dipalmitoyl-phosphatidylcholine (DPPC) lipids from different lipid aggregates has been computed using molecular dynamics simulations and umbrella sampling. The value of ΔG(des) is found to depend strongly on the local properties of the aggregate, in that both tension and curvature lead to an increase in ΔG(des). A detailed analysis shows that the increased desorption free energy for tense bilayers arises from the increased conformational entropy of the lipid tails, which reduces the favorable component -TΔS(L) of the desorption free energy.

A Single Disulfide Bond Disruption in the β3 Integrin Subunit Promotes Thiol/Disulfide Exchange, a Molecular Dynamics Study

PubMed Central

Levin, Lihie; Zelzion, Ehud; Nachliel, Esther; Gutman, Menachem; Tsfadia, Yossi; Einav, Yulia

2013-01-01

The integrins are a family of membrane receptors that attach a cell to its surrounding and play a crucial function in cell signaling. The combination of internal and external stimuli alters a folded non-active state of these proteins to an extended active configuration. The β3 subunit of the platelet αIIbβ3 integrin is made of well-structured domains rich in disulfide bonds. During the activation process some of the disulfides are re-shuffled by a mechanism requiring partial reduction of some of these bonds; any disruption in this mechanism can lead to inherent blood clotting diseases. In the present study we employed Molecular Dynamics simulations for tracing the sequence of structural fluctuations initiated by a single cysteine mutation in the β3 subunit of the receptor. These simulations showed that in-silico protein mutants exhibit major conformational deformations leading to possible disulfide exchange reactions. We suggest that any mutation that prevents Cys560 from reacting with one of the Cys567–Cys581 bonded pair, thus disrupting its ability to participate in a disulfide exchange reaction, will damage the activation mechanism of the integrin. This suggestion is in full agreement with previously published experiments. Furthermore, we suggest that rearrangement of disulfide bonds could be a part of a natural cascade of thiol/disulfide exchange reactions in the αIIbβ3 integrin, which are essential for the native activation process. PMID:23527123

Propafenone effects on the stable structures of Aβ16-22 system

NASA Astrophysics Data System (ADS)

Tran, Linh; Ngo, Son Tung; Nguyen, Minh Tho

2018-03-01

An extensive replica exchange molecular dynamics (REMD) simulation was performed to investigate the progress patterns of the molecular interactions of propafenone and Aβ16-22 system. Distinct conformational equilibrium of Aβ16-22 system with and without propafenone was analyzed in detail. Propafenone can act to prevent the Alzheimer's disease (AD) by significantly inhibiting Aβ oligomerization. Our calculated results provide insights into the inhibition mechanism of propafenone on the oligomerization process to form Aβ16-22 peptide aggregation. These findings are valuable for the development of therapeutic drugs in the AD early stage.

Underpotential deposition-mediated layer-by-layer growth of thin films

DOEpatents

Wang, Jia Xu; Adzic, Radoslav R.

2017-06-27

A method of depositing contiguous, conformal submonolayer-to-multilayer thin films with atomic-level control is described. The process involves electrochemically exchanging a mediating element on a substrate with a noble metal film by alternatingly sweeping potential in forward and reverse directions for a predetermined number of times in an electrochemical cell. By cycling the applied voltage between the bulk deposition potential for the mediating element and the material to be deposited, repeated desorption/adsorption of the mediating element during each potential cycle can be used to precisely control film growth on a layer-by-layer basis.

Elongation factor Ts directly facilitates the formation and disassembly of the Escherichia coli elongation factor Tu·GTP·aminoacyl-tRNA ternary complex.

PubMed

Burnett, Benjamin J; Altman, Roger B; Ferrao, Ryan; Alejo, Jose L; Kaur, Navdep; Kanji, Joshua; Blanchard, Scott C

2013-05-10

Aminoacyl-tRNA (aa-tRNA) enters the ribosome in a ternary complex with the G-protein elongation factor Tu (EF-Tu) and GTP. EF-Tu·GTP·aa-tRNA ternary complex formation and decay rates are accelerated in the presence of the nucleotide exchange factor elongation factor Ts (EF-Ts). EF-Ts directly facilitates the formation and disassociation of ternary complex. This system demonstrates a novel function of EF-Ts. Aminoacyl-tRNA enters the translating ribosome in a ternary complex with elongation factor Tu (EF-Tu) and GTP. Here, we describe bulk steady state and pre-steady state fluorescence methods that enabled us to quantitatively explore the kinetic features of Escherichia coli ternary complex formation and decay. The data obtained suggest that both processes are controlled by a nucleotide-dependent, rate-determining conformational change in EF-Tu. Unexpectedly, we found that this conformational change is accelerated by elongation factor Ts (EF-Ts), the guanosine nucleotide exchange factor for EF-Tu. Notably, EF-Ts attenuates the affinity of EF-Tu for GTP and destabilizes ternary complex in the presence of non-hydrolyzable GTP analogs. These results suggest that EF-Ts serves an unanticipated role in the cell of actively regulating the abundance and stability of ternary complex in a manner that contributes to rapid and faithful protein synthesis.

Microsecond kinetics in model single- and double-stranded amylose polymers.

PubMed

Sattelle, Benedict M; Almond, Andrew

2014-05-07

Amylose, a component of starch with increasing biotechnological significance, is a linear glucose polysaccharide that self-organizes into single- and double-helical assemblies. Starch granule packing, gelation and inclusion-complex formation result from finely balanced macromolecular kinetics that have eluded precise experimental quantification. Here, graphics processing unit (GPU) accelerated multi-microsecond aqueous simulations are employed to explore conformational kinetics in model single- and double-stranded amylose. The all-atom dynamics concur with prior X-ray and NMR data while surprising and previously overlooked microsecond helix-coil, glycosidic linkage and pyranose ring exchange are hypothesized. In a dodecasaccharide, single-helical collapse was correlated with linkages and rings transitioning from their expected syn and (4)C1 chair conformers. The associated microsecond exchange rates were dependent on proximity to the termini and chain length (comparing hexa- and trisaccharides), while kinetic features of dodecasaccharide linkage and ring flexing are proposed to be a good model for polymers. Similar length double-helices were stable on microsecond timescales but the parallel configuration was sturdier than the antiparallel equivalent. In both, tertiary organization restricted local chain dynamics, implying that simulations of single amylose strands cannot be extrapolated to dimers. Unbiased multi-microsecond simulations of amylose are proposed as a valuable route to probing macromolecular kinetics in water, assessing the impact of chemical modifications on helical stability and accelerating the development of new biotechnologies.

NMR experiments for the rapid identification of P=O···H-X type hydrogen bonds in nucleic acids.

PubMed

Duchardt-Ferner, Elke; Wöhnert, Jens

2017-10-01

Hydrogen bonds involving the backbone phosphate groups occur with high frequency in functional RNA molecules. They are often found in well-characterized tertiary structural motifs presenting powerful probes for the rapid identification of these motifs for structure elucidation purposes. We have shown recently that stable hydrogen bonds to the phosphate backbone can in principle be detected by relatively simple NMR-experiments, providing the identity of both the donor hydrogen and the acceptor phosphorous within the same experiment (Duchardt-Ferner et al., Angew Chem Int Ed Engl 50:7927-7930, 2011). However, for imino and hydroxyl hydrogen bond donor groups rapidly exchanging with the solvent as well as amino groups broadened by conformational exchange experimental sensitivity is severely hampered by extensive line broadening. Here, we present improved methods for the rapid identification of hydrogen bonds to phosphate groups in nucleic acids by NMR. The introduction of the SOFAST technique into 1 H, 31 P-correlation experiments as well as a BEST-HNP experiment exploiting 3h J N,P rather than 2h J H,P coupling constants enables the rapid and sensitive identification of these hydrogen bonds in RNA. The experiments are applicable for larger RNAs (up to ~ 100-nt), for donor groups influenced by conformational exchange processes such as amino groups and for hydrogen bonds with rather labile hydrogens such as 2'-OH groups as well as for moderate sample concentrations. Interestingly, the size of the through-hydrogen bond scalar coupling constants depends not only on the type of the donor group but also on the structural context. The largest coupling constants were measured for hydrogen bonds involving the imino groups of protonated cytosine nucleotides as donors.

Coulomb replica-exchange method: handling electrostatic attractive and repulsive forces for biomolecules.

PubMed

Itoh, Satoru G; Okumura, Hisashi

2013-03-30

We propose a new type of the Hamiltonian replica-exchange method (REM) for molecular dynamics (MD) and Monte Carlo simulations, which we refer to as the Coulomb REM (CREM). In this method, electrostatic charge parameters in the Coulomb interactions are exchanged among replicas while temperatures are exchanged in the usual REM. By varying the atom charges, the CREM overcomes free-energy barriers and realizes more efficient sampling in the conformational space than the REM. Furthermore, this method requires only a smaller number of replicas because only the atom charges of solute molecules are used as exchanged parameters. We performed Coulomb replica-exchange MD simulations of an alanine dipeptide in explicit water solvent and compared the results with those of the conventional canonical, replica exchange, and van der Waals REMs. Two force fields of AMBER parm99 and AMBER parm99SB were used. As a result, the CREM sampled all local-minimum free-energy states more frequently than the other methods for both force fields. Moreover, the Coulomb, van der Waals, and usual REMs were applied to a fragment of an amyloid-β peptide (Aβ) in explicit water solvent to compare the sampling efficiency of these methods for a larger system. The CREM sampled structures of the Aβ fragment more efficiently than the other methods. We obtained β-helix, α-helix, 3(10)-helix, β-hairpin, and β-sheet structures as stable structures and deduced pathways of conformational transitions among these structures from a free-energy landscape. Copyright © 2012 Wiley Periodicals, Inc.

Alternate binding modes for a ubiquitin-SH3 domain interaction studied by NMR spectroscopy.

PubMed

Korzhnev, Dmitry M; Bezsonova, Irina; Lee, Soyoung; Chalikian, Tigran V; Kay, Lewis E

2009-02-20

Surfaces of many binding domains are plastic, enabling them to interact with multiple targets. An understanding of how they bind and recognize their partners is therefore predicated on characterizing such dynamic interfaces. Yet, these interfaces are difficult to study by standard biophysical techniques that often 'freeze' out conformations or that produce data averaged over an ensemble of conformers. In this study, we used NMR spectroscopy to study the interaction between the C-terminal SH3 domain of CIN85 and ubiquitin that involves the 'classical' binding sites of these proteins. Notably, chemical shift titration data of one target with another and relaxation dispersion data that report on millisecond time scale exchange processes are both well fit to a simple binding model in which free protein is in equilibrium with a single bound conformation. However, dissociation constants and chemical shift differences between free and bound states measured from both classes of experiment are in disagreement. It is shown that the data can be reconciled by considering three-state binding models involving two distinct bound conformations. By combining titration and dispersion data, kinetic and thermodynamic parameters of the three-state binding reaction are obtained along with chemical shifts for each state. A picture emerges in which one bound conformer has increased entropy and enthalpy relative to the second and chemical shifts similar to that of the free state, suggesting a less packed interface. This study provides an example of the interplay between entropy and enthalpy to fine-tune molecular interactions involving the same binding surfaces.

Characterization of the near native conformational states of the SAM domain of Ste11 protein by NMR spectroscopy.

PubMed

Gupta, Sebanti; Bhattacharjya, Surajit

2014-11-01

The sterile alpha motif or SAM domain is one of the most frequently present protein interaction modules with diverse functional attributions. SAM domain of the Ste11 protein of budding yeast plays important roles in mitogen-activated protein kinase cascades. In the current study, urea-induced, at subdenaturing concentrations, structural, and dynamical changes in the Ste11 SAM domain have been investigated by nuclear magnetic resonance spectroscopy. Our study revealed that a number of residues from Helix 1 and Helix 5 of the Ste11 SAM domain display plausible alternate conformational states and largest chemical shift perturbations at low urea concentrations. Amide proton (H/D) exchange experiments indicated that Helix 1, loop, and Helix 5 become more susceptible to solvent exchange with increased concentrations of urea. Notably, Helix 1 and Helix 5 are directly involved in binding interactions of the Ste11 SAM domain. Our data further demonstrate that the existence of alternate conformational states around the regions involved in dimeric interactions in native or near native conditions. © 2014 Wiley Periodicals, Inc.

[The conformational dynamics of the tetramer hemoglobin molecule as revealed by hydrogen exchange. III. Influence of the heme removal].

PubMed

Abaturov, L V; Nosova, N G; Shliapnikova, S V

2006-01-01

Two main types of conformational fluctuations--local and global are characteristic of the native protein structure and revealed by hydrogen exchange. The probability of those fluctuations changes to a different extent upon hemoglobin oxygenation, changing of pH, splitting of the intersubunit contacts. To compare with the influence of the heme removal the rate of the H-D exchange of the peptide NH atoms of the human apoHb was studied at the pH range 5.5-9.0 and temperature 10-38 degrees C by the IR spectroscopy. The removal of the heme increases the rate of the H-D exchange of the 80% peptide NH atoms with the factor retardation of the exchange rate (P) in the range approximately 10(2)-10(8). For the most of the peptide NH atoms the probability of the local fluctuations weakly depends on the temperature, the enthalpy changes upon all such local conformational transitions deltaH(op) degrees are 0-15 kcal/M. Characterized by the stronger temperature dependence the global fluctuations are not arised upon the temperature increases up to 38 degrees C at pH 7.0 inspite of in these conditions the slow denaturation and aggregation of apoHb begin to occur. Upon the destabilization of the apoHb structure by the simultaneous decreasing of pH to 5.5 and temperature to 10 degrees C the global fluctuations of the apoHb native structure described by deltaH(op)o < 0 begin to intensify. The mechanism of the overall intensification of the local fluctuations upon the heme removal, the peculiarity of the heat denaturation of apoHb in conditions, close to that existing upon the selfassembly of Hb in vivo, and analogy between low temperature global fluctuations and cold denaturation of globular proteins are discussed.

Thermal and colloidal behavior of amine-treated clays: the role of amphiphilic organic cation concentration.

PubMed

Marras, S I; Tsimpliaraki, A; Zuburtikudis, I; Panayiotou, C

2007-11-15

The modification of sodium montmorillonite (NaMMT) through the insertion of amphiphilic hexadecylammonium cations into the clay's interlayer spaces has been studied. Alkylammonium concentrations equivalent to 0.15-3.00 times the cation exchange capacity of the clay were used. The conformation of the surfactant cations in the confined space of the silicate galleries was investigated by X-ray diffraction analysis and scanning electron microscopy, while the organoclay's thermal stability was examined by thermogravimetric analysis. The clay's surface properties induced by the ion-exchange process were followed by measurements of the mineral's zeta potential as a function of pH and surfactant concentration, while the coagulation rates of organoclay suspensions in water and in chloroform were examined using dynamic light scattering. All the results are consistent with showing that the overall characteristics and thus the behavior of the modified MMT particles strongly depend on the alkylammonium surfactant concentration used in the modification process. This, however, has very important implications for any attempt to incorporate the organomodified MMT particles into different media for various applications such as polymer nanocomposite preparation.

The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in Gαi1

PubMed Central

Van Eps, Ned; Thomas, Celestine J.; Hubbell, Wayne L.; Sprang, Stephen R.

2015-01-01

Heterotrimeric G proteins are activated by exchange of GDP for GTP at the G protein alpha subunit (Gα), most notably by G protein-coupled transmembrane receptors. Ric-8A is a soluble cytoplasmic protein essential for embryonic development that acts as both a guanine nucleotide exchange factor (GEF) and a chaperone for Gα subunits of the i, q, and 12/13 classes. Previous studies demonstrated that Ric-8A stabilizes a dynamically disordered state of nucleotide-free Gα as the catalytic intermediate for nucleotide exchange, but no information was obtained on the structures involved or the magnitude of the structural fluctuations. In the present study, site-directed spin labeling (SDSL) together with double electron-electron resonance (DEER) spectroscopy is used to provide global distance constraints that identify discrete members of a conformational ensemble in the Gαi1:Ric-8A complex and the magnitude of structural differences between them. In the complex, the helical and Ras-like nucleotide-binding domains of Gαi1 pivot apart to occupy multiple resolved states with displacements as large as 25 Å. The domain displacement appears to be distinct from that observed in Gαs upon binding of Gs to the β2 adrenergic receptor. Moreover, the Ras-like domain exhibits structural plasticity within and around the nucleotide-binding cavity, and the switch I and switch II regions, which are known to adopt different conformations in the GDP- and GTP-bound states of Gα, undergo structural rearrangements. Collectively, the data show that Ric-8A induces a conformationally heterogeneous state of Gαi and provide insight into the mechanism of action of a nonreceptor Gα GEF. PMID:25605908

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2011-10-17

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Atropisomerism about aryl-Csp(3) bonds: the electronic and steric influence of ortho-substituents on conformational exchange in cannabidiol and linderatin derivatives.

PubMed

Berber, Hatice; Lameiras, Pedro; Denhez, Clément; Antheaume, Cyril; Clayden, Jonathan

2014-07-03

Terpenylation reactions of substituted phenols were used to prepare cannabidiol and linderatin derivatives, and their structure and conformational behavior in solution were investigated by NMR and, for some representative examples, by DFT. VT-NMR spectra and DFT calculations were used to determine the activation energies of the conformational change arising from restricted rotation about the aryl-Csp(3) bond that lead to two unequally populated rotameric epimers. The NBO calculation was applied to explain the electronic stabilization of one conformer over another by donor-acceptor charge transfer interactions. Conformational control arises from a combination of stereoelectronic and steric effects between substituents in close contact with each other on the two rings of the endocyclic epoxide atropisomers. This study represents the first exploration of the stereoelectronic origins of atropisomerism around C(sp(2))-C(sp(3)) single bonds through theoretical calculations.

Conformational states of the full-length glucagon receptor

PubMed Central

Yang, Linlin; Yang, Dehua; de Graaf, Chris; Moeller, Arne; West, Graham M.; Dharmarajan, Venkatasubramanian; Wang, Chong; Siu, Fai Y.; Song, Gaojie; Reedtz-Runge, Steffen; Pascal, Bruce D.; Wu, Beili; Potter, Clinton S.; Zhou, Hu; Griffin, Patrick R.; Carragher, Bridget; Yang, Huaiyu; Wang, Ming-Wei; Stevens, Raymond C.; Jiang, Hualiang

2015-01-01

Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid structural biology approach together with the ECD and 7TM domain crystal structures of the glucagon receptor (GCGR), we examine the relationship between full-length receptor conformation and peptide ligand binding. Molecular dynamics (MD) and disulfide crosslinking studies suggest that apo-GCGR can adopt both an open and closed conformation associated with extensive contacts between the ECD and 7TM domain. The electron microscopy (EM) map of the full-length GCGR shows how a monoclonal antibody stabilizes the ECD and 7TM domain in an elongated conformation. Hydrogen/deuterium exchange (HDX) studies and MD simulations indicate that an open conformation is also stabilized by peptide ligand binding. The combined studies reveal the open/closed states of GCGR and suggest that glucagon binds to GCGR by a conformational selection mechanism. PMID:26227798

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

Discrete Molecular Dynamics Can Predict Helical Prestructured Motifs in Disordered Proteins

PubMed Central

Han, Kyou-Hoon; Dokholyan, Nikolay V.; Tompa, Péter; Kalmár, Lajos; Hegedűs, Tamás

2014-01-01

Intrinsically disordered proteins (IDPs) lack a stable tertiary structure, but their short binding regions termed Pre-Structured Motifs (PreSMo) can form transient secondary structure elements in solution. Although disordered proteins are crucial in many biological processes and designing strategies to modulate their function is highly important, both experimental and computational tools to describe their conformational ensembles and the initial steps of folding are sparse. Here we report that discrete molecular dynamics (DMD) simulations combined with replica exchange (RX) method efficiently samples the conformational space and detects regions populating α-helical conformational states in disordered protein regions. While the available computational methods predict secondary structural propensities in IDPs based on the observation of protein-protein interactions, our ab initio method rests on physical principles of protein folding and dynamics. We show that RX-DMD predicts α-PreSMos with high confidence confirmed by comparison to experimental NMR data. Moreover, the method also can dissect α-PreSMos in close vicinity to each other and indicate helix stability. Importantly, simulations with disordered regions forming helices in X-ray structures of complexes indicate that a preformed helix is frequently the binding element itself, while in other cases it may have a role in initiating the binding process. Our results indicate that RX-DMD provides a breakthrough in the structural and dynamical characterization of disordered proteins by generating the structural ensembles of IDPs even when experimental data are not available. PMID:24763499

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.

The structure and dynamics of rat apo-cellular retinol-binding protein II in solution: comparison with the X-ray structure.

PubMed

Lu, J; Lin, C L; Tang, C; Ponder, J W; Kao, J L; Cistola, D P; Li, E

1999-03-05

The structure and dynamics of rat apo-cellular retinol binding protein II (apo-CRBP II) in solution has been determined by multidimensional NMR analysis of uniformly enriched recombinant rat 13C, 15N-apo-CRBP II and 15N-apo-CRBP II. The final ensemble of 24 NMR structures has been calculated from 3274 conformational restraints or 24.4 restraints/residue. The average root-mean-square deviation of the backbone atoms for the final 24 structures relative to their mean structure is 1.06 A. Although the average solution structure is very similar to the crystal structure, it differs at the putative entrance to the binding cavity, which is formed by the helix-turn-helix motif, the betaC-betaD turn and the betaE-betaF turn. The mean coordinates of the main-chain atoms of amino acid residues 28-38 are displaced in the solution structure relative to the crystal structure. The side-chain of F58, located on the betaC-betaD turn, is reoriented such that it interacts with L37 and no longer blocks entry into the ligand-binding pocket. Residues 28-35, which form the second helix of the helix-turn-helix motif in the crystal structure, do not exhibit a helical conformation in the solution structure. The solution structure of apo-CRBP II exhibits discrete regions of backbone disorder which are most pronounced at residues 28-32, 37-38 and 73-76 in the betaE-betaF turn as evaluated by the consensus chemical shift index, the root-mean-square deviation, amide 1H exchange rates and 15N relaxation studies. These studies indicate that fluctuations in protein conformation occur on the microseconds to ms time-scale in these regions of the protein. Some of these exchange processes can be directly observed in the three-dimensional 15N-resolved NOESY spectrum. These results suggest that in solution, apo-CRBP II undergoes conformational changes on the microseconds to ms time-scale which result in increased access to the binding cavity. Copyright 1999 Academic Press.

Effects of force fields on the conformational and dynamic properties of amyloid β(1-40) dimer explored by replica exchange molecular dynamics simulations.

PubMed

Watts, Charles R; Gregory, Andrew; Frisbie, Cole; Lovas, Sándor

2018-03-01

The conformational space and structural ensembles of amyloid beta (Aβ) peptides and their oligomers in solution are inherently disordered and proven to be challenging to study. Optimum force field selection for molecular dynamics (MD) simulations and the biophysical relevance of results are still unknown. We compared the conformational space of the Aβ(1-40) dimers by 300 ns replica exchange MD simulations at physiological temperature (310 K) using: the AMBER-ff99sb-ILDN, AMBER-ff99sb*-ILDN, AMBER-ff99sb-NMR, and CHARMM22* force fields. Statistical comparisons of simulation results to experimental data and previously published simulations utilizing the CHARMM22* and CHARMM36 force fields were performed. All force fields yield sampled ensembles of conformations with collision cross sectional areas for the dimer that are statistically significantly larger than experimental results. All force fields, with the exception of AMBER-ff99sb-ILDN (8.8 ± 6.4%) and CHARMM36 (2.7 ± 4.2%), tend to overestimate the α-helical content compared to experimental CD (5.3 ± 5.2%). Using the AMBER-ff99sb-NMR force field resulted in the greatest degree of variance (41.3 ± 12.9%). Except for the AMBER-ff99sb-NMR force field, the others tended to under estimate the expected amount of β-sheet and over estimate the amount of turn/bend/random coil conformations. All force fields, with the exception AMBER-ff99sb-NMR, reproduce a theoretically expected β-sheet-turn-β-sheet conformational motif, however, only the CHARMM22* and CHARMM36 force fields yield results compatible with collapse of the central and C-terminal hydrophobic cores from residues 17-21 and 30-36. Although analyses of essential subspace sampling showed only minor variations between force fields, secondary structures of lowest energy conformers are different. © 2017 Wiley Periodicals, Inc.

Oligonucleotide gas-phase hydrogen/deuterium exchange with D2S in the collision cell of a quadrupole-Fourier transform ion cyclotron resonance mass spectrometer.

PubMed

Mo, Jingjie; Håkansson, Kristina

2007-10-15

We have implemented gas-phase hydrogen/deuterium exchange (HDX) experiments in the external collision cell of a hybrid quadrupole-Fourier transform ion cyclotron resonance mass spectrometer. In this configuration, multiply charged oligonucleotide anions undergo significant exchange with D(2)S at reaction intervals ranging from 0.11 to 60.1 s. For DNA homohexamers, relative exchange rates were dC(6) approximately dA(6) > dG(6) > dT(6), correlating with the gas-phase acidities of nucleobases (C > A > T > G), except for guanine. Our results are consistent with a relay mechanism in which D(2)S interacts with both a backbone phosphate group and a neutral nucleobase through hydrogen bonding. We propose that the faster exchange of polyguanosine compared to polythymidine is due to the larger size of guanine and the orientation of its labile hydrogens, which may result in gas-phase conformations more favorable for forming complexes with D(2)S. Similar trends were observed for RNA homohexamers, although their HDX rates were faster than for DNA, suggesting they can also exchange via another relay process involving the 2'-hydroxyl group. HDX of DNA duplexes further supports the involvement of nucleobase hydrogens because duplexes exchanged slower than their corresponding single strands, presumably due to the intermolecular hydrogen bonds between nucleobases. This work constitutes the first investigation of the mechanisms of oligonucleotide gas-phase HDX. Our results on duplexes show promise for application of this strategy to the characterization of structured nucleic acids.

Substrate-modulated unwinding of transmembrane helices in the NSS transporter LeuT.

PubMed

Merkle, Patrick S; Gotfryd, Kamil; Cuendet, Michel A; Leth-Espensen, Katrine Z; Gether, Ulrik; Loland, Claus J; Rand, Kasper D

2018-05-01

LeuT, a prokaryotic member of the neurotransmitter:sodium symporter (NSS) family, is an established structural model for mammalian NSS counterparts. We investigate the substrate translocation mechanism of LeuT by measuring the solution-phase structural dynamics of the transporter in distinct functional states by hydrogen/deuterium exchange mass spectrometry (HDX-MS). Our HDX-MS data pinpoint LeuT segments involved in substrate transport and reveal for the first time a comprehensive and detailed view of the dynamics associated with transition of the transporter between outward- and inward-facing configurations in a Na + - and K + -dependent manner. The results suggest that partial unwinding of transmembrane helices 1/5/6/7 drives LeuT from a substrate-bound, outward-facing occluded conformation toward an inward-facing open state. These hitherto unknown, large-scale conformational changes in functionally important transmembrane segments, observed for LeuT in detergent-solubilized form and when embedded in a native-like phospholipid bilayer, could be of physiological relevance for the translocation process.

Elongation Factor Ts Directly Facilitates the Formation and Disassembly of the Escherichia coli Elongation Factor Tu·GTP·Aminoacyl-tRNA Ternary Complex*

PubMed Central

Burnett, Benjamin J.; Altman, Roger B.; Ferrao, Ryan; Alejo, Jose L.; Kaur, Navdep; Kanji, Joshua; Blanchard, Scott C.

2013-01-01

Aminoacyl-tRNA enters the translating ribosome in a ternary complex with elongation factor Tu (EF-Tu) and GTP. Here, we describe bulk steady state and pre-steady state fluorescence methods that enabled us to quantitatively explore the kinetic features of Escherichia coli ternary complex formation and decay. The data obtained suggest that both processes are controlled by a nucleotide-dependent, rate-determining conformational change in EF-Tu. Unexpectedly, we found that this conformational change is accelerated by elongation factor Ts (EF-Ts), the guanosine nucleotide exchange factor for EF-Tu. Notably, EF-Ts attenuates the affinity of EF-Tu for GTP and destabilizes ternary complex in the presence of non-hydrolyzable GTP analogs. These results suggest that EF-Ts serves an unanticipated role in the cell of actively regulating the abundance and stability of ternary complex in a manner that contributes to rapid and faithful protein synthesis. PMID:23539628

An excited state underlies gene regulation of a transcriptional riboswitch

PubMed Central

Zhao, Bo; Guffy, Sharon L.; Williams, Benfeard; Zhang, Qi

2017-01-01

Riboswitches control gene expression through ligand-dependent structural rearrangements of the sensing aptamer domain. However, we found that the Bacillus cereus fluoride riboswitch aptamer adopts identical tertiary structures in solution with and without ligand. Using chemical exchange saturation transfer (CEST) NMR spectroscopy, we revealed that the structured ligand-free aptamer transiently accesses a low-populated (~1%) and short-lived (~3 ms) excited conformational state that unravels a conserved ‘linchpin’ base pair to signal transcription termination. Upon fluoride binding, this highly localized fleeting process is allosterically suppressed to activate transcription. We demonstrated that this mechanism confers effective fluoride-dependent gene activation over a wide range of transcription rates, which is essential for robust toxicity response across diverse cellular conditions. These results unveil a novel switching mechanism that employs ligand-dependent suppression of an aptamer excited state to coordinate regulatory conformational transitions rather than adopting distinct aptamer ground-state tertiary architectures, exemplifying a new mode of ligand-dependent RNA regulation. PMID:28719589

Conformational heterogeneity of the calmodulin binding interface

NASA Astrophysics Data System (ADS)

Shukla, Diwakar; Peck, Ariana; Pande, Vijay S.

2016-04-01

Calmodulin (CaM) is a ubiquitous Ca2+ sensor and a crucial signalling hub in many pathways aberrantly activated in disease. However, the mechanistic basis of its ability to bind diverse signalling molecules including G-protein-coupled receptors, ion channels and kinases remains poorly understood. Here we harness the high resolution of molecular dynamics simulations and the analytical power of Markov state models to dissect the molecular underpinnings of CaM binding diversity. Our computational model indicates that in the absence of Ca2+, sub-states in the folded ensemble of CaM's C-terminal domain present chemically and sterically distinct topologies that may facilitate conformational selection. Furthermore, we find that local unfolding is off-pathway for the exchange process relevant for peptide binding, in contrast to prior hypotheses that unfolding might account for binding diversity. Finally, our model predicts a novel binding interface that is well-populated in the Ca2+-bound regime and, thus, a candidate for pharmacological intervention.

Restricted amide rotation with steric hindrance induced multiple conformations

NASA Astrophysics Data System (ADS)

Krishnan, V. V.; Vazquez, Salvador; Maitra, Kalyani; Maitra, Santanu

2017-12-01

The Csbnd N bond character is dependent directly upon the resonance-contributor structure population driven by the delocalized nitrogen lone-pair of electrons. In the case of N, N-dibenzyl-ortho-toluamide (o-DBET), the molecule adopts subpopulations of conformers with distinct NMR spectral features, particularly at low temperatures. This conformational adaptation is unique to o-DBET, while the corresponding meta- and para- forms do not show such behavior. Variable-temperature (VT) NMR, two-dimensional exchange spectroscopy (EXSY), and qualitative molecular modeling studies are used to demonstrate how multiple competing interactions such as restricted amide rotation and steric hindrance effects can lead to versatile molecular adaptations in the solution state.

Large-Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin α.

PubMed

Delaforge, Elise; Milles, Sigrid; Bouvignies, Guillaume; Bouvier, Denis; Boivin, Stephane; Salvi, Nicola; Maurin, Damien; Martel, Anne; Round, Adam; Lemke, Edward A; Jensen, Malene Ringkjøbing; Hart, Darren J; Blackledge, Martin

2015-12-09

Influenza A RNA polymerase complex is formed from three components, PA, PB1, and PB2. PB2 is independently imported into the nucleus prior to polymerase reconstitution. All crystallographic structures of the PB2 C-terminus (residues 536-759) reveal two globular domains, 627 and NLS, that form a tightly packed heterodimer. The molecular basis of the affinity of 627-NLS for importins remained unclear from these structures, apparently requiring large-scale conformational changes prior to importin binding. Using a combination of solution-state NMR, small-angle neutron scattering, small-angle X-ray scattering (SAXS), and Förster resonance energy transfer (FRET), we show that 627-NLS populates a temperature-dependent dynamic equilibrium between closed and open states. The closed state is stabilized by a tripartite salt bridge involving the 627-NLS interface and the linker, that becomes flexible in the open state, with 627 and NLS dislocating into a highly dynamic ensemble. Activation enthalpies and entropies associated with the rupture of this interface were derived from simultaneous analysis of temperature-dependent chemical exchange saturation transfer measurements, revealing a strong temperature dependence of both open-state population and exchange rate. Single-molecule FRET and SAXS demonstrate that only the open-form is capable of binding to importin α and that, upon binding, the 627 domain samples a dynamic conformational equilibrium in the vicinity of the C-terminus of importin α. This intrinsic large-scale conformational flexibility therefore enables 627-NLS to bind importin through conformational selection from a temperature-dependent equilibrium comprising both functional forms of the protein.

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Calcium Binding Properties and Allosteric Regulation of Downstream Regulatory Element Antagonist Modulator (DREAM).

PubMed

Zhang, Jun; Li, Jing; Craig, Theodore A; Kumar, Rajiv; Gross, Michael L

2017-07-18

Downstream regulatory element antagonist modulator (DREAM) is an EF-hand Ca 2+ -binding protein that also binds to a specific DNA sequence, downstream regulatory elements (DRE), and thereby regulates transcription in a calcium-dependent fashion. DREAM binds to DRE in the absence of Ca 2+ but detaches from DRE under Ca 2+ stimulation, allowing gene expression. The Ca 2+ binding properties of DREAM and the consequences of the binding on protein structure are key to understanding the function of DREAM. Here we describe the application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis to investigate the Ca 2+ binding properties and the subsequent conformational changes of full-length DREAM. We demonstrate that all EF-hands undergo large conformation changes upon calcium binding even though the EF-1 hand is not capable of binding to Ca 2+ . Moreover, EF-2 is a lower-affinity site compared to EF-3 and -4 hands. Comparison of HDX profiles between wild-type DREAM and two EF-1 mutated constructs illustrates that the conformational changes in the EF-1 hand are induced by long-range structural interactions. HDX analyses also reveal a conformational change in an N-terminal leucine-charged residue-rich domain (LCD) remote from Ca 2+ -binding EF-hands. This LCD domain is responsible for the direct interaction between DREAM and cAMP response element-binding protein (CREB) and regulates the recruitment of the co-activator, CREB-binding protein. These long-range interactions strongly suggest how conformational changes transmit the Ca 2+ signal to CREB-mediated gene transcription.

Hydrogen exchange mass spectrometry of functional membrane-bound chemotaxis receptor complexes.

PubMed

Koshy, Seena S; Eyles, Stephen J; Weis, Robert M; Thompson, Lynmarie K

2013-12-10

The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended membrane-bound arrays. Previous studies have shown that attractant binding induces a small (∼2 Å) piston displacement of one helix of the periplasmic and transmembrane domains toward the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 Å away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston motion could propagate through a dynamic domain to control CheA kinase activity. To address this, we have developed a method for measuring dynamics of the receptor cytoplasmic fragment (CF) in functional complexes with CheA and CheW. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) measurements of global exchange of the CF demonstrate that the CF exhibits significantly slower exchange in functional complexes than in solution. Because the exchange rates in functional complexes are comparable to those of other proteins with similar structures, the CF appears to be a well-structured protein within these complexes, which is compatible with its role in propagating a signal that appears to be a tiny conformational change in the periplasmic and transmembrane domains of the receptor. We also demonstrate the feasibility of this protocol for local exchange measurements by incorporating a pepsin digest step to produce peptides with 87% sequence coverage and only 20% back exchange. This method extends HDX-MS to membrane-bound functional complexes without detergents that may perturb the stability or structure of the system.

Hydrogen Exchange Mass Spectrometry of Functional Membrane-bound Chemotaxis Receptor Complexes

PubMed Central

Koshy, Seena S.; Eyles, Stephen J.; Weis, Robert M.; Thompson, Lynmarie K.

2014-01-01

The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended membrane-bound arrays. Previous studies have shown that attractant binding induces a small (~2 Å) piston displacement of one helix of the periplasmic and transmembrane domains towards the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 Å away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston motion could propagate through a dynamic domain to control CheA kinase activity. To address this, we have developed a method for measuring dynamics of the receptor cytoplasmic fragment (CF) in functional complexes with CheA and CheW. Hydrogen exchange mass spectrometry (HDX-MS) measurements of global exchange of CF demonstrate that CF exhibits significantly slower exchange in functional complexes than in solution. Since the exchange rates in functional complexes are comparable to that of other proteins of similar structure, the CF appears to be a well-structured protein within these complexes, which is compatible with its role in propagating a signal that appears to be a tiny conformational change in the periplasmic and transmembrane domains of the receptor. We also demonstrate the feasibility of this protocol for local exchange measurements, by incorporating a pepsin digest step to produce peptides with 87% sequence coverage and only 20% back exchange. This method extends HDX-MS to membrane-bound functional complexes without detergents that may perturb the stability or structure of the system. PMID:24274333

Analysis of Protein Conformation and Dynamics by Hydrogen/Deuterium Exchange MS

PubMed Central

Engen, John R.

2009-01-01

synopsis Recent technological advances hydrogen exchange MS have led to improvements in the technique’s ability to analyze the shape and movements of proteins. John Engen of Northeastern University gives a much needed update on the field. The cover, created by Engen, shows proteins “swimming” in an H2O/D2O solution with a sample mass spectrum in the background. PMID:19788312

Chemical exchange in biomacromolecules: Past, present, and future

PubMed Central

Palmer, Arthur G.

2014-01-01

The perspective reviews quantitative investigations of chemical exchange phenomena in proteins and other biological macromolecules using NMR spectroscopy, particularly relaxation dispersion methods. The emphasis is on techniques and applications that quantify the populations, interconversion kinetics, and structural features of sparsely populated conformational states in equilibrium with a highly populated ground state. Applications to folding, mol ecular recognition, catalysis, and allostery by proteins and nucleic acids are highlighted. PMID:24656076

Efficient Conformational Sampling in Explicit Solvent Using a Hybrid Replica Exchange Molecular Dynamics Method

DTIC Science & Technology

2011-12-01

REMD while reproducing the energy landscape of explicit solvent simulations . ’ INTRODUCTION Molecular dynamics (MD) simulations of proteins can pro...Mongan, J.; McCammon, J. A. Accelerated molecular dynamics : a promising and efficient simulation method for biomolecules. J. Chem. Phys. 2004, 120 (24...Chemical Theory and Computation ARTICLE (8) Abraham,M. J.; Gready, J. E. Ensuringmixing efficiency of replica- exchange molecular dynamics simulations . J

NMR detection of pH-dependent histidine-water proton exchange reveals the conduction mechanism of a transmembrane proton channel.

PubMed

Hu, Fanghao; Schmidt-Rohr, Klaus; Hong, Mei

2012-02-29

The acid-activated proton channel formed by the influenza M2 protein is important for the life cycle of the virus. A single histidine, His37, in the M2 transmembrane domain (M2TM) is responsible for pH activation and proton selectivity of the channel. Recent studies suggested three models for how His37 mediates proton transport: a shuttle mechanism involving His37 protonation and deprotonation, a H-bonded imidazole-imidazolium dimer model, and a transporter model involving large protein conformational changes in synchrony with proton conduction. Using magic-angle-spinning (MAS) solid-state NMR spectroscopy, we examined the proton exchange and backbone conformational dynamics of M2TM in a virus-envelope-mimetic membrane. At physiological temperature and pH, (15)N NMR spectra show fast exchange of the imidazole (15)N between protonated and unprotonated states. To quantify the proton exchange rates, we measured the (15)N T(2) relaxation times and simulated them for chemical-shift exchange and fluctuating N-H dipolar fields under (1)H decoupling and MAS. The exchange rate is 4.5 × 10(5) s(-1) for Nδ1 and 1.0 × 10(5) s(-1) for Nε2, which are approximately synchronized with the recently reported imidazole reorientation. Binding of the antiviral drug amantadine suppressed both proton exchange and ring motion, thus interfering with the proton transfer mechanism. By measuring the relative concentrations of neutral and cationic His as a function of pH, we determined the four pK(a) values of the His37 tetrad in the viral membrane. Fitting the proton current curve using the charge-state populations from these pK(a)'s, we obtained the relative conductance of the five charge states, which showed that the +3 channel has the highest time-averaged unitary conductance. At physiologically relevant pH, 2D correlation spectra indicated that the neutral and cationic histidines do not have close contacts, ruling out the H-bonded dimer model. Moreover, a narrowly distributed nonideal helical structure coexists with a broadly distributed ideal helical conformation without interchange on the sub-10 ms time scale, thus excluding the transporter model in the viral membrane. These data support the shuttle mechanism of proton conduction, whose essential steps involve His-water proton exchange facilitated by imidazole ring reorientations. © 2011 American Chemical Society

Effects of the interaction range on structural phases of flexible polymers.

PubMed

Gross, J; Neuhaus, T; Vogel, T; Bachmann, M

2013-02-21

We systematically investigate how the range of interaction between non-bonded monomers influences the formation of structural phases of elastic, flexible polymers. Massively parallel replica-exchange simulations of a generic, coarse-grained model, performed partly on graphics processing units and in multiple-gaussian modified ensembles, pave the way for the construction of the structural phase diagram, parametrized by interaction range and temperature. Conformational transitions between gas-like, liquid, and diverse solid (pseudo) phases are identified by microcanonical statistical inflection-point analysis. We find evidence for finite-size effects that cause the crossover of "collapse" and "freezing" transitions for very short interaction ranges.

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.

Conformational change of Sos-derived proline-rich peptide upon binding Grb2 N-terminal SH3 domain probed by NMR

NASA Astrophysics Data System (ADS)

Ogura, Kenji; Okamura, Hideyasu

2013-10-01

Growth factor receptor-bound protein 2 (Grb2) is a small adapter protein composed of a single SH2 domain flanked by two SH3 domains. The N-terminal SH3 (nSH3) domain of Grb2 binds a proline-rich region present in the guanine nucleotide releasing factor, son of sevenless (Sos). Using NMR relaxation dispersion and chemical shift analysis methods, we investigated the conformational change of the Sos-derived proline-rich peptide during the transition between the free and Grb2 nSH3-bound states. The chemical shift analysis revealed that the peptide does not present a fully random conformation but has a relatively rigid structure. The relaxation dispersion analysis detected conformational exchange of several residues of the peptide upon binding to Grb2 nSH3.

Probing the Gaseous Structure of a β-Hairpin Peptide with H/D Exchange and Electron Capture Dissociation

NASA Astrophysics Data System (ADS)

Straus, Rita N.; Jockusch, Rebecca A.

2017-02-01

An improved understanding of the extent to which native protein structure is retained upon transfer to the gas phase promises to enhance biological mass spectrometry, potentially streamlining workflows and providing fundamental insights into hydration effects. Here, we investigate the gaseous conformation of a model β-hairpin peptide using gas-phase hydrogen-deuterium (H/D) exchange with subsequent electron capture dissociation (ECD). Global gas-phase H/D exchange levels, and residue-specific exchange levels derived from ECD data, are compared among the wild type 16-residue peptide GB1p and several variants. High protection from H/D exchange observed for GB1p, but not for a truncated version, is consistent with the retention of secondary structure of GB1p in the gas phase or its refolding into some other compact structure. Four alanine mutants that destabilize the hairpin in solution show levels of protection similar to that of GB1p, suggesting collapse or (re)folding of these peptides upon transfer to the gas phase. These results offer a starting point from which to understand how a key secondary structural element, the β-hairpin, is affected by transfer to the gas phase. This work also demonstrates the utility of a much-needed addition to the tool set that is currently available for the investigation of the gaseous conformation of biomolecules, which can be employed in the future to better characterize gaseous proteins and protein complexes.

Probing the Gaseous Structure of a β-Hairpin Peptide with H/D Exchange and Electron Capture Dissociation.

PubMed

Straus, Rita N; Jockusch, Rebecca A

2017-02-01

An improved understanding of the extent to which native protein structure is retained upon transfer to the gas phase promises to enhance biological mass spectrometry, potentially streamlining workflows and providing fundamental insights into hydration effects. Here, we investigate the gaseous conformation of a model β-hairpin peptide using gas-phase hydrogen-deuterium (H/D) exchange with subsequent electron capture dissociation (ECD). Global gas-phase H/D exchange levels, and residue-specific exchange levels derived from ECD data, are compared among the wild type 16-residue peptide GB1p and several variants. High protection from H/D exchange observed for GB1p, but not for a truncated version, is consistent with the retention of secondary structure of GB1p in the gas phase or its refolding into some other compact structure. Four alanine mutants that destabilize the hairpin in solution show levels of protection similar to that of GB1p, suggesting collapse or (re)folding of these peptides upon transfer to the gas phase. These results offer a starting point from which to understand how a key secondary structural element, the β-hairpin, is affected by transfer to the gas phase. This work also demonstrates the utility of a much-needed addition to the tool set that is currently available for the investigation of the gaseous conformation of biomolecules, which can be employed in the future to better characterize gaseous proteins and protein complexes. Graphical Abstract ᅟ.

Investigating the Role of Large-Scale Domain Dynamics in Protein-Protein Interactions.

PubMed

Delaforge, Elise; Milles, Sigrid; Huang, Jie-Rong; Bouvier, Denis; Jensen, Malene Ringkjøbing; Sattler, Michael; Hart, Darren J; Blackledge, Martin

2016-01-01

Intrinsically disordered linkers provide multi-domain proteins with degrees of conformational freedom that are often essential for function. These highly dynamic assemblies represent a significant fraction of all proteomes, and deciphering the physical basis of their interactions represents a considerable challenge. Here we describe the difficulties associated with mapping the large-scale domain dynamics and describe two recent examples where solution state methods, in particular NMR spectroscopy, are used to investigate conformational exchange on very different timescales.

Investigating the Role of Large-Scale Domain Dynamics in Protein-Protein Interactions

PubMed Central

Delaforge, Elise; Milles, Sigrid; Huang, Jie-rong; Bouvier, Denis; Jensen, Malene Ringkjøbing; Sattler, Michael; Hart, Darren J.; Blackledge, Martin

2016-01-01

Intrinsically disordered linkers provide multi-domain proteins with degrees of conformational freedom that are often essential for function. These highly dynamic assemblies represent a significant fraction of all proteomes, and deciphering the physical basis of their interactions represents a considerable challenge. Here we describe the difficulties associated with mapping the large-scale domain dynamics and describe two recent examples where solution state methods, in particular NMR spectroscopy, are used to investigate conformational exchange on very different timescales. PMID:27679800

Witten diagrams revisited: the AdS geometry of conformal blocks

DOE PAGES

Hijano, Eliot; Kraus, Per; Perlmutter, Eric; ...

2016-01-25

Here, we develop a new method for decomposing blocks. The steps involved are elementary, requiring no explicit integration, and operate directly in position space. Central to this construction is an appealingly simple answer to the question: what object in AdS computes a conformal block? The answer is a "geodesic Witten diagram", which is essentially an ordinary exchange Witten diagram, except that the cubic vertices are not integrated over all of AdS, but only over bulk geodesics connecting the boundary operators. In particular, we also consider the case of four-point functions of scalar operators, and show how to easily reproduce existingmore » results for the relevant conformal blocks in arbitrary dimension.« less

Local energy decomposition analysis of hydrogen-bonded dimers within a domain-based pair natural orbital coupled cluster study.

PubMed

Altun, Ahmet; Neese, Frank; Bistoni, Giovanni

2018-01-01

The local energy decomposition (LED) analysis allows for a decomposition of the accurate domain-based local pair natural orbital CCSD(T) [DLPNO-CCSD(T)] energy into physically meaningful contributions including geometric and electronic preparation, electrostatic interaction, interfragment exchange, dynamic charge polarization, and London dispersion terms. Herein, this technique is employed in the study of hydrogen-bonding interactions in a series of conformers of water and hydrogen fluoride dimers. Initially, DLPNO-CCSD(T) dissociation energies for the most stable conformers are computed and compared with available experimental data. Afterwards, the decay of the LED terms with the intermolecular distance ( r ) is discussed and results are compared with the ones obtained from the popular symmetry adapted perturbation theory (SAPT). It is found that, as expected, electrostatic contributions slowly decay for increasing r and dominate the interaction energies in the long range. London dispersion contributions decay as expected, as r -6 . They significantly affect the depths of the potential wells. The interfragment exchange provides a further stabilizing contribution that decays exponentially with the intermolecular distance. This information is used to rationalize the trend of stability of various conformers of the water and hydrogen fluoride dimers.

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.

Adjustment of Conformational Flexibility is a Key Event in the Thermal Adaptation of Proteins

NASA Astrophysics Data System (ADS)

Zavodszky, Peter; Kardos, Jozsef; Svingor, Adam; Petsko, Gregory A.

1998-06-01

3-Isopropylmalate dehydrogenase (IPMDH, E.C. 1.1.1.85) from the thermophilic bacterium Thermus thermophilus HB8 is homologous to IPMDH from the mesophilic Escherichia coli, but has an approximately 17 degrees C higher melting temperature. Its temperature optimum is 22-25 degrees C higher than that of the E. coli enzyme; however, it is hardly active at room temperature. The increased conformational rigidity required to stabilize the thermophilic enzyme against heat denaturation might explain its different temperature-activity profile. Hydrogen/deuterium exchange studies were performed on this thermophilic-mesophilic enzyme pair to compare their conformational flexibilities. It was found that Th. thermophilus IPMDH is significantly more rigid at room temperature than E. coli IPMDH, whereas the enzymes have nearly identical flexibilities under their respective optimal working conditions, suggesting that evolutionary adaptation tends to maintain a ``corresponding state'' regarding conformational flexibility. These observations confirm that conformational fluctuations necessary for catalytic function are restricted at room temperature in the thermophilic enzyme, suggesting a close relationship between conformational flexibility and enzyme function.

Thermodynamic contribution of backbone conformational entropy in the binding between SH3 domain and proline-rich motif.

PubMed

Zeng, Danyun; Shen, Qingliang; Cho, Jae-Hyun

2017-02-26

Biological functions of intrinsically disordered proteins (IDPs), and proteins containing intrinsically disordered regions (IDRs) are often mediated by short linear motifs, like proline-rich motifs (PRMs). Upon binding to their target proteins, IDPs undergo a disorder-to-order transition which is accompanied by a large conformational entropy penalty. Hence, the molecular mechanisms underlying control of conformational entropy are critical for understanding the binding affinity and selectivity of IDPs-mediated protein-protein interactions (PPIs). Here, we investigated the backbone conformational entropy change accompanied by binding of the N-terminal SH3 domain (nSH3) of CrkII and PRM derived from guanine nucleotide exchange factor 1 (C3G). In particular, we focused on the estimation of conformational entropy change of disordered PRM upon binding to the nSH3 domain. Quantitative characterization of conformational dynamics of disordered peptides like PRMs is limited. Hence, we combined various methods, including NMR model-free analysis, δ2D, DynaMine, and structure-based calculation of entropy loss. This study demonstrates that the contribution of backbone conformational entropy change is significant in the PPIs mediated by IDPs/IDRs. Copyright © 2017 Elsevier Inc. All rights reserved.

Painting proteins with covalent labels: what's in the picture?

PubMed

Fitzgerald, Michael C; West, Graham M

2009-06-01

Knowledge about the structural and biophysical properties of proteins when they are free in solution and/or in complexes with other molecules is essential for understanding the biological processes that proteins regulate. Such knowledge is also important to drug discovery efforts, particularly those focused on the development of therapeutic agents with protein targets. In the last decade a variety of different covalent labeling techniques have been used in combination with mass spectrometry to probe the solution-phase structures and biophysical properties of proteins and protein-ligand complexes. Highlighted here are five different mass spectrometry-based covalent labeling strategies including: continuous hydrogen/deuterium (H/D) exchange labeling, hydroxyl radical-mediated footprinting, SUPREX (stability of unpurified proteins from rates of H/D exchange), PLIMSTEX (protein-ligand interaction by mass spectrometry, titration, and H/D exchange), and SPROX (stability of proteins from rates of oxidation). The basic experimental protocols used in each of the above-cited methods are summarized along with the kind of biophysical information they generate. Also discussed are the relative strengths and weaknesses of the different methods for probing the wide range of conformational states that proteins and protein-ligand complexes can adopt when they are in solution.

Temperature-induced conformational change at the catalytic site of Sulfolobus solfataricus alcohol dehydrogenase highlighted by Asn249Tyr substitution. A hydrogen/deuterium exchange, kinetic, and fluorescence quenching study.

PubMed

Secundo, Francesco; Russo, Consiglia; Giordano, Antonietta; Carrea, Giacomo; Rossi, Mosè; Raia, Carlo A

2005-08-23

A combination of hydrogen/deuterium exchange, fluorescence quenching, and kinetic studies was used to acquire experimental evidence for the crystallographically hypothesized increase in local flexibility which occurs in thermophilic NAD(+)-dependent Sulfolobus solfataricus alcohol dehydrogenase (SsADH) upon substitution Asn249Tyr. The substitution, located at the adenine-binding site, proved to decrease the affinity for both coenzyme and substrate, rendering the mutant enzyme 6-fold more active when compared to the wild-type enzyme [Esposito et al. (2003) FEBS Lett. 539, 14-18]. The amide H/D exchange data show that the wild-type and mutant enzymes have similar global flexibility at 22 and 60 degrees C. However, the temperature dependence of the Stern-Volmer constant determined by acrylamide quenching shows that the increase in temperature affects the local flexibility differently, since the K(SV) increment is significantly higher for the wild-type than for the mutant enzyme over the range 18-45 degrees C. Interestingly, the corresponding van't Hoff plot (log K(SV) vs 1/T) proves nonlinear for the apo and holo wild-type and apo mutant enzymes, with a break at approximately 45 degrees C in all three cases due to a conformational change affecting the tryptophan microenvironment experienced by the quencher molecules. The Arrhenius and van't Hoff plots derived from the k(cat) and K(M) thermodependence measured with cyclohexanol and NAD(+) at different temperatures display an abrupt change of slope at 45-50 degrees C. This proves more pronounced in the case of the mutant enzyme compared to the wild-type enzyme due to a conformational change in the structure rather than to an overlapping of two or more rate-limiting reaction steps with different temperature dependencies of their rate constants. Three-dimensional analysis indicates that the observed conformational change induced by temperature is associated with the flexible loops directly involved in the substrate and coenzyme binding.

The Structural Basis of Oncogenic Mutations G12, G13 and Q61 in Small GTPase K-Ras4B

NASA Astrophysics Data System (ADS)

Lu, Shaoyong; Jang, Hyunbum; Nussinov, Ruth; Zhang, Jian

2016-02-01

Ras mediates cell proliferation, survival and differentiation. Mutations in K-Ras4B are predominant at residues G12, G13 and Q61. Even though all impair GAP-assisted GTP → GDP hydrolysis, the mutation frequencies of K-Ras4B in human cancers vary. Here we aim to figure out their mechanisms and differential oncogenicity. In total, we performed 6.4 μs molecular dynamics simulations on the wild-type K-Ras4B (K-Ras4BWT-GTP/GDP) catalytic domain, the K-Ras4BWT-GTP-GAP complex, and the mutants (K-Ras4BG12C/G12D/G12V-GTP/GDP, K-Ras4BG13D-GTP/GDP, K-Ras4BQ61H-GTP/GDP) and their complexes with GAP. In addition, we simulated ‘exchanged’ nucleotide states. These comprehensive simulations reveal that in solution K-Ras4BWT-GTP exists in two, active and inactive, conformations. Oncogenic mutations differentially elicit an inactive-to-active conformational transition in K-Ras4B-GTP; in K-Ras4BG12C/G12D-GDP they expose the bound nucleotide which facilitates the GDP-to-GTP exchange. These mechanisms may help elucidate the differential mutational statistics in K-Ras4B-driven cancers. Exchanged nucleotide simulations reveal that the conformational transition is more accessible in the GTP-to-GDP than in the GDP-to-GTP exchange. Importantly, GAP not only donates its R789 arginine finger, but stabilizes the catalytically-competent conformation and pre-organizes catalytic residue Q61; mutations disturb the R789/Q61 organization, impairing GAP-mediated GTP hydrolysis. Together, our simulations help provide a mechanistic explanation of key mutational events in one of the most oncogenic proteins in cancer.

Molecular dynamics simulations of biological membranes and membrane proteins using enhanced conformational sampling algorithms☆

PubMed Central

Mori, Takaharu; Miyashita, Naoyuki; Im, Wonpil; Feig, Michael; Sugita, Yuji

2016-01-01

This paper reviews various enhanced conformational sampling methods and explicit/implicit solvent/membrane models, as well as their recent applications to the exploration of the structure and dynamics of membranes and membrane proteins. Molecular dynamics simulations have become an essential tool to investigate biological problems, and their success relies on proper molecular models together with efficient conformational sampling methods. The implicit representation of solvent/membrane environments is reasonable approximation to the explicit all-atom models, considering the balance between computational cost and simulation accuracy. Implicit models can be easily combined with replica-exchange molecular dynamics methods to explore a wider conformational space of a protein. Other molecular models and enhanced conformational sampling methods are also briefly discussed. As application examples, we introduce recent simulation studies of glycophorin A, phospholamban, amyloid precursor protein, and mixed lipid bilayers and discuss the accuracy and efficiency of each simulation model and method. This article is part of a Special Issue entitled: Membrane Proteins. Guest Editors: J.C. Gumbart and Sergei Noskov. PMID:26766517

Asymmetry of inverted-topology repeats in the AE1 anion exchanger suggests an elevator-like mechanism

PubMed Central

Faraldo-Gómez, José D.

2017-01-01

The membrane transporter anion exchanger 1 (AE1), or band 3, is a key component in the processes of carbon-dioxide transport in the blood and urinary acidification in the renal collecting duct. In both erythrocytes and the basolateral membrane of the collecting-duct α-intercalated cells, the role of AE1 is to catalyze a one-for-one exchange of chloride for bicarbonate. After decades of biochemical and functional studies, the structure of the transmembrane region of AE1, which catalyzes the anion-exchange reaction, has finally been determined. Each protomer of the AE1 dimer comprises two repeats with inverted transmembrane topologies, but the structures of these repeats differ. This asymmetry causes the putative substrate-binding site to be exposed only to the extracellular space, consistent with the expectation that anion exchange occurs via an alternating-access mechanism. Here, we hypothesize that the unknown, inward-facing conformation results from inversion of this asymmetry, and we propose a model of this state constructed using repeat-swap homology modeling. By comparing this inward-facing model with the outward-facing experimental structure, we predict that the mechanism of AE1 involves an elevator-like motion of the substrate-binding domain relative to the nearly stationary dimerization domain and to the membrane plane. This hypothesis is in qualitative agreement with a wide range of biochemical and functional data, which we review in detail, and suggests new avenues of experimentation. PMID:29167180

Exploring the folding free energy landscape of insulin using bias exchange metadynamics.

PubMed

Todorova, Nevena; Marinelli, Fabrizio; Piana, Stefano; Yarovsky, Irene

2009-03-19

The bias exchange metadynamics (BE-META) technique was applied to investigate the folding mechanism of insulin, one of the most studied and biologically important proteins. The BE-META simulations were performed starting from an extended conformation of chain B of insulin, using only eight replicas and seven reaction coordinates. The folded state, together with the intermediate states along the folding pathway were identified and their free energy was determined. Three main basins were found separated from one another by a large free energy barrier. The characteristic native fold of chain B was observed in one basin, while the other two most populated basins contained "molten-globule" conformations stabilized by electrostatic and hydrophobic interactions, respectively. Transitions between the three basins occur on the microsecond time scale. The implications and relevance of this finding to the folding mechanisms of insulin were investigated.

Effect of bisecting GlcNAc and core fucosylation on conformational properties of biantennary complex-type N-glycans in solution.

PubMed

Nishima, Wataru; Miyashita, Naoyuki; Yamaguchi, Yoshiki; Sugita, Yuji; Re, Suyong

2012-07-26

The introduction of bisecting GlcNAc and core fucosylation in N-glycans is essential for fine functional regulation of glycoproteins. In this paper, the effect of these modifications on the conformational properties of N-glycans is examined at the atomic level by performing replica-exchange molecular dynamics (REMD) simulations. We simulate four biantennary complex-type N-glycans, namely, unmodified, two single-substituted with either bisecting GlcNAc or core fucose, and disubstituted forms. By using REMD as an enhanced sampling technique, five distinct conformers in solution, each of which is characterized by its local orientation of the Manα1-6Man glycosidic linkage, are observed for all four N-glycans. The chemical modifications significantly change their conformational equilibria. The number of major conformers is reduced from five to two and from five to four upon the introduction of bisecting GlcNAc and core fucosylation, respectively. The population change is attributed to specific inter-residue hydrogen bonds, including water-mediated ones. The experimental NMR data, including nuclear Overhauser enhancement and scalar J-coupling constants, are well reproduced taking the multiple conformers into account. Our structural model supports the concept of "conformer selection", which emphasizes the conformational flexibility of N-glycans in protein-glycan interactions.

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.

Characterizing slow chemical exchange in nucleic acids by carbon CEST and low spin-lock field R(1ρ) NMR spectroscopy.

PubMed

Zhao, Bo; Hansen, Alexandar L; Zhang, Qi

2014-01-08

Quantitative characterization of dynamic exchange between various conformational states provides essential insights into the molecular basis of many regulatory RNA functions. Here, we present an application of nucleic-acid-optimized carbon chemical exchange saturation transfer (CEST) and low spin-lock field R(1ρ) relaxation dispersion (RD) NMR experiments in characterizing slow chemical exchange in nucleic acids that is otherwise difficult if not impossible to be quantified by the ZZ-exchange NMR experiment. We demonstrated the application on a 47-nucleotide fluoride riboswitch in the ligand-free state, for which CEST and R(1ρ) RD profiles of base and sugar carbons revealed slow exchange dynamics involving a sparsely populated (p ~ 10%) and shortly lived (τ ~ 10 ms) NMR "invisible" state. The utility of CEST and low spin-lock field R(1ρ) RD experiments in studying slow exchange was further validated in characterizing an exchange as slow as ~60 s(-1).

Characterizing Slow Chemical Exchange in Nucleic Acids by Carbon CEST and Low Spin-Lock Field R1ρ NMR Spectroscopy

PubMed Central

Zhao, Bo; Hansen, Alexandar L.; Zhang, Qi

2016-01-01

Quantitative characterization of dynamic exchange between various conformational states provides essential insights into the molecular basis of many regulatory RNA functions. Here, we present an application of nucleic-acid-optimized carbon chemical exchange saturation transfer (CEST) and low spin-lock field R1ρ relaxation dispersion (RD) NMR experiments in characterizing slow chemical exchange in nucleic acids that is otherwise difficult if not impossible to be quantified by the ZZ-exchange NMR experiment. We demonstrated the application on a 47-nucleotide fluoride riboswitch in the ligand-free state, for which CEST and R1ρ RD profiles of base and sugar carbons revealed slow exchange dynamics involving a sparsely populated (p ~ 10%) and shortly lived (τ ~ 10 ms) NMR “invisible” state. The utility of CEST and low spin-lock field R1ρ RD experiments in studying slow exchange was further validated in characterizing an exchange as slow as ~60 s−1. PMID:24299272

Intramolecular mobility of η(5)-ligands in chiral zirconocene complexes and the enantioselectivity of alkene functionalization by organoaluminum compounds.

PubMed

Parfenova, Lyudmila V; Zakirova, Irina V; Kovyazin, Pavel V; Karchevsky, Stanislav G; Istomina, Galina P; Khalilov, Leonard M; Dzhemilev, Usein M

2016-08-09

The effect of solvent nature (CD2Cl2, d8-toluene, d8-THF) on the conformational behavior of neomenthyl-substituted zirconocenes CpInd*ZrCl2 (Cp = η(5)-C5H5, Ind* = η(5)-neomenthylindenyl), CpCp'ZrCl2 (Cp = η(5)-C5H5, Cp' = η(5)-neomenthyl-4,5,6,7-tetrahydroindenyl), and Ind*2ZrCl2 (Ind* = η(5)-neomenthylindenyl) was shown by means of dynamic NMR spectroscopy, and the constants and thermodynamic parameters of conformer exchange were determined. The experimental conformational composition of the complexes was compared with structures obtained by quantum chemical modeling using the DFT methods PBE/3ζ and M06-2X/cc-pVDZ(H, C, Cl)/cc-pVDZ-PP(Zr), which predicted three rotamers in the case of both CpInd*ZrCl2 and CpCp'ZrCl2, and seven rotational isomers for Ind*2ZrCl2, three of these being C2-symmetric and the others being asymmetric. The enantioselectivity of the conformationally mobile complex Ind*2ZrCl2 in the reactions of terminal alkenes with AlR3 (R = Me, Et) was compared with that of rigid ansa-complexes, rac-p-S, p-S-[Y(η(5)-C9H10)2]ZrX2 (Y = SiMe2, C2H4; X = S-binaphtholate). Faster exchange between the conformers of Ind*2ZrCl2 in a chlorinated solvent gives the structural isomer of catalytically active sites, which affords higher substrate conversion and reaction enantioselectivity. Binding of the ligands to ansa-zirconocenes prevents the rotational isomerism of the complexes, providing the same configuration of the β-stereogenic center in the methyl- and ethylalumination products (unlike the conformationally mobile complex Ind*2ZrCl2) with an enantiomeric purity of 50-65%.

Manufacture of Regularly Shaped Sol-Gel Pellets

NASA Technical Reports Server (NTRS)

Leventis, Nicholas; Johnston, James C.; Kinder, James D.

2006-01-01

An extrusion batch process for manufacturing regularly shaped sol-gel pellets has been devised as an improved alternative to a spray process that yields irregularly shaped pellets. The aspect ratio of regularly shaped pellets can be controlled more easily, while regularly shaped pellets pack more efficiently. In the extrusion process, a wet gel is pushed out of a mold and chopped repetitively into short, cylindrical pieces as it emerges from the mold. The pieces are collected and can be either (1) dried at ambient pressure to xerogel, (2) solvent exchanged and dried under ambient pressure to ambigels, or (3) supercritically dried to aerogel. Advantageously, the extruded pellets can be dropped directly in a cross-linking bath, where they develop a conformal polymer coating around the skeletal framework of the wet gel via reaction with the cross linker. These pellets can be dried to mechanically robust X-Aerogel.

Plasticity of 150-loop in influenza neuraminidase explored by Hamiltonian replica exchange molecular dynamics simulations.

PubMed

Han, Nanyu; Mu, Yuguang

2013-01-01

Neuraminidase (NA) of influenza is a key target for antiviral inhibitors, and the 150-cavity in group-1 NA provides new insight in treating this disease. However, NA of 2009 pandemic influenza (09N1) was found lacking this cavity in a crystal structure. To address the issue of flexibility of the 150-loop, Hamiltonian replica exchange molecular dynamics simulations were performed on different groups of NAs. Free energy landscape calculated based on the volume of 150-cavity indicates that 09N1 prefers open forms of 150-loop. The turn A (residues 147-150) of the 150-loop is discovered as the most dynamical motif which induces the inter-conversion of this loop among different conformations. In the turn A, the backbone dynamic of residue 149 is highly related with the shape of 150-loop, thus can function as a marker for the conformation of 150-loop. As a contrast, the closed conformation of 150-loop is more energetically favorable in N2, one of group-2 NAs. The D147-H150 salt bridge is found having no correlation with the conformation of 150-loop. Instead the intimate salt bridge interaction between the 150 and 430 loops in N2 variant contributes the stabilizing factor for the closed form of 150-loop. The clustering analysis elaborates the structural plasticity of the loop. This enhanced sampling simulation provides more information in further structural-based drug discovery on influenza virus.

Plasticity of 150-Loop in Influenza Neuraminidase Explored by Hamiltonian Replica Exchange Molecular Dynamics Simulations

PubMed Central

Han, Nanyu; Mu, Yuguang

2013-01-01

Neuraminidase (NA) of influenza is a key target for antiviral inhibitors, and the 150-cavity in group-1 NA provides new insight in treating this disease. However, NA of 2009 pandemic influenza (09N1) was found lacking this cavity in a crystal structure. To address the issue of flexibility of the 150-loop, Hamiltonian replica exchange molecular dynamics simulations were performed on different groups of NAs. Free energy landscape calculated based on the volume of 150-cavity indicates that 09N1 prefers open forms of 150-loop. The turn A (residues 147–150) of the 150-loop is discovered as the most dynamical motif which induces the inter-conversion of this loop among different conformations. In the turn A, the backbone dynamic of residue 149 is highly related with the shape of 150-loop, thus can function as a marker for the conformation of 150-loop. As a contrast, the closed conformation of 150-loop is more energetically favorable in N2, one of group-2 NAs. The D147-H150 salt bridge is found having no correlation with the conformation of 150-loop. Instead the intimate salt bridge interaction between the 150 and 430 loops in N2 variant contributes the stabilizing factor for the closed form of 150-loop. The clustering analysis elaborates the structural plasticity of the loop. This enhanced sampling simulation provides more information in further structural-based drug discovery on influenza virus. PMID:23593372

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

Real-time observation of the conformational dynamics of mitochondrial Hsp70 by spFRET.

PubMed

Sikor, Martin; Mapa, Koyeli; von Voithenberg, Lena Voith; Mokranjac, Dejana; Lamb, Don C

2013-05-29

The numerous functions of the important class of molecular chaperones, heat shock proteins 70 (Hsp70), rely on cycles of intricate conformational changes driven by ATP-hydrolysis and regulated by cochaperones and substrates. Here, we used Förster resonance energy transfer to study the conformational dynamics of individual molecules of Ssc1, a mitochondrial Hsp70, in real time. The intrinsic dynamics of the substrate-binding domain of Ssc1 was observed to be uncoupled from the dynamic interactions between substrate- and nucleotide-binding domains. Analysis of the fluctuations in the interdomain separation revealed frequent transitions to a nucleotide-free state. The nucleotide-exchange factor Mge1 did not induce ADP release, as expected, but rather facilitated binding of ATP. These results indicate that the conformational cycle of Ssc1 is more elaborate than previously thought and provide insight into how the Hsp70s can perform a wide variety of functions.

Conformational change of adenosine deaminase during ligand-exchange in a crystal.

PubMed

Kinoshita, Takayoshi; Tada, Toshiji; Nakanishi, Isao

2008-08-15

Adenosine deaminase (ADA) perpetuates chronic inflammation by degrading extracellular adenosine which is toxic for lymphocytes. ADA has two distinct conformations: open form and closed form. From the crystal structures with various ligands, the non-nucleoside type inhibitors bind to the active site occupying the critical water-binding-position and sustain the open form of apo-ADA. In contrast, substrate mimics do not occupy the critical position, and induce the large conformational change to the closed form. However, it is difficult to predict the binding of (+)-erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), as it possesses characteristic parts of both the substrate and the non-nucleoside inhibitors. The crystal structure shows that EHNA binds to the open form through a novel recognition of the adenine base accompanying conformational change from the closed form of the PR-ADA complex in crystalline state.

N-propyl nitrate vibrational spectrum analysis using DFT B3LYP quantum-chemical method

NASA Astrophysics Data System (ADS)

Shaikhullina, R. M.; Hrapkovsky, G. M.; Shaikhullina, M. M.

2018-05-01

Calculation of a molecular structure, conformation and related vibrational spectra of the n- propyl nitrate C3H7NO3 was carried out by means of density functional theory (DFT) by employing the Gaussian 03 package. The molecular geometries were fully optimized by using the Becker's three-parameter hybrid exchange functional combined with the Lee–Yang–Parr correlation functional (B3LYP) and using the 6-31G(d) basis set. By scanning the dihedral angles around C-O and C-C bonds, five energetically most favorable conformers of n-propyl nitrate - TG, TT, GT, GG and G´G forms were found. Vibrational spectra of the most energetically favorable conformers were calculated. The comparative analysis of calculated and experimental spectra is carried out, the spectral features of the conformational state of n-propyl nitrate and the spectral effects of formation of intramolecular hydrogen bonds are established.

Deuteration and selective labeling of alanine methyl groups of β2-adrenergic receptor expressed in a baculovirus-insect cell expression system.

PubMed

Kofuku, Yutaka; Yokomizo, Tomoki; Imai, Shunsuke; Shiraishi, Yutaro; Natsume, Mei; Itoh, Hiroaki; Inoue, Masayuki; Nakata, Kunio; Igarashi, Shunsuke; Yamaguchi, Hideyuki; Mizukoshi, Toshimi; Suzuki, Ei-Ichiro; Ueda, Takumi; Shimada, Ichio

2018-03-08

G protein-coupled receptors (GPCRs) exist in equilibrium between multiple conformations, and their populations and exchange rates determine their functions. However, analyses of the conformational dynamics of GPCRs in lipid bilayers are still challenging, because methods for observations of NMR signals of large proteins expressed in a baculovirus-insect cell expression system (BVES) are limited. Here, we report a method to incorporate methyl- 13 C 1 H 3 -labeled alanine with > 45% efficiency in highly deuterated proteins expressed in BVES. Application of the method to the NMR observations of β 2 -adrenergic receptor in micelles and in nanodiscs revealed the ligand-induced conformational differences throughout the transmembrane region of the GPCR.

Theoretical calculations of Electron Paramagnetic Resonance parameters of liquid phase Orotic acid radical

NASA Astrophysics Data System (ADS)

Sarikaya, Ebru Karakaş; Dereli, Ömer

2017-02-01

To obtain liquid phase molecular structure, conformational analysis of Orotic acid was performed and six conformers were determined. For these conformations, eight possible radicals were modelled by using Density Functional Theory computations with respect to molecular structure. Electron Paramagnetic Resonance parameters of these model radicals were calculated and then they were compared with the experimental ones. Geometry optimizations of the molecule and modeled radicals were performed using Becke's three-parameter hybrid-exchange functional combined with the Lee-Yang-Parr correlation functional of Density Functional Theory and 6-311++G(d,p) basis sets in p-dioxane solution. Because Orotic acid can be mutagenic in mammalian somatic cells and it is also mutagenic for bacteria and yeast, it has been studied.

Analytical aspects of hydrogen exchange mass spectrometry

PubMed Central

Engen, John R.; Wales, Thomas E.

2016-01-01

The analytical aspects of measuring hydrogen exchange by mass spectrometry are reviewed. The nature of analytical selectivity in hydrogen exchange is described followed by review of the analytical tools required to accomplish fragmentation, separation, and the mass spectrometry measurements under restrictive exchange quench conditions. In contrast to analytical quantitation that relies on measurements of peak intensity or area, quantitation in hydrogen exchange mass spectrometry depends on measuring a mass change with respect to an undeuterated or deuterated control, resulting in a value between zero and the maximum amount of deuterium that could be incorporated. Reliable quantitation is a function of experimental fidelity and to achieve high measurement reproducibility, a large number of experimental variables must be controlled during sample preparation and analysis. The method also reports on important qualitative aspects of the sample, including conformational heterogeneity and population dynamics. PMID:26048552

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.

Proline cis-trans isomerization and its implications for the dimerization of analogues of cyclopeptide stylostatin 1: a combined computational and experimental study.

PubMed

López-Martínez, C; Flores-Morales, P; Cruz, M; González, T; Feliz, M; Diez, A; Campanera, Josep M

2016-05-14

Cis and trans proline conformers are often associated with dramatic changes in the biological function of peptides. A slow equilibrium between cis and trans Ile-Pro amide bond conformers occurs in constrained derivatives of the native marine cyclic heptapeptide stylostatin 1 (cyclo-(NSLAIPF)), a potential anticancer agent. In this work, four cyclopeptides, cyclo-(NSTAIPF), cyclo-(KSTAIPF), cyclo-(RSTAIPF) and cyclo-(DSTAIPF), which are structurally related to stylostatin 1, are experimentally and computationally examined in order to assess the effect of residue mutations on the cis-trans conformational ratio and the apparent capacity to form dimeric aggregates. Primarily, cyclo-(KSTAIPF) and cyclo-(RSTAIPF) showed specific trends in circular dichroism, MALDI-TOF and HPLC purification experiments, which suggests the occurrence of peptide dimerization. Meanwhile, the NMR spectrum of cyclo-(KSTAIPF) indicates that this cyclopeptide exists in the two slow-exchange families of conformations mentioned above. Molecular dynamics simulations combined with quantum mechanical calculations have shed light on the factors governing the cis/trans conformational ratio. In particular, we have found that residue mutations affect the internal hydrogen bond pattern which ultimately tunes the cis/trans conformational ratio and that only trans conformers are capable of aggregating due to the shape complementarity of the two subunits.

Conformity and statistical tolerancing

NASA Astrophysics Data System (ADS)

Leblond, Laurent; Pillet, Maurice

2018-02-01

Statistical tolerancing was first proposed by Shewhart (Economic Control of Quality of Manufactured Product, (1931) reprinted 1980 by ASQC), in spite of this long history, its use remains moderate. One of the probable reasons for this low utilization is undoubtedly the difficulty for designers to anticipate the risks of this approach. The arithmetic tolerance (worst case) allows a simple interpretation: conformity is defined by the presence of the characteristic in an interval. Statistical tolerancing is more complex in its definition. An interval is not sufficient to define the conformance. To justify the statistical tolerancing formula used by designers, a tolerance interval should be interpreted as the interval where most of the parts produced should probably be located. This tolerance is justified by considering a conformity criterion of the parts guaranteeing low offsets on the latter characteristics. Unlike traditional arithmetic tolerancing, statistical tolerancing requires a sustained exchange of information between design and manufacture to be used safely. This paper proposes a formal definition of the conformity, which we apply successively to the quadratic and arithmetic tolerancing. We introduce a concept of concavity, which helps us to demonstrate the link between tolerancing approach and conformity. We use this concept to demonstrate the various acceptable propositions of statistical tolerancing (in the space decentring, dispersion).

Structural confirmation and spectroscopic study of a biomolecule: Norepinephrine.

PubMed

Yadav, T; Mukherjee, V

2018-05-21

The present work deals with the conformational and vibrational spectroscopic study of an important bio-molecule named norepinephrine in gas phase. The FTIR and FTRaman spectrum of norepinephrine in amorphous form were recorded in wavenumber range 4000-400 cm -1 and 4000-50 cm -1 respectively. We have investigated twenty-seven stable conformational structures of norepinephrine molecule. All the calculations have been done using Density Functional Theory with exchange functional B3LYP incorporated with the 6-31++G(d, p) basis set. The effect of hydrochloride on different bond lengths, bond angles and dihedral angles in the most stable conformer has also been studied. The total potential energy distribution for both the most stable conformer and the most stable conformer in hydrochloride was performed with the help Normal coordinate analysis method. Most of the calculated vibrational frequencies are in good agreement with the experimental frequencies. The natural bond orbital analysis was also performed to ensure the stability of electronic structures of norepinephrine. To know chemical reactivity of norepinephrine molecule we have calculated the energy gap between HOMO and LUMO orbitals and it has found above 5 eV in all the conformers. Copyright © 2018 Elsevier B.V. All rights reserved.

Investigation of the redox-dependent modulation of structure and dynamics in human cytochrome c

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

Imai, Mizue; Saio, Tomohide; Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810

2016-01-22

Redox-dependent changes in the structure and dynamics of human cytochrome c (Cyt c) were investigated by solution NMR. We found significant structural changes in several regions, including residues 23–28 (loop 3), which were further corroborated by chemical shift differences between the reduced and oxidized states of Cyt c. These differences are essential for discriminating redox states in Cyt c by cytochrome c oxidase (CcO) during electron transfer reactions. Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments identified that the region around His33 undergoes conformational exchanges on the μs-ms timescale, indicating significant redox-dependent structural changes. Because His33 is not part of the interaction sitemore » for CcO, our data suggest that the dynamic properties of the region, which is far from the interaction site for CcO, contribute to conformational changes during electron transfer to CcO. - Highlights: • Solution structure and dynamics analysis for human Cyt c by NMR. • Structural changes responsible for the discrimination of the redox state in Cyt c. • Conformational exchange in the region outside of the interaction site for CcO. • Less flexibility and rigid structure of the interaction site on Cyt c for CcO.« less

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

Structural study of human growth hormone-releasing factor fragment (1?29) by vibrational spectroscopy

NASA Astrophysics Data System (ADS)

Carmona, P.; Molina, M.; Lasagabaster, A.

1995-05-01

The conformational structure of fragment 1-29 of human growth hormone releasing factor, hGHRF (1-29), in aqueous solution and in the solid state is investigated by infrared and Raman spectroscopy. The polypeptide backbone is found to be unordered in the solid state. However, the spectra of the peptide prepared as 5% (w/w) aqueous solutions show that approximately 28% of the peptide is involved in intermolecular β-sheet aggregation. The remainder of the peptide exists largely as disordered and β-sheet conformations with a small portion of α-helices. Tyrosine residues are found to be exposed to the solvent. The secondary structures are quantitatively examined through infrared spectroscopy, the conformational percentages being near those obtained by HONDAet al. [ Biopolymers31, 869 (1991)] using circular dichroism. The fast hydrogen/deuterium exchange in peptide groups and the absence of any NMR sign indicative of ordered structure [ G. M. CLOREet al., J. Molec. Biol.191, 553 (1986)] support that the solution conformations of the non-aggregated peptide interconvert in dynamic equilibrium. Some physiological advantages that may derive from this conformational flexibility are also discussed

Molecular dynamics simulations of biological membranes and membrane proteins using enhanced conformational sampling algorithms.

PubMed

Mori, Takaharu; Miyashita, Naoyuki; Im, Wonpil; Feig, Michael; Sugita, Yuji

2016-07-01

This paper reviews various enhanced conformational sampling methods and explicit/implicit solvent/membrane models, as well as their recent applications to the exploration of the structure and dynamics of membranes and membrane proteins. Molecular dynamics simulations have become an essential tool to investigate biological problems, and their success relies on proper molecular models together with efficient conformational sampling methods. The implicit representation of solvent/membrane environments is reasonable approximation to the explicit all-atom models, considering the balance between computational cost and simulation accuracy. Implicit models can be easily combined with replica-exchange molecular dynamics methods to explore a wider conformational space of a protein. Other molecular models and enhanced conformational sampling methods are also briefly discussed. As application examples, we introduce recent simulation studies of glycophorin A, phospholamban, amyloid precursor protein, and mixed lipid bilayers and discuss the accuracy and efficiency of each simulation model and method. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Modulation of phase transition of thermosensitive liposomes with leucine zipper-structured lipopeptides.

PubMed

Xu, Xiejun; Xiao, Xingqing; Wang, Yiming; Xu, Shouhong; Liu, Honglai

2018-06-13

Targeted therapy for cancer requires thermosensitive components in drug carriers for controlled drug release against viral cells. The conformational transition characteristic of leucine zipper-structured lipopeptides is utilized in our lab to modulate the phase transition temperature of liposomes, thus achieving temperature-responsive control. In this study, we computationally examined the conformational transition behaviors of leucine zipper-structured lipopeptides that were modified at the N-terminus by distinct functional groups. The conformational transition temperatures of these lipopeptides were determined by structural analysis of the implicit-solvent replica exchange molecular dynamics simulation trajectories using the dihedral angle principal component analysis and the dictionary of protein secondary structure method. Our calculations revealed that the computed transition temperatures of the lipopeptides are in good agreement with the experimental measurements. The effect of hydrogen bonds on the conformational stability of the lipopeptide dimers was examined in conventional explicit-solvent molecular dynamics simulations. A quantitative correlation of the degree of structural dissociation of the dimers and their binding strength is well described by an exponential fit of the binding free energies to the conformation transition temperatures of the lipopeptides.

Hydraulic Failure Defines the Recovery and Point of Death in Water-Stressed Conifers[OA

PubMed Central

Brodribb, Tim J.; Cochard, Hervé

2009-01-01

This study combines existing hydraulic principles with recently developed methods for probing leaf hydraulic function to determine whether xylem physiology can explain the dynamic response of gas exchange both during drought and in the recovery phase after rewatering. Four conifer species from wet and dry forests were exposed to a range of water stresses by withholding water and then rewatering to observe the recovery process. During both phases midday transpiration and leaf water potential (Ψleaf) were monitored. Stomatal responses to Ψleaf were established for each species and these relationships used to evaluate whether the recovery of gas exchange after drought was limited by postembolism hydraulic repair in leaves. Furthermore, the timing of gas-exchange recovery was used to determine the maximum survivable water stress for each species and this index compared with data for both leaf and stem vulnerability to water-stress-induced dysfunction measured for each species. Recovery of gas exchange after water stress took between 1 and >100 d and during this period all species showed strong 1:1 conformity to a combined hydraulic-stomatal limitation model (r2 = 0.70 across all plants). Gas-exchange recovery time showed two distinct phases, a rapid overnight recovery in plants stressed to <50% loss of leaf hydraulic conductance (Kleaf) and a highly Ψleaf-dependent phase in plants stressed to >50% loss of Kleaf. Maximum recoverable water stress (Ψmin) corresponded to a 95% loss of Kleaf. Thus, we conclude that xylem hydraulics represents a direct limit to the drought tolerance of these conifer species. PMID:19011001

Hydraulic failure defines the recovery and point of death in water-stressed conifers.

PubMed

Brodribb, Tim J; Cochard, Hervé

2009-01-01

This study combines existing hydraulic principles with recently developed methods for probing leaf hydraulic function to determine whether xylem physiology can explain the dynamic response of gas exchange both during drought and in the recovery phase after rewatering. Four conifer species from wet and dry forests were exposed to a range of water stresses by withholding water and then rewatering to observe the recovery process. During both phases midday transpiration and leaf water potential (Psileaf) were monitored. Stomatal responses to Psileaf were established for each species and these relationships used to evaluate whether the recovery of gas exchange after drought was limited by postembolism hydraulic repair in leaves. Furthermore, the timing of gas-exchange recovery was used to determine the maximum survivable water stress for each species and this index compared with data for both leaf and stem vulnerability to water-stress-induced dysfunction measured for each species. Recovery of gas exchange after water stress took between 1 and >100 d and during this period all species showed strong 1:1 conformity to a combined hydraulic-stomatal limitation model (r2 = 0.70 across all plants). Gas-exchange recovery time showed two distinct phases, a rapid overnight recovery in plants stressed to <50% loss of leaf hydraulic conductance (Kleaf) and a highly Psileaf-dependent phase in plants stressed to >50% loss of Kleaf. Maximum recoverable water stress (Psimin) corresponded to a 95% loss of Kleaf. Thus, we conclude that xylem hydraulics represents a direct limit to the drought tolerance of these conifer species.

Pressurized bellows flat contact heat exchanger interface

NASA Technical Reports Server (NTRS)

Voss, Fred E. (Inventor); Howell, Harold R. (Inventor); Winkler, Roger V. (Inventor)

1990-01-01

Disclosed is an interdigitated plate-type heat exchanger interface. The interface includes a modular interconnect to thermally connect a pair or pairs of plate-type heat exchangers to a second single or multiple plate-type heat exchanger. The modular interconnect comprises a series of parallel, plate-type heat exchangers arranged in pairs to form a slot therebetween. The plate-type heat exchangers of the second heat exchanger insert into the slots of the modular interconnect. Bellows are provided between the pairs of fins of the modular interconnect so that when the bellows are pressurized, they drive the plate-type heat exchangers of the modular interconnect toward one another, thus closing upon the second heat exchanger plates. Each end of the bellows has a part thereof a thin, membrane diaphragm which readily conforms to the contours of the heat exchanger plates of the modular interconnect when the bellows is pressurized. This ensures an even distribution of pressure on the heat exchangers of the modular interconnect thus creating substantially planar contact between the two heat exchangers. The effect of the interface of the present invention is to provide a dry connection between two heat exchangers whereby the rate of heat transfer can be varied by varying the pressure within the bellows.

Parallel Tempering of Dark Matter from the Ebola Virus Proteome: Comparison of CHARMM36m and CHARMM22 Force Fields with Implicit Solvent.

PubMed

Olson, Mark A

2018-01-22

Intrinsically disordered proteins are characterized by their large manifold of thermally accessible conformations and their related statistical weights, making them an interesting target of simulation studies. To assess the development of a computational framework for modeling this distinct class of proteins, this work examines temperature-based replica-exchange simulations to generate a conformational ensemble of a 28-residue peptide from the Ebola virus protein VP35. Starting from a prefolded helix-β-turn-helix topology observed in a crystallographic assembly, the simulation strategy tested is the recently refined CHARMM36m force field combined with a generalized Born solvent model. A comparison of two replica-exchange methods is provided, where one is a traditional approach with a fixed set of temperatures and the other is an adaptive scheme in which the thermal windows are allowed to move in temperature space. The assessment is further extended to include a comparison with equivalent CHARMM22 simulation data sets. The analysis finds CHARMM36m to shift the minimum in the potential of mean force (PMF) to a lower fractional helicity compared with CHARMM22, while the latter showed greater conformational plasticity along the helix-forming reaction coordinate. Among the simulation models, only the adaptive tempering method with CHARMM36m found an ensemble of conformational heterogeneity consisting of transitions between α-helix-β-hairpin folds and unstructured states that produced a PMF of fractional fold propensity in qualitative agreement with circular dichroism experiments reporting a disordered peptide.

A Potential Yeast Actin Allosteric Conduit Dependent on Hydrophobic Core Residues Val-76 and Trp-79*

PubMed Central

Wen, Kuo-Kuang; McKane, Melissa; Stokasimov, Ema; Fields, Jonathon; Rubenstein, Peter A.

2010-01-01

Intramolecular allosteric interactions responsible for actin conformational regulation are largely unknown. Previous work demonstrated that replacing yeast actin Val-76 with muscle actin Ile caused decreased nucleotide exchange. Residue 76 abuts Trp-79 in a six-residue linear array beginning with Lys-118 on the surface and ending with His-73 in the nucleotide cleft. To test if altering the degree of packing of these two residues would affect actin dynamics, we constructed V76I, W79F, and W79Y single mutants as well as the Ile-76/Phe-79 and Ile-76/Tyr-79 double mutants. Tyr or Phe should decrease crowding and increase protein flexibility. Subsequent introduction of Ile should restore packing and dampen changes. All mutants showed decreased growth in liquid medium. W79Y alone was severely osmosensitive and exhibited vacuole abnormalities. Both properties were rescued by Ile-76. Phe-79 or Tyr decreased the thermostability of actin and increased its nucleotide exchange rate. These effects, generally greater for Tyr than for Phe, were reversed by introduction of Ile-76. HD exchange showed that the mutations caused propagated conformational changes to all four subdomains. Based on results from phosphate release and light-scattering assays, single mutations affected polymerization in the order of Ile, Phe, and Tyr from least to most. Introduction of Ile-76 partially rescued the polymerization defects caused by either Tyr-79 or Phe-79. Thus, alterations in crowding of the 76–79 residue pair can strongly affect actin conformation and behavior, and these results support the theory that the amino acid array in which they are located may play a central role in actin regulation. PMID:20442407

Satisfying patient-consumer principles for health information exchange: evidence from California case studies.

PubMed

Miller, Robert H

2012-03-01

In June 2010 sixteen organizations representing California patients and consumers adopted nine principles for electronically exchanging health information among and within provider organizations. The principles were formulated with the goal of improving patient and population health care by increasing the availability and use of patient data while protecting patients' privacy. This study assesses to what extent five health care organizations-all in different stages of increasing their capacity for health information exchange-conformed to the principles in early 2011. Although an increasing amount of electronic data has been exchanged among organizations and with patients, progress has been modest, and patients still have little control over their data. For organizations to comply with all nine patient and consumer principles, clear "rules of the road" for information sharing must be defined, and patient education in health information exchange and control over personal data must be increased.

NASA geometry data exchange specification for computational fluid dynamics (NASA IGES)

NASA Technical Reports Server (NTRS)

Blake, Matthew W.; Kerr, Patricia A.; Thorp, Scott A.; Jou, Jin J.

1994-01-01

This document specifies a subset of an existing product data exchange specification that is widely used in industry and government. The existing document is called the Initial Graphics Exchange Specification. This document, a subset of IGES, is intended for engineers analyzing product performance using tools such as computational fluid dynamics (CFD) software. This document specifies how to define mathematically and exchange the geometric model of an object. The geometry is represented utilizing nonuniform rational B-splines (NURBS) curves and surfaces. Only surface models are represented; no solid model representation is included. This specification does not include most of the other types of product information available in IGES (e.g., no material properties or surface finish properties) and does not provide all the specific file format details of IGES. The data exchange protocol specified in this document is fully conforming to the American National Standard (ANSI) IGES 5.2.

Replica Exchange with Solute Tempering: Efficiency in Large Scale Systems

PubMed Central

Huang, Xuhui; Hagen, Morten; Kim, Byungchan; Friesner, Richard A.; Zhou, Ruhong; Berne, B. J.

2009-01-01

We apply the recently developed replica exchange with solute tempering (REST) to three large solvated peptide systems: an α-helix, a β-hairpin, and a TrpCage, with these peptides defined as the “central group”. We find that our original implementation of REST is not always more efficient than the replica exchange method (REM). Specifically, we find that exchanges between folded (F) and unfolded (U) conformations with vastly different structural energies are greatly reduced by the nonappearance of the water self-interaction energy in the replica exchange acceptance probabilities. REST, however, is expected to remain useful for a large class of systems for which the energy gap between the two states is not large, such as weakly bound protein–ligand complexes. Alternatively, a shell of water molecules can be incorporated into the central group, as discussed in the original paper. PMID:17439169

A Probabilistic Framework for Constructing Temporal Relations in Replica Exchange Molecular Trajectories.

PubMed

Chattopadhyay, Aditya; Zheng, Min; Waller, Mark Paul; Priyakumar, U Deva

2018-05-23

Knowledge of the structure and dynamics of biomolecules is essential for elucidating the underlying mechanisms of biological processes. Given the stochastic nature of many biological processes, like protein unfolding, it's almost impossible that two independent simulations will generate the exact same sequence of events, which makes direct analysis of simulations difficult. Statistical models like Markov Chains, transition networks etc. help in shedding some light on the mechanistic nature of such processes by predicting long-time dynamics of these systems from short simulations. However, such methods fall short in analyzing trajectories with partial or no temporal information, for example, replica exchange molecular dynamics or Monte Carlo simulations. In this work we propose a probabilistic algorithm, borrowing concepts from graph theory and machine learning, to extract reactive pathways from molecular trajectories in the absence of temporal data. A suitable vector representation was chosen to represent each frame in the macromolecular trajectory (as a series of interaction and conformational energies) and dimensionality reduction was performed using principal component analysis (PCA). The trajectory was then clustered using a density-based clustering algorithm, where each cluster represents a metastable state on the potential energy surface (PES) of the biomolecule under study. A graph was created with these clusters as nodes with the edges learnt using an iterative expectation maximization algorithm. The most reactive path is conceived as the widest path along this graph. We have tested our method on RNA hairpin unfolding trajectory in aqueous urea solution. Our method makes the understanding of the mechanism of unfolding in RNA hairpin molecule more tractable. As this method doesn't rely on temporal data it can be used to analyze trajectories from Monte Carlo sampling techniques and replica exchange molecular dynamics (REMD).

Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination.

PubMed

Fornander, Louise H; Frykholm, Karolin; Reymer, Anna; Renodon-Cornière, Axelle; Takahashi, Masayuki; Nordén, Bengt

2012-06-01

Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca(2+) than of Mg(2+), we have investigated the effect of these ions on the structure of HsRad51 filament complexes with single- and double-stranded DNA, the reaction intermediates. Flow linear dichroism spectroscopy shows that the two ionic conditions induce significantly different structures in the HsRad51/single-stranded DNA complex, while the HsRad51/double-stranded DNA complex does not demonstrate this ionic dependence. In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca(2+) induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg(2+), ADP/Mg(2+) or ADP/Ca(2+) does not. A high strand exchange activity is observed for the filament formed with ATP/Ca(2+), whereas the other filaments exhibit lower activity. Molecular modelling suggests that the structural variation is caused by the divalent cation interfering with the L2 loop close to the DNA-binding site. It is proposed that the larger Ca(2+) stabilizes the loop conformation and thereby the protein-DNA interaction. A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange.

Replica exchange molecular dynamics simulation of structure variation from α/4β-fold to 3α-fold protein.

PubMed

Lazim, Raudah; Mei, Ye; Zhang, Dawei

2012-03-01

Replica exchange molecular dynamics (REMD) simulation provides an efficient conformational sampling tool for the study of protein folding. In this study, we explore the mechanism directing the structure variation from α/4β-fold protein to 3α-fold protein after mutation by conducting REMD simulation on 42 replicas with temperatures ranging from 270 K to 710 K. The simulation began from a protein possessing the primary structure of GA88 but the tertiary structure of GB88, two G proteins with "high sequence identity." Albeit the large Cα-root mean square deviation (RMSD) of the folded protein (4.34 Å at 270 K and 4.75 Å at 304 K), a variation in tertiary structure was observed. Together with the analysis of secondary structure assignment, cluster analysis and principal component, it provides insights to the folding and unfolding pathway of 3α-fold protein and α/4β-fold protein respectively paving the way toward the understanding of the ongoings during conformational variation.

Molecular dynamics simulations using temperature-enhanced essential dynamics replica exchange.

PubMed

Kubitzki, Marcus B; de Groot, Bert L

2007-06-15

Today's standard molecular dynamics simulations of moderately sized biomolecular systems at full atomic resolution are typically limited to the nanosecond timescale and therefore suffer from limited conformational sampling. Efficient ensemble-preserving algorithms like replica exchange (REX) may alleviate this problem somewhat but are still computationally prohibitive due to the large number of degrees of freedom involved. Aiming at increased sampling efficiency, we present a novel simulation method combining the ideas of essential dynamics and REX. Unlike standard REX, in each replica only a selection of essential collective modes of a subsystem of interest (essential subspace) is coupled to a higher temperature, with the remainder of the system staying at a reference temperature, T(0). This selective excitation along with the replica framework permits efficient approximate ensemble-preserving conformational sampling and allows much larger temperature differences between replicas, thereby considerably enhancing sampling efficiency. Ensemble properties and sampling performance of the method are discussed using dialanine and guanylin test systems, with multi-microsecond molecular dynamics simulations of these test systems serving as references.

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

Exploring the binding pathways of the 14-3-3ζ protein: Structural and free-energy profiles revealed by Hamiltonian replica exchange molecular dynamics with distancefield distance restraints

PubMed Central

Nagy, Gabor; Oostenbrink, Chris; Hritz, Jozef

2017-01-01

The 14-3-3 protein family performs regulatory functions in eukaryotic organisms by binding to a large number of phosphorylated protein partners. Whilst the binding mode of the phosphopeptides within the primary 14-3-3 binding site is well established based on the crystal structures of their complexes, little is known about the binding process itself. We present a computational study of the process by which phosphopeptides bind to the 14-3-3ζ protein. Applying a novel scheme combining Hamiltonian replica exchange molecular dynamics and distancefield restraints allowed us to map and compare the most likely phosphopeptide-binding pathways to the 14-3-3ζ protein. The most important structural changes to the protein and peptides involved in the binding process were identified. In order to bind phosphopeptides to the primary interaction site, the 14-3-3ζ adopted a newly found wide-opened conformation. Based on our findings we additionally propose a secondary interaction site on the inner surface of the 14-3-3ζ dimer, and a direct interference on the binding process by the flexible C-terminal tail. A minimalistic model was designed to allow for the efficient calculation of absolute binding affinities. Binding affinities calculated from the potential of mean force along the binding pathway are in line with the available experimental estimates for two of the studied systems. PMID:28727767

Real-time observation of the conformational dynamics of mitochondrial Hsp70 by spFRET

PubMed Central

Sikor, Martin; Mapa, Koyeli; von Voithenberg, Lena Voith; Mokranjac, Dejana; Lamb, Don C

2013-01-01

The numerous functions of the important class of molecular chaperones, heat shock proteins 70 (Hsp70), rely on cycles of intricate conformational changes driven by ATP-hydrolysis and regulated by cochaperones and substrates. Here, we used Förster resonance energy transfer to study the conformational dynamics of individual molecules of Ssc1, a mitochondrial Hsp70, in real time. The intrinsic dynamics of the substrate-binding domain of Ssc1 was observed to be uncoupled from the dynamic interactions between substrate- and nucleotide-binding domains. Analysis of the fluctuations in the interdomain separation revealed frequent transitions to a nucleotide-free state. The nucleotide-exchange factor Mge1 did not induce ADP release, as expected, but rather facilitated binding of ATP. These results indicate that the conformational cycle of Ssc1 is more elaborate than previously thought and provide insight into how the Hsp70s can perform a wide variety of functions. PMID:23624933

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

CHUGH, Devesh; Gluesenkamp, Kyle R; Abdelaziz, Omar

In this study, development of a novel system for combined water heating, dehumidification, and space evaporative cooling is discussed. Ambient water vapor is used as a working fluid in an open system. First, water vapor is absorbed from an air stream into an absorbent solution. The latent heat of absorption is transferred into the process water that cools the absorber. The solution is then regenerated in the desorber, where it is heated by a heating fluid. The water vapor generated in the desorber is condensed and its heat of phase change is transferred to the process water in the condenser.more » The condensed water can then be used in an evaporative cooling process to cool the dehumidified air exiting the absorber, or it can be drained if primarily dehumidification is desired. Essentially, this open absorption cycle collects space heat and transfers it to process water. This technology is enabled by a membrane-based absorption/desorption process in which the absorbent is constrained by hydrophobic vapor-permeable membranes. Constraining the absorbent film has enabled fabrication of the absorber and desorber in a plate-and-frame configuration. An air stream can flow against the membrane at high speed without entraining the absorbent, which is a challenge in conventional dehumidifiers. Furthermore, the absorption and desorption rates of an absorbent constrained by a membrane are greatly enhanced. Isfahani and Moghaddam (Int. J. Heat Mass Transfer, 2013) demonstrated absorption rates of up to 0.008 kg/m2s in a membrane-based absorber and Isfahani et al. (Int. J. Multiphase Flow, 2013) have reported a desorption rate of 0.01 kg/m2s in a membrane-based desorber. The membrane-based architecture also enables economical small-scale systems, novel cycle configurations, and high efficiencies. The absorber, solution heat exchanger, and desorber are fabricated on a single metal sheet. In addition to the open arrangement and membrane-based architecture, another novel feature of the cycle is recovery of the solution heat energy exiting the desorber by process water (a process-solution heat exchanger ) rather than the absorber exiting solution (the conventional solution heat exchanger ). This approach has enabled heating the process water from an inlet temperature of 15 C to 57 C (conforming to the DOE water heater test standard) and interfacing the process water with absorbent on the opposite side of a single metal sheet encompassing the absorber, process-solution heat exchanger, and desorber. The system under development has a 3.2 kW water heating capacity and a target thermal coefficient of performance (COP) of 1.6.« less

Rho proteins of plants--functional cycle and regulation of cytoskeletal dynamics.

PubMed

Mucha, Elena; Fricke, Inka; Schaefer, Antje; Wittinghofer, Alfred; Berken, Antje

2011-11-01

Rho-related ROP proteins are molecular switches that essentially regulate a wide variety of processes. Of central interest is their influence on the plant cytoskeleton by which they affect vital processes like cell division, growth, morphogenesis, and pathogen defense. ROPs switch between GTP- and GDP-bound conformations by strictly regulated nucleotide exchange and GTP-hydrolysis, and only the active GTP-form interacts with downstream effectors to ultimately provoke a biological response. However, the mode of action of the engaged regulators and effectors as well as their upstream and downstream interaction partners have long been largely unknown. As opposed to analogous systems in animals and fungi, plants use specific GTPase activating proteins (RopGAPs) with a unique domain composition and novel guanine nucleotide exchange factors (RopGEFs) with a probable link to cell surface receptors. Moreover, plants comprise novel effector molecules and adapters connecting ROPs to mostly unknown downstream targets on the route to the cytoskeleton. This review aims to summarize recent knowledge on the molecular mechanisms and reaction cascades involved in ROP dependent cytoskeletal rearrangements, addressing the structure and function of the unusual RopGAPs, RopGEFs and effectors, and the upstream and downstream pathways linking ROPs to cell receptor-like kinases, actin filaments, and microtubules. Copyright © 2010 Elsevier GmbH. All rights reserved.

A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers

PubMed Central

Noble, Geoffrey P.; Wang, Daphne W.; Walsh, Daniel J.; Barone, Justin R.; Miller, Michael B.; Nishina, Koren A.; Li, Sheng; Supattapone, Surachai

2015-01-01

Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure. PMID:26125623

Anharmonic Vibrational Analyses of Pentapeptide Conformations Explored with Enhanced Sampling Simulations.

PubMed

Otaki, Hiroki; Yagi, Kiyoshi; Ishiuchi, Shun-Ichi; Fujii, Masaaki; Sugita, Yuji

2016-10-06

An accurate theoretical prediction of the vibrational spectrum of polypeptides remains to be a challenge due to (1) their conformational flexibility and (2) non-negligible anharmonic effects. The former makes the search for conformers that contribute to the spectrum difficult, and the latter requires an expensive, quantum mechanical calculation for both electrons and vibrations. Here, we propose a new theoretical approach, which implements an enhanced conformational sampling by the replica-exchange molecular dynamics method, a structural clustering to identify distinct conformations, and a vibrational structure calculation by the second-order vibrational quasi-degenerate perturbation theory (VQDPT2). A systematic mode-selection scheme is developed to reduce the cost of VQDPT2 and the generation of a potential energy surface by the electronic structure calculation. The proposed method is applied to a pentapeptide, SIVSF-NH 2 , for which the infrared spectrum has recently been measured in the gas phase with high resolution in the OH and NH stretching region. The theoretical spectrum of the lowest energy conformer is obtained with a mean absolute deviation of 11.2 cm -1 from the experimental spectrum. Furthermore, the NH stretching frequencies of the five lowest energy conformers are found to be consistent with the literature values measured for small peptides with a similar secondary structure. Therefore, the proposed method is a promising way to analyze the vibrational spectrum of polypeptides.

Aspects of AdS/CFT: Conformal Deformations and the Goldstone Equivalence Theorem

NASA Astrophysics Data System (ADS)

Cantrell, Sean Andrew

The AdS/CFT correspondence provides a map from the states of theories situated in AdSd+1 to those in dual conformal theories in a d-dimensional space. The correspondence can be used to establish certain universal properties of some theories in one space by examining the behave of general objects in the other. In this thesis, we develop various formal aspects of AdS/CFT. Conformal deformations manifest in the AdS/CFT correspondence as boundary conditions on the AdS field. Heretofore, double-trace deformations have been the primary focus in this context. To better understand multitrace deformations, we revisit the relationship between the generating AdS partition function for a free bulk theory and the boundary CFT partition function subject to arbitrary conformal deformations. The procedure leads us to a formalism that constructs bulk fields from boundary operators. We independently replicate the holographic RG flow narrative to go on to interpret the brane used to regulate the AdS theory as a renormalization scale. The scale-dependence of the dilatation spectrum of a boundary theory in the presence of general deformations can be thus understood on the AdS side using this formalism. The Goldstone equivalence theorem allows one to relate scattering amplitudes of massive gauge fields to those of scalar fields in the limit of large scattering energies. We generalize this theorem under the framework of the AdS/CFT correspondence. First, we obtain an expression of the equivalence theorem in terms of correlation functions of creation and annihilation operators by using an AdS wave function approach to the AdS/CFT dictionary. It is shown that the divergence of the non-conserved conformal current dual to the bulk gauge field is approximately primary when computing correlators for theories in which the masses of all the exchanged particles are sufficiently large. The results are then generalized to higher spin fields. We then go on to generalize the theorem using conformal blocks in two and four-dimensional CFTs. We show that when the scaling dimensions of the exchanged operators are large compared to both their spins and the dimension of the current, the conformal blocks satisfy an equivalence theorem.

Ligand adsorption and exchange on pegylated gold nanoparticles

USDA-ARS?s Scientific Manuscript database

Previous researchers proposed that thiolated poly(ethylene glycol) (PEG-SH) adopts a “mushroom-like” conformation on gold nanoparticles (AuNPs) in water. However, information regarding the size and permeability of the PEG-SH mushroom caps and surface area passivated by the PEG-SH mushroom stems are ...

Effects of subtle differences in ligand constitution and conformation in metallo-supramolecular self-assembled polygons.

PubMed

Brusilowskij, Boris; Dzyuba, Egor V; Troff, Ralf W; Schalley, Christoph A

2011-12-07

3,3'-Bis(pyridin-[n]-ylethynyl)biphenyl (n = 3, 4) and the corresponding 2,2'-bipyridines assemble with (dppp)Pt(II) triflate into metallo-supramolecular polygons. Depending on the position of the terminal pyridine N atoms, the assembly reaction leads to different equilibrium products. With the slow ligand exchange on Pt(II) complexes, the equilibrium is reached on a many-hour time-scale. During the assembly process, larger polygons form under kinetic control. This was confirmed by time-dependent (1)H and (31)P NMR spectroscopy in line with complementary ESI mass spectrometric experiments. The constitutional difference in the pyridine N-atom position is reflected in the tandem mass spectra of the complex ions. In addition, a highly specific fragmentation process of mass-selected M(3)L(3) ions was observed, which proceeds through a ring contraction yielding smaller M(2)L(2) ions.

Characterizing Solution Surface Loop Conformational Flexibility of the GM2 Activator Protein

PubMed Central

2015-01-01

GM2AP has a β-cup topology with numerous X-ray structures showing multiple conformations for some of the surface loops, revealing conformational flexibility that may be related to function, where function is defined as either membrane binding associated with ligand binding and extraction or interaction with other proteins. Here, site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and molecular dynamic (MD) simulations are used to characterize the mobility and conformational flexibility of various structural regions of GM2AP. A series of 10 single cysteine amino acid substitutions were generated, and the constructs were chemically modified with the methanethiosulfonate spin label. Continuous wave (CW) EPR line shapes were obtained and subsequently simulated using the microscopic order macroscopic disorder (MOMD) program. Line shapes for sites that have multiple conformations in the X-ray structures required two spectral components, whereas spectra of the remaining sites were adequately fit with single-component parameters. For spin labeled sites L126C and I66C, spectra were acquired as a function of temperature, and simulations provided for the determination of thermodynamic parameters associated with conformational change. Binding to GM2 ligand did not alter the conformational flexibility of the loops, as evaluated by EPR and NMR spectroscopies. These results confirm that the conformational flexibility observed in the surface loops of GM2AP crystals is present in solution and that the exchange is slow on the EPR time scale (>ns). Furthermore, MD simulation results are presented and agree well with the conformational heterogeneity revealed by SDSL. PMID:25127419

Conformation and Dynamics of Human Urotensin II and Urotensin Related Peptide in Aqueous Solution.

PubMed

Haensele, Elke; Mele, Nawel; Miljak, Marija; Read, Christopher M; Whitley, David C; Banting, Lee; Delépée, Carla; Sopkova-de Oliveira Santos, Jana; Lepailleur, Alban; Bureau, Ronan; Essex, Jonathan W; Clark, Timothy

2017-02-27

Conformation and dynamics of the vasoconstrictive peptides human urotensin II (UII) and urotensin related peptide (URP) have been investigated by both unrestrained and enhanced-sampling molecular-dynamics (MD) simulations and NMR spectroscopy. These peptides are natural ligands of the G-protein coupled urotensin II receptor (UTR) and have been linked to mammalian pathophysiology. UII and URP cannot be characterized by a single structure but exist as an equilibrium of two main classes of ring conformations, open and folded, with rapidly interchanging subtypes. The open states are characterized by turns of various types centered at K 8 Y 9 or F 6 W 7 predominantly with no or only sparsely populated transannular hydrogen bonds. The folded conformations show multiple turns stabilized by highly populated transannular hydrogen bonds comprising centers F 6 W 7 K 8 or W 7 K 8 Y 9 . Some of these conformations have not been characterized previously. The equilibrium populations that are experimentally difficult to access were estimated by replica-exchange MD simulations and validated by comparison of experimental NMR data with chemical shifts calculated with density-functional theory. UII exhibits approximately 72% open:28% folded conformations in aqueous solution. URP shows very similar ring conformations as UII but differs in an open:folded equilibrium shifted further toward open conformations (86:14) possibly arising from the absence of folded N-terminal tail-ring interaction. The results suggest that the different biological effects of UII and URP are not caused by differences in ring conformations but rather by different interactions with UTR.

Spectroscopic studies on the conformational transitions of a bovine growth hormone releasing factor analog

NASA Astrophysics Data System (ADS)

Sarver, Ronald W.; Friedman, Alan R.; Thamann, Thomas J.

1997-10-01

The secondary structure of the bovine growth hormone releasing factor analog, [Ile 2, Ser 8,28, Ala 15, Leu 27, Hse 30] bGRF(1-30)-NH-Ethyl, acetate salt (U-90699F) was studied in solution by Fourier transform infrared and Raman spectroscopies. Spectroscopic studies revealed that concentrated aqueous solutions of U-90699F (100 mg ml -1) undergo a secondary structure transition from disordered coil/α-helix to intermolecular β-sheet. Disordered coil and α-helical structure were grouped together in the infrared and Raman studies since the amide I vibrations are close in frequency and overlap in assignments was possible. Before the conformational transition, the facile exchange of the peptide's amide hydrogens for deuterium indicated that the majority of amide hydrogens were readily accessible to solvent. The kinetics of the conformational transition coincided with an increase in solution viscosity and turbidity. An initiation phase preceded the conformational transition during which only minor spectral changes were observed by infrared spectroscopy. The initiation phase and reaction kinetics were consistent with a highly cooperative nucleation ultimately leading to a network of intermolecular β-sheet structure and gel formation. Increased temperature accelerated the conformational transition. The conformational transition was thermally irreversible but the β-sheet structure of aggregated or gelled peptide could be disrupted by dilution and agitation.

Accelerating the Conformational Sampling of Intrinsically Disordered Proteins.

PubMed

Do, Trang Nhu; Choy, Wing-Yiu; Karttunen, Mikko

2014-11-11

Intrinsically disordered proteins (IDPs) are a class of proteins lacking a well-defined secondary structure. Instead, they are able to attain multiple conformations, bind to multiple targets, and respond to changes in their surroundings. Functionally, IDPs have been associated with molecular recognition, cell regulation, and signal transduction. The dynamic conformational ensemble of IDPs is highly environmental and binding partner dependent, rendering the characterization of IDPs extremely challenging. Here, we compare the sampling efficiencies of conventional molecular dynamics (MD), well-tempered metadynamics (WT-META), and bias-exchange metadynamics (BE-META). The total simulation time was over 10 μs, and a 20-mer peptide derived from the Neh2 domain of the Nuclear factor erythroid 2-related factor 2 (Nrf2) protein was simulated. BE-META, with a neutral replica and seven biased replicas employing a set of seven relevant collective variables (CVs), provided the most reliable and efficient sampling. Finally, we propose a free-energy reconstruction method based on the probability distribution of the secondary structure contents. This postprocessing analysis confirms the presence of not only the β-hairpin conformation of the free Neh2 peptide but also its rare bound-state-like conformation, both of that have been experimentally observed. In addition, our simulations also predict other possible conformations to be verified with future experiments.

Conformational Ensemble of hIAPP Dimer: Insight into the Molecular Mechanism by which a Green Tea Extract inhibits hIAPP Aggregation

NASA Astrophysics Data System (ADS)

Mo, Yuxiang; Lei, Jiangtao; Sun, Yunxiang; Zhang, Qingwen; Wei, Guanghong

2016-09-01

Small oligomers formed early along human islet amyloid polypeptide (hIAPP) aggregation is responsible for the cell death in Type II diabetes. The epigallocatechin gallate (EGCG), a green tea extract, was found to inhibit hIAPP fibrillation. However, the inhibition mechanism and the conformational distribution of the smallest hIAPP oligomer - dimer are mostly unknown. Herein, we performed extensive replica exchange molecular dynamic simulations on hIAPP dimer with and without EGCG molecules. Extended hIAPP dimer conformations, with a collision cross section value similar to that observed by ion mobility-mass spectrometry, were observed in our simulations. Notably, these dimers adopt a three-stranded antiparallel β-sheet and contain the previously reported β-hairpin amyloidogenic precursor. We find that EGCG binding strongly blocks both the inter-peptide hydrophobic and aromatic-stacking interactions responsible for inter-peptide β-sheet formation and intra-peptide interaction crucial for β-hairpin formation, thus abolishes the three-stranded β-sheet structures and leads to the formation of coil-rich conformations. Hydrophobic, aromatic-stacking, cation-π and hydrogen-bonding interactions jointly contribute to the EGCG-induced conformational shift. This study provides, on atomic level, the conformational ensemble of hIAPP dimer and the molecular mechanism by which EGCG inhibits hIAPP aggregation.

Data processing in neutron protein crystallography using positron-sensitive detectors

NASA Astrophysics Data System (ADS)

Schoenborn, B. P.

Neutrons provide a unique probe for localizing hydrogen atoms and for distinguishing hydrogen from deuterons. Hydrogen atoms largely determine the three dimensional structure of proteins and are responsible for many catalytic reactions. The study of hydrogen bonding and hydrogen exchange will therefore give insight into reaction mechanisms and conformational fluctuations. In addition, neutrons provide the ability to distinguish N from C and O and to allow correct orientation of groups such as histidine and glutamine. To take advantage of these unique features of neutron crystallography, one needs accurate Fourier maps depicting atomic structure to a high precision. Special attention is given to subtraction of the high background associated with hydrogen containing molecules, which produces a disproportionately large statistical error.

Collider effects of unparticle interactions in multiphoton signals

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

Aliev, T. M.; Frank, Mariana; Turan, Ismail

2009-12-01

A new model of physics, with a hidden conformal sector which manifests itself as an unparticle coupling to standard model particles effectively through higher dimensional operators, predicts strong collider signals due to unparticle self-interactions. We perform a complete analysis of the most spectacular of these signals at the hadron collider, pp(p){yields}{gamma}{gamma}{gamma}{gamma} and {gamma}{gamma}gg. These processes can go through the three-point unparticle self-interactions as well as through some s and t channel diagrams with one and/or two unparticle exchanges. We study the contributions of individual diagrams classified with respect to the number of unparticle exchanges and discuss their effect on themore » cross sections at the Tevatron and the LHC. We also restrict the Tevatron bound on the unknown coefficient of the three-point unparticle correlator. With the availability of data from the Tevatron, and the advent of the data emerging from the LHC, these interactions can provide a clear and strong indication of unparticle physics and distinguish this model from other beyond the standard model scenarios.« less

NMR Stratagems for the Study of Multiple Kinetic Hydrogen/Deuterium Isotope Effectsof Proton Exchange. Example: Di-p-fluorophenylformamidine/THF

NASA Astrophysics Data System (ADS)

Limbach, Hans-Heinrich; Meschede, Ludger; Scherer, Gerd

1989-05-01

Stratagems are presented for the determination of kinetic isotope effects of proton exchange reactions by dynamic NMR spectroscopy. In such experiments, lineshape analyses and/or polarization transfer experiments are performed on the exchanging protons or deuterons as well as on remote spins, as a function of the deuterium fraction in the mobile proton sites. These methods are NMR analogs of previous proton inventory techniques involving classical kinetic methods. A theory is developed in order to derive the kinetic isotope effects as well as the number of transferred protons from the experimental NMR spectra. The technique is then applied to the problem of proton exchange in the system 15N,15N'-di-p-fluorophenylibrmamidine, a nitrogen analog of formic acid, dissolved in tetrahydrofuran-d8 (THF). DFFA forms two conformers in THF to which s-trans and s-cis structures have been assigned. Only the s-trans conformer is able to dimerize and exchange protons. Lineshape simulations and magnetization transfer experiments were carried out at 189,2 K, at a concentration of 0.02 mol l-1, as a function of the deuterium fraction D in the 1H-15N sites. Using 1H NMR spectroscopy, a linear dependence of the inverse proton lifetimes on D was observed. From this it was concluded that two protons are transported in the rate limiting step of the proton exchange. This result is expected for a double proton transfer in an s-trans dimer with a cyclic structure. The full kinetic HH/HD/DD isotope effects of 233:11:1 at 189 K were determined through 19F NMR experiments on the same samples. The deviation from the rule of geometric mean, although substantial, is much smaller than found in previous studies of intramolecular HH transfer reactions. Possible causes of this effect are discussed.

Dynamic regulation of GDP binding to G proteins revealed by magnetic field-dependent NMR relaxation analyses

PubMed Central

Toyama, Yuki; Kano, Hanaho; Mase, Yoko; Yokogawa, Mariko; Osawa, Masanori; Shimada, Ichio

2017-01-01

Heterotrimeric guanine-nucleotide-binding proteins (G proteins) serve as molecular switches in signalling pathways, by coupling the activation of cell surface receptors to intracellular responses. Mutations in the G protein α-subunit (Gα) that accelerate guanosine diphosphate (GDP) dissociation cause hyperactivation of the downstream effector proteins, leading to oncogenesis. However, the structural mechanism of the accelerated GDP dissociation has remained unclear. Here, we use magnetic field-dependent nuclear magnetic resonance relaxation analyses to investigate the structural and dynamic properties of GDP bound Gα on a microsecond timescale. We show that Gα rapidly exchanges between a ground-state conformation, which tightly binds to GDP and an excited conformation with reduced GDP affinity. The oncogenic D150N mutation accelerates GDP dissociation by shifting the equilibrium towards the excited conformation. PMID:28223697

Dynamic regulation of GDP binding to G proteins revealed by magnetic field-dependent NMR relaxation analyses.

PubMed

Toyama, Yuki; Kano, Hanaho; Mase, Yoko; Yokogawa, Mariko; Osawa, Masanori; Shimada, Ichio

2017-02-22

Heterotrimeric guanine-nucleotide-binding proteins (G proteins) serve as molecular switches in signalling pathways, by coupling the activation of cell surface receptors to intracellular responses. Mutations in the G protein α-subunit (Gα) that accelerate guanosine diphosphate (GDP) dissociation cause hyperactivation of the downstream effector proteins, leading to oncogenesis. However, the structural mechanism of the accelerated GDP dissociation has remained unclear. Here, we use magnetic field-dependent nuclear magnetic resonance relaxation analyses to investigate the structural and dynamic properties of GDP bound Gα on a microsecond timescale. We show that Gα rapidly exchanges between a ground-state conformation, which tightly binds to GDP and an excited conformation with reduced GDP affinity. The oncogenic D150N mutation accelerates GDP dissociation by shifting the equilibrium towards the excited conformation.

Syntheses, structural analyses and redox kinetics of four-coordinate [CuL2]2+ and five-coordinate [CuL2(solvent)]2+ complexes (L = 6,6'-dimethyl-2,2'-bipyridine or 2,9-dimethyl-1,10-phenanthroline): completely gated reduction reaction of [Cu(dmp)2]2+ in nitromethane.

PubMed

Itoh, Sumitaka; Kishikawa, Nobuyuki; Suzuki, Takayoshi; Takagi, Hideo D

2005-03-21

[Cu(2,9-dimethyl-1,10-phenanthroline)(2)](2+) and [Cu(6,6'-dimethyl-2,2'-bipyridine)(2)](2+/+) complexes with no coordinated solvent molecule were synthesized and the crystal structures were analyzed: the coordination geometry around the Cu(i) center was in the D(2d) symmetry while a D(2) structure was observed for the four-coordinate Cu(ii) complexes. Coordination of a water or an acetonitrile molecule was found in the trigonal plane of the five-coordinate Cu(ii) complex in the Tbp(trigonal bipyramidal) structure. Spectrophotometric analyses revealed that the D(2) structure of the Cu(ii) complex was retained in nitromethane, although a five-coordinate Tbp species (green in color), was readily formed upon dissolution of the solid (reddish brown) in acetonitrile. The electron self-exchange reaction between D(2d)-Cu(I) and D(2)-Cu(II), observed by the NMR method, was very rapid with k(ex)=(1.1 +/- 0.2) x 10(5) kg mol(-1) s(-1) at 25 degrees C (DeltaH*= 15.6 +/- 1.3 kJ mol(-1) and DeltaS*=-96 +/- 4 J mol(-1) K(-1)), which was more than 10 times larger than that reported for the self-exchange reaction between D(2d)-Cu(I) and Tbp-Cu(II) in acetonitrile. The cross reduction reactions of D(2)-Cu(ii) by ferrocene and decamethylferrocene in nitromethane exhibited a completely gated behavior, while the oxidation reaction of D(2d)-Cu(i) by [Ni(1,4,7-triazacyclononane)(2)](3+) in nitromethane estimated an identically large self-exchange rate constant to that directly obtained by the NMR method. The electron self-exchange rate constant estimated from the oxidation cross reaction in 50% v/v acetonitrile-nitromethane mixture was 10 times smaller than that observed in pure nitromethane. On the basis of the Principle of the Least Motion (PLM) and the Symmetry Rules, it was concluded that gated behaviors observed for the reduction reactions of the five-coordinate Cu(ii)-polypyridine complexes are related to the high-energy C(2v)--> D(2d) conformational change around Cu(ii), and that the electron self-exchange reactions of the Cu(ii)/(i) couples are always adiabatic through the C(2v) structures for both Cu(ii) and Cu(i) since the conformational changes between D(2d), D(2) and C(2v) structures for Cu(i) as well as the conformational change between Tbp and C(2v) structures for Cu(ii) are symmetry-allowed. The completely gated behavior observed for the reduction reactions of D(2)-Cu(ii) species in nitromethane was attributed to the very slow conformational change from the ground-state D(2) to the entatic D(2d) structure that is symmetry-forbidden for d(9) metal complexes: the very slow back reaction, the forbidden conformational change from entatic D(2d) to the ground-state D(2) structure, ensures that the rate of the reduction reaction is independent of the concentration of the reducing reagent.

Guanidinium/ammonium competition and proton transfer in the interaction of the amino acid arginine with the tetracarboxylic 18-crown-6 ionophore.

PubMed

Avilés-Moreno, Juan Ramón; Berden, Giel; Oomens, Jos; Martínez-Haya, Bruno

2018-02-07

The recognition of arginine plays a central role in modern proteomics and genomics. Arginine is unique among natural amino acids due to the high basicity of its guanidinium side chain, which sustains specific interactions and proton exchange biochemical processes. The search for suitable macrocyclic ionophores constitutes a promising route towards the development of arginine receptors. This study evaluates the conformational features involved in the binding of free arginine by the polyether macrocycle (18-crown-6)-tetracarboxylic acid. Infrared action vibrational spectroscopy and quantum-chemical computations are combined to characterize the complexes with net charges +1 and +2. The spectrum of the +1 complex can be explained in terms of a configuration predominantly stabilized by a robust bidentate coordination of guanidinium with a carboxylate group formed from the deprotonation of one side group of the crown ether. The released proton is transferred to the amino terminus of arginine, which then coordinates with the crown ether ring. In an alternative type of conformation, partly consistent with experiment, the amino terminus is neutral and the guanidinium group inserts into the crown ether cavity. In the +2 complexes, arginine is always doubly protonated and the most stable conformations are characterized by a tripodal coordination of the ammonium -NH 3 + group of arginine with the oxygen atoms of the macrocycle ring, while the interactions of the amino acid with the side carboxylic acid groups of the crown ether acquire a remarkable lesser role.

Polymorphism at 129 dictates metastable conformations of the human prion protein N-terminal β-sheet† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc03275c Click here for additional data file.

PubMed Central

Paz, S. Alexis; Vanden-Eijnden, Eric

2017-01-01

We study the thermodynamic stability of the native state of the human prion protein using a new free-energy method, replica-exchange on-the-fly parameterization. This method is designed to overcome hidden-variable sampling limitations to yield nearly error-free free-energy profiles along a conformational coordinate. We confirm that all four (M129V, D178N) polymorphs have a ground-state conformation with three intact β-sheet hydrogen bonds. Additionally, they are observed to have distinct metastabilities determined by the side-chain at position 129. We rationalize these findings with reference to the prion “strain” hypothesis, which links the variety of transmissible spongiform encephalopathy phenotypes to conformationally distinct infectious prion forms and classifies distinct phenotypes of sporadic Creutzfeldt-Jakob disease based solely on the 129 polymorphism. Because such metastable structures are not easily observed in structural experiments, our approach could potentially provide new insights into the conformational origins of prion diseases and other pathologies arising from protein misfolding and aggregation. PMID:28451263

Effect of Proline Mutations on the Monomer Conformations of Amylin

PubMed Central

Chiu, Chi-cheng; Singh, Sadanand; de Pablo, Juan J.

2013-01-01

The formation of human islet amyloid polypeptide (hIAPP) is implicated in the loss of pancreatic β-cells in type II diabetes. Rat amylin, which differs from human amylin at six residues, does not lead to formation of amyloid fibrils. Pramlintide is a synthetic analog of human amylin that shares three proline substitutions with rat amylin. Pramlintide has a much smaller propensity to form amyloid aggregates and has been widely prescribed in amylin replacement treatment. It is known that the three prolines attenuate β-sheet formation. However, the detailed effects of these proline substitutions on full-length hIAPP remain poorly understood. In this work, we use molecular simulations and bias-exchange metadynamics to investigate the effect of proline substitutions on the conformation of the hIAPP monomer. Our results demonstrate that hIAPP can adopt various β-sheet conformations, some of which have been reported in experiments. The proline substitutions perturb the formation of long β-sheets and reduce their stability. More importantly, we find that all three proline substitutions of pramlintide are required to inhibit β conformations and stabilize the α-helical conformation. Fewer substitutions do not have a significant inhibiting effect. PMID:24010666

Steady-State Ion Beam Modeling with MICHELLE

NASA Astrophysics Data System (ADS)

Petillo, John

2003-10-01

There is a need to efficiently model ion beam physics for ion implantation, chemical vapor deposition, and ion thrusters. Common to all is the need for three-dimensional (3D) simulation of volumetric ion sources, ion acceleration, and optics, with the ability to model charge exchange of the ion beam with a background neutral gas. The two pieces of physics stand out as significant are the modeling of the volumetric source and charge exchange. In the MICHELLE code, the method for modeling the plasma sheath in ion sources assumes that the electron distribution function is a Maxwellian function of electrostatic potential over electron temperature. Charge exchange is the process by which a neutral background gas with a "fast" charged particle streaming through exchanges its electron with the charged particle. An efficient method for capturing this is essential, and the model presented is based on semi-empirical collision cross section functions. This appears to be the first steady-state 3D algorithm of its type to contain multiple generations of charge exchange, work with multiple species and multiple charge state beam/source particles simultaneously, take into account the self-consistent space charge effects, and track the subsequent fast neutral particles. The solution used by MICHELLE is to combine finite element analysis with particle-in-cell (PIC) methods. The basic physics model is based on the equilibrium steady-state application of the electrostatic particle-in-cell (PIC) approximation employing a conformal computational mesh. The foundation stems from the same basic model introduced in codes such as EGUN. Here, Poisson's equation is used to self-consistently include the effects of space charge on the fields, and the relativistic Lorentz equation is used to integrate the particle trajectories through those fields. The presentation will consider the complexity of modeling ion thrusters.

Backbone resonance assignment of an insect arylalkylamine N-acetyltransferase from Bombyx mori reveals conformational heterogeneity.

PubMed

Aboalroub, Adam A; Zhang, Ziming; Keramisanou, Dimitra; Gelis, Ioannis

2017-04-01

Arylalkylamine N-acetyltransferases (AANATs) catalyze the transfer of an acetyl group from the acetyl-group donor, acetyl-CoA, to an arylalkylamine acceptor. Although a single AANAT has been identified in mammals, insects utilize multiple AANATs in a diverse array of biological processes. AANATs belong to the GCN5-related acetyltransferase (GNAT) superfamily of enzymes, which despite their overall very low sequence homology, are characterized by a well conserved catalytic core domain. The structural properties of many GNATs have been extensively studied by X-ray crystallography that revealed common features during the catalytic cycle. Here we report the 1 H, 13 C and 15 N backbone NMR resonance assignment of the 24 kDa AANAT3 from Bombyx mori (bmAANAT3) as a first step towards understanding the role of protein dynamics in the catalytic properties of AANATs. Our preliminary solution NMR studies reveal that bmAANAT3 is well-folded in solution. The P-loop, which is responsible for cofactor binding, is flexible in the free-state, while a large region of the enzyme interconverts between two distinct conformations in the slow exchange regime.

Drawing dependent structures, mechanical properties and cyclization behaviors of polyacrylonitrile and polyacrylonitrile/carbon nanotube composite fibers prepared by plasticized spinning.

PubMed

Li, Xiang; Qin, Aiwen; Zhao, Xinzhen; Liu, Dapeng; Wang, Haiye; He, Chunju

2015-09-14

Drawing to change the structural properties and cyclization behaviors of the polyacrylonitrile (PAN) chains in crystalline and amorphous regions is carried out on PAN and PAN/carbon nanotube (CNT) composite fibers. Various characterization methods including Fourier transform infrared spectroscopy, differential scanning calorimetry, X-ray diffraction and thermal gravimetric analysis are used to monitor the structural evolution and cyclization behaviors of the fibers. With an increase of the draw ratio during the plasticized spinning process, the structural parameters of the fibers, i.e. crystallinity and planar zigzag conformation, are decreased at first, and then increased, which are associated with the heat exchange rate and the oriented-crystallization rate. A possible mechanism for plasticized spinning is proposed to explain the changing trends of crystallinity and planar zigzag conformation. PAN and PAN/CNT fibers exhibit various cyclization behaviors induced by drawing, e.g., the initiation temperature for the cyclization (Ti) of PAN fibers is increased with increasing draw ratio, while Ti of PAN/CNT fibers is decreased. Drawing also facilitates cyclization and lowers the percentage of β-amino nitrile for PAN/CNT fibers during the stabilization.

Structural and motional contributions of the Bacillus subtilis ClpC N-domain in adaptor protein interactions

PubMed Central

Kojetin, Douglas J.; McLaughlin, Patrick D.; Thompson, Richele J.; Dubnau, David; Prepiak, Peter; Rance, Mark; Cavanagh, John

2009-01-01

Summary The AAA+ superfamily protein ClpC is a key regulator of cell development in Bacillus subtilis. As part of a large oligomeric complex, ClpC controls an array of cellular processes by recognizing, unfolding, and providing misfolded and aggregated proteins as substrates for the ClpP peptidase. ClpC is unique compared to other HSP100/Clp proteins, as it requires an adaptor protein for all fundamental activities. The NMR solution structure of the N-terminal repeat domain of ClpC (N-ClpCR) comprises two structural repeats of a four-helix motif. NMR experiments used to map the MecA adaptor protein interaction surface of N-ClpCR reveal that regions involved in the interaction possess conformational flexibility, as well as conformational exchange on the μs-ms time-scale. The electrostatic surface of N-ClpCR differs substantially compared to the N-domain of Escherichia coli ClpA and ClpB, suggesting that the electrostatic surface characteristics of HSP100/Clp N-domains may play a role in adaptor protein and substrate interaction specificity, and perhaps contribute to the unique adaptor protein requirement of ClpC. PMID:19361434

Do TFSA anions slither? Pressure exposes the role of TFSA conformational exchange in self-diffusion

DOE PAGES

Suarez, Sophia N.; Wishart, James F.; Rua, Armando; ...

2015-10-28

Multi-nuclear ( 1H, 2H, and 19F) magnetic resonance spectroscopy techniques as functions of temperature and pressure were applied to the study of selectively deuterated 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)amide (EMIM TFSA) ionic liquid isotopologues and related ionic liquids. For EMIM TFSA, temperature-dependent 2H T 1 data indicate stronger electric field gradients in the alkyl chain region compared to the imidazolium ring. Most significantly, the pressure dependences of the EMIM and TFSA self-diffusion coefficients revealed that the displacements of the cations and anions are independent, with diffusion of the TFSA anions being slowed much more by increasing pressure than for the EMIM cations, asmore » shown by their respective activation volumes (28.8 ± 2.5 cm³/mol for TFSA vs. 14.6 ± 1.3 cm³/mol for EMIM). Increasing pressure may lower the mobility of the TFSA anion by hindering its interconversion between trans and cis conformers, a process that is coupled to diffusion according to published molecular dynamics simulations. Measured activation volumes (ΔV ‡) for ion self-diffusion in EMIM bis(fluoromethylsulfonyl)amide and EMIM tetrafluoroborate support this hypothesis.« less

Do TFSA anions slither? Pressure exposes the role of TFSA conformational exchange in self-diffusion

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

Suarez, Sophia N.; Wishart, James F.; Rua, Armando

Multi-nuclear ( 1H, 2H, and 19F) magnetic resonance spectroscopy techniques as functions of temperature and pressure were applied to the study of selectively deuterated 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)amide (EMIM TFSA) ionic liquid isotopologues and related ionic liquids. For EMIM TFSA, temperature-dependent 2H T 1 data indicate stronger electric field gradients in the alkyl chain region compared to the imidazolium ring. Most significantly, the pressure dependences of the EMIM and TFSA self-diffusion coefficients revealed that the displacements of the cations and anions are independent, with diffusion of the TFSA anions being slowed much more by increasing pressure than for the EMIM cations, asmore » shown by their respective activation volumes (28.8 ± 2.5 cm³/mol for TFSA vs. 14.6 ± 1.3 cm³/mol for EMIM). Increasing pressure may lower the mobility of the TFSA anion by hindering its interconversion between trans and cis conformers, a process that is coupled to diffusion according to published molecular dynamics simulations. Measured activation volumes (ΔV ‡) for ion self-diffusion in EMIM bis(fluoromethylsulfonyl)amide and EMIM tetrafluoroborate support this hypothesis.« less

Replica Exchange Molecular Dynamics in the Age of Heterogeneous Architectures

NASA Astrophysics Data System (ADS)

Roitberg, Adrian

2014-03-01

The rise of GPU-based codes has allowed MD to reach timescales only dreamed of only 5 years ago. Even within this new paradigm there is still need for advanced sampling techniques. Modern supercomputers (e.g. Blue Waters, Titan, Keeneland) have made available to users a significant number of GPUS and CPUS, which in turn translate into amazing opportunities for dream calculations. Replica-exchange based methods can optimally use tis combination of codes and architectures to explore conformational variabilities in large systems. I will show our recent work in porting the program Amber to GPUS, and the support for replica exchange methods, where the replicated dimension could be Temperature, pH, Hamiltonian, Umbrella windows and combinations of those schemes.

Molecular Dynamics Simulations Provide Atomistic Insight into Hydrogen Exchange Mass Spectrometry Experiments.

PubMed

Petruk, Ariel A; Defelipe, Lucas A; Rodríguez Limardo, Ramiro G; Bucci, Hernán; Marti, Marcelo A; Turjanski, Adrian G

2013-01-08

It is now clear that proteins are flexible entities that in solution switch between conformations to achieve their function. Hydrogen/Deuterium Exchange Mass Spectrometry (HX/MS) is an invaluable tool to understand dynamic changes in proteins modulated by cofactor binding, post-transductional modifications, or protein-protein interactions. ERK2MAPK, a protein involved in highly conserved signal transduction pathways of paramount importance for normal cellular function, has been extensively studied by HX/MS. Experiments of the ERK2MAPK in the inactive and active states (in the presence or absence of bound ATP) have provided valuable information on the plasticity of the MAPK domain. However, interpretation of the HX/MS data is difficult, and changes are mostly explained in relation to available X-ray structures, precluding a complete atomic picture of protein dynamics. In the present work, we have used all atom Molecular Dynamics simulations (MD) to provide a theoretical framework for the interpretation of HX/MS data. Our results show that detailed analysis of protein-solvent interaction along the MD simulations allows (i) prediction of the number of protons exchanged for each peptide in the HX/MS experiments, (ii) rationalization of the experimentally observed changes in exchange rates in different protein conditions at the residue level, and (iii) that at least for ERK2MAPK, most of the functionally observed differences in protein dynamics are related to what can be considered the native state conformational ensemble. In summary, the combination of HX/MS experiments with all atom MD simulations emerges as a powerful approach to study protein native state dynamics with atomic resolution.

Holographic reconstruction of AdS exchanges from crossing symmetry

DOE PAGES

Alday, Luis F.; Bissi, Agnese; Perlmutter, Eric

2017-08-31

Motivated by AdS/CFT, we address the following outstanding question in large N conformal field theory: given the appearance of a single-trace operator in the O x O OPE of a scalar primary O, what is its total contribution to the vacuum four-point function (OOOO) as dictated by crossing symmetry? We solve this problem in 4d conformal field theories at leading order in 1/N. Viewed holographically, this provides a field theory reconstruction of crossing-symmetric, four-point exchange amplitudes in AdS 5. Our solution takes the form of a resummation of the large spin solution to the crossing equations, supplemented by corrections atmore » finite spin, required by crossing. The method can be applied to the exchange of operators of arbitrary twist τ and spin s, although it vastly simplifies for even-integer twist, where we give explicit results. The output is the set of OPE data for the exchange of all double-trace operators [OO] n,ℓ. We find that the double-trace anomalous dimensions γ n,ℓ are negative, monotonic and convex functions of ℓ, for all n and all ℓ > s. This constitutes a holographic signature of bulk causality and classical dynamics of even-spin fields. We also find that the “derivative relation” between double-trace anomalous dimensions and OPE coefficients does not hold in general, and derive the explicit form of the deviation in several cases. Finally, we study large n limits of γ n,ℓ, relevant for the Regge and bulk-point regimes.« less

Structural and kinetic mapping of side-chain exposure onto the protein energy landscape.

PubMed

Bernstein, Rachel; Schmidt, Kierstin L; Harbury, Pehr B; Marqusee, Susan

2011-06-28

Identification and characterization of structural fluctuations that occur under native conditions is crucial for understanding protein folding and function, but such fluctuations are often rare and transient, making them difficult to study. Native-state hydrogen exchange (NSHX) has been a powerful tool for identifying such rarely populated conformations, but it generally reveals no information about the placement of these species along the folding reaction coordinate or the barriers separating them from the folded state and provides little insight into side-chain packing. To complement such studies, we have performed native-state alkyl-proton exchange, a method analogous to NSHX that monitors cysteine modification rather than backbone amide exchange, to examine the folding landscape of Escherichia coli ribonuclease H, a protein well characterized by hydrogen exchange. We have chosen experimental conditions such that the rate-limiting barrier acts as a kinetic partition: residues that become exposed only upon crossing the unfolding barrier are modified in the EX1 regime (alkylation rates report on the rate of unfolding), while those exposed on the native side of the barrier are modified predominantly in the EX2 regime (alkylation rates report on equilibrium populations). This kinetic partitioning allows for identification and placement of partially unfolded forms along the reaction coordinate. Using this approach we detect previously unidentified, rarely populated conformations residing on the native side of the barrier and identify side chains that are modified only upon crossing the unfolding barrier. Thus, in a single experiment under native conditions, both sides of the rate-limiting barrier are investigated.

Structural and kinetic mapping of side-chain exposure onto the protein energy landscape

PubMed Central

Bernstein, Rachel; Schmidt, Kierstin L.; Harbury, Pehr B.; Marqusee, Susan

2011-01-01

Identification and characterization of structural fluctuations that occur under native conditions is crucial for understanding protein folding and function, but such fluctuations are often rare and transient, making them difficult to study. Native-state hydrogen exchange (NSHX) has been a powerful tool for identifying such rarely populated conformations, but it generally reveals no information about the placement of these species along the folding reaction coordinate or the barriers separating them from the folded state and provides little insight into side-chain packing. To complement such studies, we have performed native-state alkyl-proton exchange, a method analogous to NSHX that monitors cysteine modification rather than backbone amide exchange, to examine the folding landscape of Escherichia coli ribonuclease H, a protein well characterized by hydrogen exchange. We have chosen experimental conditions such that the rate-limiting barrier acts as a kinetic partition: residues that become exposed only upon crossing the unfolding barrier are modified in the EX1 regime (alkylation rates report on the rate of unfolding), while those exposed on the native side of the barrier are modified predominantly in the EX2 regime (alkylation rates report on equilibrium populations). This kinetic partitioning allows for identification and placement of partially unfolded forms along the reaction coordinate. Using this approach we detect previously unidentified, rarely populated conformations residing on the native side of the barrier and identify side chains that are modified only upon crossing the unfolding barrier. Thus, in a single experiment under native conditions, both sides of the rate-limiting barrier are investigated. PMID:21670244

Microstructural Characteristic of the Al-Fe-Cu Alloy During High-Speed Repetitive Continuous Extrusion Forming

NASA Astrophysics Data System (ADS)

Hu, Jiamin; Teng, Jie; Ji, Xiankun; Kong, Xiangxin; Jiang, Fulin; Zhang, Hui

2016-11-01

High-speed repetitive continuous extrusion forming process (R-Conform process) was performed on the Al-Fe-Cu alloy. The microstructural evolution and mechanical properties were studied by x-ray diffraction, electron backscatter diffraction, transmission electron microscopy and tensile testing. The results show that a significant improvement of tensile ductility concurs with a considerable loss of tensile strength before four passes, after that the process on mechanical properties variation tends to be steady, indicating an accelerated mechanical softening occurs when comparing to low-speed R-Conform process. Microstructure characterization indicates that the accumulated strain promotes the transformation of low angle boundaries to high angle boundaries, thus leading to the acceleration of continuous dynamic recrystallization process, and the precipitates are broken, spheroidized and homogeneously distribute in Al matrix as increasing R-Conform passes. Massive microshear bands are observed after initial passes of R-Conform process, which may promote continuous dynamic recrystallization and further grain refinement during high-speed R-Conform process.

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.

Conformations of the Huntingtin N-term in aqueous solution from atomistic simulations.

PubMed

Rossetti, Giulia; Cossio, Pilar; Laio, Alessandro; Carloni, Paolo

2011-10-03

The first 17 amino acids of Huntingtin protein (N17) play a crucial role in the protein's aggregation. Here we predict its free energy landscape in aqueous solution by using bias exchange metadynamics. All our findings are consistent with experimental data. N17 populates four main kinetic basins, which interconvert on the microsecond time-scale. The most populated basin (about 75%) is a random coil, with an extended flat exposed hydrophobic surface. This might create a hydrophobic seed promoting Huntingtin aggregation. The other main populated basins contain helical conformations, which could facilitate N17 binding on its cellular targets. Copyright © 2011. Published by Elsevier B.V.

Adsorption and conformations of lysozyme and α-lactalbumin at a water-octane interface

NASA Astrophysics Data System (ADS)

Cheung, David L.

2017-11-01

As proteins contain both hydrophobic and hydrophilic amino acids, they will readily adsorb onto interfaces between water and hydrophobic fluids such as oil. This adsorption normally causes changes in the protein structure, which can result in loss of protein function and irreversible adsorption, leading to the formation of protein interfacial films. While this can be advantageous in some applications (e.g., food technology), in most cases it limits our ability to exploit protein functionality at interfaces. To understand and control protein interfacial adsorption and function, it is necessary to understand the microscopic conformation of proteins at liquid interfaces. In this paper, molecular dynamics simulations are used to investigate the adsorption and conformation of two similar proteins, lysozyme and α-lactalbumin, at a water-octane interface. While they both adsorb onto the interface, α-lactalbumin does so in a specific orientation, mediated by two amphipathic helices, while lysozyme adsorbs in a non-specific manner. Using replica exchange simulations, both proteins are found to possess a number of distinct interfacial conformations, with compact states similar to the solution conformation being most common for both proteins. Decomposing the different contributions to the protein energy at oil-water interfaces suggests that conformational change for α-lactalbumin, unlike lysozyme, is driven by favourable protein-oil interactions. Revealing these differences between the factors that govern the conformational change at interfaces in otherwise similar proteins can give insight into the control of protein interfacial adsorption, aggregation, and function.

Free-energy landscape of a hyperstable RNA tetraloop.

PubMed

Miner, Jacob C; Chen, Alan A; García, Angel E

2016-06-14

We report the characterization of the energy landscape and the folding/unfolding thermodynamics of a hyperstable RNA tetraloop obtained through high-performance molecular dynamics simulations at microsecond timescales. Sampling of the configurational landscape is conducted using temperature replica exchange molecular dynamics over three isochores at high, ambient, and negative pressures to determine the thermodynamic stability and the free-energy landscape of the tetraloop. The simulations reveal reversible folding/unfolding transitions of the tetraloop into the canonical A-RNA conformation and the presence of two alternative configurations, including a left-handed Z-RNA conformation and a compact purine Triplet. Increasing hydrostatic pressure shows a stabilizing effect on the A-RNA conformation and a destabilization of the left-handed Z-RNA. Our results provide a comprehensive description of the folded free-energy landscape of a hyperstable RNA tetraloop and highlight the significant advances of all-atom molecular dynamics in describing the unbiased folding of a simple RNA secondary structure motif.

Computational investigation of the conformational profile of the four stereomers of Ac-L-Pro-c3Phe-NHMe (c3Phe= 2,3-methanophenylalanine).

PubMed

Rodriguez, Alejandro; Canto, Josep; Corcho, Francesc J; Perez, Juan J

2009-01-01

The present report regards a computational study aimed at assessing the conformational profile of the four stereoisomers of the peptide Ace-Pro-c3Phe-NMe, previously reported to exhibit beta-turn structures in dichloromethane with different type I/type II beta-turn profiles. Molecular systems were represented at the molecular mechanics level using the parm96 parameterization of the AMBER force field. Calculations were carried out in dichloromethane using an implicit solvent approach. Characterization of the conformational features of the peptide analogs was carried out using simulated annealing (SA), molecular dynamics (MD) and replica exchange molecular dynamics (REMD). Present results show that MD calculations do not provide a reasonable sampling after 300 ns. In contrast, both SA and REMD provide similar results and agree well with experimental observations. Copyright 2009 Wiley Periodicals, Inc.

Conformal Bootstrap in Mellin Space

NASA Astrophysics Data System (ADS)

Gopakumar, Rajesh; Kaviraj, Apratim; Sen, Kallol; Sinha, Aninda

2017-02-01

We propose a new approach towards analytically solving for the dynamical content of conformal field theories (CFTs) using the bootstrap philosophy. This combines the original bootstrap idea of Polyakov with the modern technology of the Mellin representation of CFT amplitudes. We employ exchange Witten diagrams with built-in crossing symmetry as our basic building blocks rather than the conventional conformal blocks in a particular channel. Demanding consistency with the operator product expansion (OPE) implies an infinite set of constraints on operator dimensions and OPE coefficients. We illustrate the power of this method in the ɛ expansion of the Wilson-Fisher fixed point by reproducing anomalous dimensions and, strikingly, obtaining OPE coefficients to higher orders in ɛ than currently available using other analytic techniques (including Feynman diagram calculations). Our results enable us to get a somewhat better agreement between certain observables in the 3D Ising model and the precise numerical values that have been recently obtained.

17 CFR 270.8b-11 - Number of copies; signatures; binding.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies; signatures... (CONTINUED) RULES AND REGULATIONS, INVESTMENT COMPANY ACT OF 1940 § 270.8b-11 Number of copies; signatures... manner prescribed by the appropriate form. Unsigned copies shall be conformed. If the signature of any...

18 CFR 35.31 - Commission review.

Code of Federal Regulations, 2011 CFR

2011-04-01

... determine whether the ASC set by BPA for the applicable exchange period was determined in accordance with... Commission will order that BPA amend the ASC to conform with the methodology. If the ASC is in accord with... by the Commission will be at the rate charged to BPA by the U.S. Treasury during that period, unless...

18 CFR 35.31 - Commission review.

Code of Federal Regulations, 2012 CFR

2012-04-01

... determine whether the ASC set by BPA for the applicable exchange period was determined in accordance with... Commission will order that BPA amend the ASC to conform with the methodology. If the ASC is in accord with... by the Commission will be at the rate charged to BPA by the U.S. Treasury during that period, unless...

18 CFR 35.31 - Commission review.

Code of Federal Regulations, 2010 CFR

2010-04-01

... determine whether the ASC set by BPA for the applicable exchange period was determined in accordance with... Commission will order that BPA amend the ASC to conform with the methodology. If the ASC is in accord with... by the Commission will be at the rate charged to BPA by the U.S. Treasury during that period, unless...

18 CFR 35.31 - Commission review.

Code of Federal Regulations, 2013 CFR

2013-04-01

... determine whether the ASC set by BPA for the applicable exchange period was determined in accordance with... Commission will order that BPA amend the ASC to conform with the methodology. If the ASC is in accord with... by the Commission will be at the rate charged to BPA by the U.S. Treasury during that period, unless...

18 CFR 35.31 - Commission review.

Code of Federal Regulations, 2014 CFR

2014-04-01

... determine whether the ASC set by BPA for the applicable exchange period was determined in accordance with... Commission will order that BPA amend the ASC to conform with the methodology. If the ASC is in accord with... by the Commission will be at the rate charged to BPA by the U.S. Treasury during that period, unless...

21 CFR 173.20 - Ion-exchange membranes.

Code of Federal Regulations, 2014 CFR

2014-04-01

... subjecting a polyethylene base conforming to § 177.1520 of this chapter to polymerization with styrene until the polystyrene phase of the base is not less than 16 percent nor more than 30 percent by weight. The base is then modified by reaction with chloromethyl methyl ether, and by subsequent amination with tri...

Situational and Dispositional Factors as Antecedents of Ingratiatory Behaviors in Organizational Settings

ERIC Educational Resources Information Center

Kacmar, K. Michele; Carlson, Dawn S.; Bratton, Virginia K.

2004-01-01

This study examined both situational and dispositional antecedents of four ingratiatory behaviors: other-enhancing, opinion conformity, favor rendering, and self-promotion. The two situational variables (i.e., role ambiguity and leader-member exchange) and the four dispositional variables (i.e., self-esteem, need for power, job involvement, and…

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

Low molecular weight oligomers of amyloid peptides display β-barrel conformations: A replica exchange molecular dynamics study in explicit solvent

NASA Astrophysics Data System (ADS)

De Simone, Alfonso; Derreumaux, Philippe

2010-04-01

The self-assembly of proteins and peptides into amyloid fibrils is connected to over 40 pathological conditions including neurodegenerative diseases and systemic amyloidosis. Diffusible, low molecular weight protein and peptide oligomers that form in the early steps of aggregation appear to be the harmful cytotoxic species in the molecular etiology of these diseases. So far, the structural characterization of these oligomers has remained elusive owing to their transient and dynamic features. We here address, by means of full atomistic replica exchange molecular dynamics simulations, the energy landscape of heptamers of the amyloidogenic peptide NHVTLSQ from the beta-2 microglobulin protein. The simulations totaling 5 μs show that low molecular weight oligomers in explicit solvent consist of β-barrels in equilibrium with amorphous states and fibril-like assemblies. The results, also accounting for the influence of the pH on the conformational properties, provide a strong evidence of the formation of transient β-barrel assemblies in the early aggregation steps of amyloid-forming systems. Our findings are discussed in terms of oligomers cytotoxicity.

A magnesium-induced triplex pre-organizes the SAM-II riboswitch

PubMed Central

Roy, Susmita; Lammert, Heiko; Dayie, T. Kwaku; Sanbonmatsu, Karissa Y.

2017-01-01

Our 13C- and 1H-chemical exchange saturation transfer (CEST) experiments previously revealed a dynamic exchange between partially closed and open conformations of the SAM-II riboswitch in the absence of ligand. Here, all-atom structure-based molecular simulations, with the electrostatic effects of Manning counter-ion condensation and explicit magnesium ions are employed to calculate the folding free energy landscape of the SAM-II riboswitch. We use this analysis to predict that magnesium ions remodel the landscape, shifting the equilibrium away from the extended, partially unfolded state towards a compact, pre-organized conformation that resembles the ligand-bound state. Our CEST and SAXS experiments, at different magnesium ion concentrations, quantitatively confirm our simulation results, demonstrating that magnesium ions induce collapse and pre-organization. Agreement between theory and experiment bolsters microscopic interpretation of our simulations, which shows that triplex formation between helix P2b and loop L1 is highly sensitive to magnesium and plays a key role in pre-organization. Pre-organization of the SAM-II riboswitch allows rapid detection of ligand with high selectivity, which is important for biological function. PMID:28248966

On the Helix Propensity in Generalized Born Solvent Descriptions of Modeling the Dark Proteome

PubMed Central

Olson, Mark A.

2017-01-01

Intrinsically disordered proteins that populate the so-called “Dark Proteome” offer challenging benchmarks of atomistic simulation methods to accurately model conformational transitions on a multidimensional energy landscape. This work explores the application of parallel tempering with implicit solvent models as a computational framework to capture the conformational ensemble of an intrinsically disordered peptide derived from the Ebola virus protein VP35. A recent X-ray crystallographic study reported a protein-peptide interface where the VP35 peptide underwent a folding transition from a disordered form to a helix-β-turn-helix topological fold upon molecular association with the Ebola protein NP. An assessment is provided of the accuracy of two generalized Born solvent models (GBMV2 and GBSW2) using the CHARMM force field and applied with temperature-based replica exchange dynamics to calculate the disorder propensity of the peptide and its probability density of states in a continuum solvent. A further comparison is presented of applying an explicit/implicit solvent hybrid replica exchange simulation of the peptide to determine the effect of modeling water interactions at the all-atom resolution. PMID:28197405

On the Helix Propensity in Generalized Born Solvent Descriptions of Modeling the Dark Proteome.

PubMed

Olson, Mark A

2017-01-01

Intrinsically disordered proteins that populate the so-called "Dark Proteome" offer challenging benchmarks of atomistic simulation methods to accurately model conformational transitions on a multidimensional energy landscape. This work explores the application of parallel tempering with implicit solvent models as a computational framework to capture the conformational ensemble of an intrinsically disordered peptide derived from the Ebola virus protein VP35. A recent X-ray crystallographic study reported a protein-peptide interface where the VP35 peptide underwent a folding transition from a disordered form to a helix-β-turn-helix topological fold upon molecular association with the Ebola protein NP. An assessment is provided of the accuracy of two generalized Born solvent models (GBMV2 and GBSW2) using the CHARMM force field and applied with temperature-based replica exchange dynamics to calculate the disorder propensity of the peptide and its probability density of states in a continuum solvent. A further comparison is presented of applying an explicit/implicit solvent hybrid replica exchange simulation of the peptide to determine the effect of modeling water interactions at the all-atom resolution.

Allosteric Coupling of CARMIL and V-1 Binding to Capping Protein Revealed by Hydrogen-Deuterium Exchange.

PubMed

Johnson, Britney; McConnell, Patrick; Kozlov, Alex G; Mekel, Marlene; Lohman, Timothy M; Gross, Michael L; Amarasinghe, Gaya K; Cooper, John A

2018-05-29

Actin assembly is important for cell motility. The ability of actin subunits to join or leave filaments via the barbed end is critical to actin dynamics. Capping protein (CP) binds to barbed ends to prevent subunit gain and loss and is regulated by proteins that include V-1 and CARMIL. V-1 inhibits CP by sterically blocking one binding site for actin. CARMILs bind at a distal site and decrease the affinity of CP for actin, suggested to be caused by conformational changes. We used hydrogen-deuterium exchange with mass spectrometry (HDX-MS) to probe changes in structural dynamics induced by V-1 and CARMIL binding to CP. V-1 and CARMIL induce changes in both proteins' binding sites on the surface of CP, along with a set of internal residues. Both also affect the conformation of CP's ββ subunit "tentacle," a second distal actin-binding site. Concerted regulation of actin assembly by CP occurs through allosteric couplings between CP modulator and actin binding sites. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Bias-Exchange Metadynamics Simulation of Membrane Permeation of 20 Amino Acids.

PubMed

Cao, Zanxia; Bian, Yunqiang; Hu, Guodong; Zhao, Liling; Kong, Zhenzhen; Yang, Yuedong; Wang, Jihua; Zhou, Yaoqi

2018-03-16

Thermodynamics of the permeation of amino acids from water to lipid bilayers is an important first step for understanding the mechanism of cell-permeating peptides and the thermodynamics of membrane protein structure and stability. In this work, we employed bias-exchange metadynamics simulations to simulate the membrane permeation of all 20 amino acids from water to the center of a dipalmitoylphosphatidylcholine (DPPC) membrane (consists of 256 lipids) by using both directional and torsion angles for conformational sampling. The overall accuracy for the free energy profiles obtained is supported by significant correlation coefficients (correlation coefficient at 0.5-0.6) between our results and previous experimental or computational studies. The free energy profiles indicated that (1) polar amino acids have larger free energy barriers than nonpolar amino acids; (2) negatively charged amino acids are the most difficult to enter into the membrane; and (3) conformational transitions for many amino acids during membrane crossing is the key for reduced free energy barriers. These results represent the first set of simulated free energy profiles of membrane crossing for all 20 amino acids.

Conformational Dynamics of Insulin

PubMed Central

Hua, Qing-Xin; Jia, Wenhua; Weiss, Michael A.

2011-01-01

We have exploited a prandial insulin analog to elucidate the underlying structure and dynamics of insulin as a monomer in solution. A model was provided by insulin lispro (the active component of Humalog®; Eli Lilly and Co.). Whereas NMR-based modeling recapitulated structural relationships of insulin crystals (T-state protomers), dynamic anomalies were revealed by amide-proton exchange kinetics in D2O. Surprisingly, the majority of hydrogen bonds observed in crystal structures are only transiently maintained in solution, including key T-state-specific inter-chain contacts. Long-lived hydrogen bonds (as defined by global exchange kinetics) exist only at a subset of four α-helical sites (two per chain) flanking an internal disulfide bridge (cystine A20–B19); these sites map within the proposed folding nucleus of proinsulin. The anomalous flexibility of insulin otherwise spans its active surface and may facilitate receptor binding. Because conformational fluctuations promote the degradation of pharmaceutical formulations, we envisage that “dynamic re-engineering” of insulin may enable design of ultra-stable formulations for humanitarian use in the developing world. PMID:22649374

Efficient evaluation of sampling quality of molecular dynamics simulations by clustering of dihedral torsion angles and Sammon mapping.

PubMed

Frickenhaus, Stephan; Kannan, Srinivasaraghavan; Zacharias, Martin

2009-02-01

A direct conformational clustering and mapping approach for peptide conformations based on backbone dihedral angles has been developed and applied to compare conformational sampling of Met-enkephalin using two molecular dynamics (MD) methods. Efficient clustering in dihedrals has been achieved by evaluating all combinations resulting from independent clustering of each dihedral angle distribution, thus resolving all conformational substates. In contrast, Cartesian clustering was unable to accurately distinguish between all substates. Projection of clusters on dihedral principal component (PCA) subspaces did not result in efficient separation of highly populated clusters. However, representation in a nonlinear metric by Sammon mapping was able to separate well the 48 highest populated clusters in just two dimensions. In addition, this approach also allowed us to visualize the transition frequencies between clusters efficiently. Significantly, higher transition frequencies between more distinct conformational substates were found for a recently developed biasing-potential replica exchange MD simulation method allowing faster sampling of possible substates compared to conventional MD simulations. Although the number of theoretically possible clusters grows exponentially with peptide length, in practice, the number of clusters is only limited by the sampling size (typically much smaller), and therefore the method is well suited also for large systems. The approach could be useful to rapidly and accurately evaluate conformational sampling during MD simulations, to compare different sampling strategies and eventually to detect kinetic bottlenecks in folding pathways.

The analytic structure of conformal blocks and the generalized Wilson-Fisher fixed points

DOE PAGES

Gliozzi, Ferdinando; Guerrieri, Andrea L.; Petkou, Anastasios C.; ...

2017-04-11

Here, we describe in detail the method used in our previous work arXiv:1611.10344 to study the Wilson-Fisher critical points nearby generalized free CFTs, exploiting the analytic structure of conformal blocks as functions of the conformal dimension of the exchanged operator. Our method is equivalent to the mechanism of conformal multiplet recombination set up by null states. We also compute, to the first non-trivial order in the ε-expansion, the anomalous dimensions and the OPE coefficients of infinite classes of scalar local operators using just CFT data. We study single-scalar and O(N)-invariant theories, as well as theories with multiple deformations. When availablemore » we agree with older results, but we also produce a wealth of new ones. Furthermore, unitarity and crossing symmetry are not used in our approach and we are able to apply our method to non-unitary theories as well. Some implications of our results for the study of the non-unitary theories containing partially conserved higher-spin currents are briefly mentioned.« less

Synchrotron based infrared imaging and spectroscopy via focal plane array on live fibroblasts in D2O enriched medium

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

Quaroni, Luca; Zlateva, Theodora; Sarafimov, Blagoj

2014-03-26

We tested the viability of using synchrotron based infrared imaging to study biochemical processes inside living cells. As a model system, we studied fibroblast cells exposed to a medium highly enriched with D2O. We could show that the experimental technique allows us to reproduce at the cellular level measurements that are normally performed on purified biological molecules. We can obtain information about lipid conformation and distribution, kinetics of hydrogen/deuterium exchange, and the formation of concentration gradients of H and O isotopes in water that are associated with cell metabolism. The implementation of the full field technique in a sequential imagingmore » format gives a description of cellular biochemistry and biophysics that contains both spatial and temporal information.« less

Easy design of colorimetric logic gates based on nonnatural base pairing and controlled assembly of gold nanoparticles.

PubMed

Zhang, Li; Wang, Zhong-Xia; Liang, Ru-Ping; Qiu, Jian-Ding

2013-07-16

Utilizing the principles of metal-ion-mediated base pairs (C-Ag-C and T-Hg-T), the pH-sensitive conformational transition of C-rich DNA strand, and the ligand-exchange process triggered by DL-dithiothreitol (DTT), a system of colorimetric logic gates (YES, AND, INHIBIT, and XOR) can be rationally constructed based on the aggregation of the DNA-modified Au NPs. The proposed logic operation system is simple, which consists of only T-/C-rich DNA-modified Au NPs, and it is unnecessary to exquisitely design and alter the DNA sequence for different multiple molecular logic operations. The nonnatural base pairing combined with unique optical properties of Au NPs promises great potential in multiplexed ion sensing, molecular-scale computers, and other computational logic devices.

Investigation of the binding of a carbohydrate-mimetic peptide to its complementary anticarbohydrate antibody by STD-NMR spectroscopy and molecular-dynamics simulations.

PubMed

Szczepina, Monica G; Bleile, Dustin W; Pinto, B Mario

2011-10-04

Saturation transfer difference (STD)-NMR spectroscopy was used to probe experimentally the bioactive solution conformation of the carbohydrate mimic MDWNMHAA 1 of the O-polysaccharide of Shigella flexneri Y when bound to its complementary antibody, mAb SYA/J6. Molecular dynamics simulations using the ZymeCAD™ Molecular Dynamics platform were also undertaken to give a more accurate picture of the conformational flexibility and the possibilities for bound ligand conformations. The ligand topology, or the dynamic epitope, was mapped with the CORCEMA-ST (COmplete Relaxation and Conformational Exchange Matrix Analysis of Saturation Transfer) program that calculates a total matrix analysis of relaxation and exchange effects to generate predicted STD-NMR intensities from simulation. The comparison of these predicted STD enhancements with experimental data was used to select a representative binding mode. A protocol that employed theoretical STD effects calculated at snapshots during the entire course of a molecular dynamics (MD) trajectory of the peptide bound to the Fv portion of the antibody, and not the averaged atomic positions of receptor-ligand complexes, was also examined. In addition, the R factor was calculated on the basis of STD (fit) to avoid T1 bias, and an effective R factor, R(eff), was defined such that if the calculated STD (fit) for proton k was within error of the experimental STD (fit) for proton k, then that calculated STD (fit) for proton k was not included in the calculation of the R factor. This protocol was effective in deriving the antibody-bound solution conformation of the peptide which also differed from the bound conformation determined by X-ray crystallography; however, several discrepancies between experimental and calculated STD (fit) values were observed. The bound conformation was therefore further refined with a simulated annealing refinement protocol known as STD-NMR intensity-restrained CORCEMA optimization (SICO) to give a more accurate representation of the bound peptide epitope. Further optimization was required in this case, but a satisfactory correlation between experimental and calculated STD values was obtained. Attempts were also made to obtain STD enhancements with a synthetic pentasaccharide hapten, corresponding to the O-polysaccharide, while bound to the antibody. However, unfavorable kinetics of binding in this system prevented sufficient STD build-up, which, in turn, hindered a rigorous analysis via full STD build-up curves. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of major sources controlling groundwater chemistry from a hard rock terrain — A case study from Mettur taluk, Salem district, Tamil Nadu, India

NASA Astrophysics Data System (ADS)

Srinivasamoorthy, K.; Chidambaram, S.; Prasanna, M. V.; Vasanthavihar, M.; Peter, John; Anandhan, P.

2008-02-01

The study area Mettur forms an important industrial town situated NW of Salem district. The geology of the area is mainly composed of Archean crystalline metamorphic complexes. To identify the major process activated for controlling the groundwater chemistry an attempt has been made by collecting a total of 46 groundwater samples for two different seasons, viz., pre-monsoon and post-monsoon. The groundwater chemistry is dominated by silicate weathering and (Na + Mg) and (Cl + SO4) accounts of about 90% of cations and anions. The contribution of (Ca + Mg) and (Na + K) to total cations and HCO3 indicates the domination of silicate weathering as major sources for cations. The plot for Na to Cl indicates higher Cl in both seasons, derived from Anthropogenic (human) sources from fertilizer, road salt, human and animal waste, and industrial applications, minor representations of Na also indicates source from weathering of silicate-bearing minerals. The plot for Na/Cl to EC indicates Na released from silicate weathering process which is also supported by higher HCO3 values in both the seasons. Ion exchange process is also activated in the study area which is indicated by shifting to right in plot for Ca + Mg to SO4 + HCO3. The plot of Na-Cl to Ca + Mg-HCO3-SO4 confirms that Ca, Mg and Na concentrations in groundwater are derived from aquifer materials. Thermodynamic plot indicates that groundwater is in equilibrium with kaolinite, muscovite and chlorite minerals. Saturation index of silicate and carbonate minerals indicate oversaturation during pre-monsoon and undersaturation during post-monsoon, conforming dissolution and dilution process. In general, water chemistry is guided by complex weathering process, ion exchange along with influence of Cl ions from anthropogenic impact.

Degradation of Redox-Sensitive Proteins including Peroxiredoxins and DJ-1 is Promoted by Oxidation-induced Conformational Changes and Ubiquitination

NASA Astrophysics Data System (ADS)

Song, In-Kang; Lee, Jae-Jin; Cho, Jin-Hwan; Jeong, Jihye; Shin, Dong-Hae; Lee, Kong-Joo

2016-10-01

Reactive oxygen species (ROS) are key molecules regulating various cellular processes. However, what the cellular targets of ROS are and how their functions are regulated is unclear. This study explored the cellular proteomic changes in response to oxidative stress using H2O2 in dose- and recovery time-dependent ways. We found discernible changes in 76 proteins appearing as 103 spots on 2D-PAGE. Of these, Prxs, DJ-1, UCH-L3 and Rla0 are readily oxidized in response to mild H2O2 stress, and then degraded and active proteins are newly synthesized during recovery. In studies designed to understand the degradation process, multiple cellular modifications of redox-sensitive proteins were identified by peptide sequencing with nanoUPLC-ESI-q-TOF tandem mass spectrometry and the oxidative structural changes of Prx2 explored employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that hydrogen/deuterium exchange rate increased in C-terminal region of oxidized Prx2, suggesting the exposure of this region to solvent under oxidation. We also found that Lys191 residue in this exposed C-terminal region of oxidized Prx2 is polyubiquitinated and the ubiquitinated Prx2 is readily degraded in proteasome and autophagy. These findings suggest that oxidation-induced ubiquitination and degradation can be a quality control mechanism of oxidized redox-sensitive proteins including Prxs and DJ-1.

Effect of proline mutations on the monomer conformations of amylin.

PubMed

Chiu, Chi-cheng; Singh, Sadanand; de Pablo, Juan J

2013-09-03

The formation of human islet amyloid polypeptide (hIAPP) is implicated in the loss of pancreatic β-cells in type II diabetes. Rat amylin, which differs from human amylin at six residues, does not lead to formation of amyloid fibrils. Pramlintide is a synthetic analog of human amylin that shares three proline substitutions with rat amylin. Pramlintide has a much smaller propensity to form amyloid aggregates and has been widely prescribed in amylin replacement treatment. It is known that the three prolines attenuate β-sheet formation. However, the detailed effects of these proline substitutions on full-length hIAPP remain poorly understood. In this work, we use molecular simulations and bias-exchange metadynamics to investigate the effect of proline substitutions on the conformation of the hIAPP monomer. Our results demonstrate that hIAPP can adopt various β-sheet conformations, some of which have been reported in experiments. The proline substitutions perturb the formation of long β-sheets and reduce their stability. More importantly, we find that all three proline substitutions of pramlintide are required to inhibit β conformations and stabilize the α-helical conformation. Fewer substitutions do not have a significant inhibiting effect. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Geometric analysis characterizes molecular rigidity in generic and non-generic protein configurations

PubMed Central

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

2015-01-01

Proteins operate and interact with partners by dynamically exchanging between functional substates of a conformational ensemble on a rugged free energy landscape. Understanding how these substates are linked by coordinated, collective motions requires exploring a high-dimensional space, which remains a tremendous challenge. While molecular dynamics simulations can provide atomically detailed insight into the dynamics, computational demands to adequately sample conformational ensembles of large biomolecules and their complexes often require tremendous resources. Kinematic models can provide high-level insights into conformational ensembles and molecular rigidity beyond the reach of molecular dynamics by reducing the dimensionality of the search space. Here, we model a protein as a kinematic linkage and present a new geometric method to characterize molecular rigidity from the constraint manifold Q and its tangent space Q at the current configuration q. In contrast to methods based on combinatorial constraint counting, our method is valid for both generic and non-generic, e.g., singular configurations. Importantly, our geometric approach provides an explicit basis for collective motions along floppy modes, resulting in an efficient procedure to probe conformational space. An atomically detailed structural characterization of coordinated, collective motions would allow us to engineer or allosterically modulate biomolecules by selectively stabilizing conformations that enhance or inhibit function with broad implications for human health. PMID:26213417

Geometric analysis characterizes molecular rigidity in generic and non-generic protein configurations

NASA Astrophysics Data System (ADS)

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

2015-10-01

Proteins operate and interact with partners by dynamically exchanging between functional substates of a conformational ensemble on a rugged free energy landscape. Understanding how these substates are linked by coordinated, collective motions requires exploring a high-dimensional space, which remains a tremendous challenge. While molecular dynamics simulations can provide atomically detailed insight into the dynamics, computational demands to adequately sample conformational ensembles of large biomolecules and their complexes often require tremendous resources. Kinematic models can provide high-level insights into conformational ensembles and molecular rigidity beyond the reach of molecular dynamics by reducing the dimensionality of the search space. Here, we model a protein as a kinematic linkage and present a new geometric method to characterize molecular rigidity from the constraint manifold Q and its tangent space Tq Q at the current configuration q. In contrast to methods based on combinatorial constraint counting, our method is valid for both generic and non-generic, e.g., singular configurations. Importantly, our geometric approach provides an explicit basis for collective motions along floppy modes, resulting in an efficient procedure to probe conformational space. An atomically detailed structural characterization of coordinated, collective motions would allow us to engineer or allosterically modulate biomolecules by selectively stabilizing conformations that enhance or inhibit function with broad implications for human health.

Use of pressure in reversed-phase liquid chromatography to study protein conformational changes by differential deuterium exchange.

PubMed

Makarov, Alexey A; Schafer, Wes A; Helmy, Roy

2015-02-17

The market of protein therapeutics is exploding, and characterization methods for proteins are being further developed to understand and explore conformational structures with regards to function and activity. There are several spectroscopic techniques that allow for analyzing protein secondary structure in solution. However, a majority of these techniques need to use purified protein, concentrated enough in the solution to produce a relevant spectrum. In this study, we describe a novel approach which uses ultrahigh pressure liquid chromatography (UHPLC) coupled with mass-spectrometry (MS) to explore compressibility of the secondary structure of proteins under increasing pressure detected by hydrogen-deuterium exchange (HDX). Several model proteins were used for these studies. The studies were conducted with UHPLC in isocratic mode at constant flow rate and temperature. The pressure was modified by a backpressure regulator up to about 1200 bar. It was found that the increase of retention factors upon pressure increase, at constant flow rate and temperature, was based on reduction of the proteins' molecular molar volume. The change in the proteins' molecular molar volume was caused by changes in protein folding, as was revealed by differential deuterium exchange. The degree of protein folding under certain UHPLC conditions can be controlled by pressure, at constant temperature and flow rate. By modifying pressure during UHPLC separation, it was possible to achieve changes in protein folding, which were manifested as changes in the number of labile protons exchanged to deuterons, or vice versa. Moreover, it was demonstrated with bovine insulin that a small difference in the number of protons exchanged to deuterons (based on protein folding under pressure) could be observed between batches obtained from different sources. The use of HDX during UHPLC separation allowed one to examine protein folding by pressure at constant flow rate and temperature in a mixture of sample solution with minimal amounts of sample used for analysis.

Structurally controlled deposition of silicon onto nanowires

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

Wang, Weijie; Liu, Zuqin; Han, Song

Provided herein are nanostructures for lithium ion battery electrodes and methods of fabrication. In some embodiments, a nanostructure template coated with a silicon coating is provided. The silicon coating may include a non-conformal, more porous layer and a conformal, denser layer on the non-conformal, more porous layer. In some embodiments, two different deposition processes, e.g., a PECVD layer to deposit the non-conformal layer and a thermal CVD process to deposit the conformal layer, are used. Anodes including the nanostructures have longer cycle lifetimes than anodes made using either a PECVD or thermal CVD method alone.

Simulated Tempering Distributed Replica Sampling, Virtual Replica Exchange, and Other Generalized-Ensemble Methods for Conformational Sampling.

PubMed

Rauscher, Sarah; Neale, Chris; Pomès, Régis

2009-10-13

Generalized-ensemble algorithms in temperature space have become popular tools to enhance conformational sampling in biomolecular simulations. A random walk in temperature leads to a corresponding random walk in potential energy, which can be used to cross over energetic barriers and overcome the problem of quasi-nonergodicity. In this paper, we introduce two novel methods: simulated tempering distributed replica sampling (STDR) and virtual replica exchange (VREX). These methods are designed to address the practical issues inherent in the replica exchange (RE), simulated tempering (ST), and serial replica exchange (SREM) algorithms. RE requires a large, dedicated, and homogeneous cluster of CPUs to function efficiently when applied to complex systems. ST and SREM both have the drawback of requiring extensive initial simulations, possibly adaptive, for the calculation of weight factors or potential energy distribution functions. STDR and VREX alleviate the need for lengthy initial simulations, and for synchronization and extensive communication between replicas. Both methods are therefore suitable for distributed or heterogeneous computing platforms. We perform an objective comparison of all five algorithms in terms of both implementation issues and sampling efficiency. We use disordered peptides in explicit water as test systems, for a total simulation time of over 42 μs. Efficiency is defined in terms of both structural convergence and temperature diffusion, and we show that these definitions of efficiency are in fact correlated. Importantly, we find that ST-based methods exhibit faster temperature diffusion and correspondingly faster convergence of structural properties compared to RE-based methods. Within the RE-based methods, VREX is superior to both SREM and RE. On the basis of our observations, we conclude that ST is ideal for simple systems, while STDR is well-suited for complex systems.

Structures and mechanisms in clay nanopore trapping of structurally-different fluoroquinolone antimicrobials.

PubMed

Okaikue-Woodi, Fanny E K; Kelch, Sabrina E; Schmidt, Michael P; Enid Martinez, Carmen; Youngman, Randall E; Aristilde, Ludmilla

2018-03-01

Smectite clay nanoparticles are implicated in the retention of antimicrobials within soils and sediments; these clays are also inspected as drug carriers in physiological systems. Cation exchange is considered the primary adsorption mechanism of antimicrobials within smectite nanopores. However, a dual role of acid-base chemistry and adsorptive structures is speculated by recent studies. Using the prototypical smectite clay montmorillonite, we employed a combination of X-ray diffraction (XRD), nuclear magnetic resonance, attenuated total reflectance-Fourier transform infrared spectroscopy, and molecular dynamics simulations to investigate the interlayer nanopore trapping of two structurally-different fluoroquinolone (FQ) antimicrobials with similar acid-base chemistry: ciprofloxacin (a first-generation FQ) and moxifloxacin (a third-generation FQ). Greater sorption at pH 5.0 than at pH 7.0 for both FQs was consistent with cation-exchange of positively-charged species. However, the clay exhibited a near twofold higher sorption capacity for moxifloxacin than for ciprofloxacin. This difference was shown by the XRD data to be accompanied by enhanced trapping of moxifloxacin within the clay interlayers. Using the XRD-determined nanopore sizes, we performed molecular dynamics simulations of thermodynamically-favorable model adsorbates, which revealed that ciprofloxacin was adsorbed parallel to the clay surface but moxifloxacin adopted a tilted conformation across the nanopore. These conformations resulted in more slowly-exchanged than quickly-exchanged Na complexes with ciprofloxacin compared with moxifloxacin. These different Na populations were also captured by 23 Na nuclear magnetic resonance. Furthermore, the simulated adsorbates uncovered different complexation interactions that were corroborated by infrared spectroscopy. Therefore, beyond acid-base chemistry, our findings imply that distinct adsorbate structures control antimicrobial trapping within clay nanopores, which can promote persistence in environmental matrices and stable delivery in biological systems. Copyright © 2017 Elsevier Inc. All rights reserved.

Prediction of protein loop conformations using multiscale modeling methods with physical energy scoring functions.

PubMed

Olson, Mark A; Feig, Michael; Brooks, Charles L

2008-04-15

This article examines ab initio methods for the prediction of protein loops by a computational strategy of multiscale conformational sampling and physical energy scoring functions. Our approach consists of initial sampling of loop conformations from lattice-based low-resolution models followed by refinement using all-atom simulations. To allow enhanced conformational sampling, the replica exchange method was implemented. Physical energy functions based on CHARMM19 and CHARMM22 parameterizations with generalized Born (GB) solvent models were applied in scoring loop conformations extracted from the lattice simulations and, in the case of all-atom simulations, the ensemble of conformations were generated and scored with these models. Predictions are reported for 25 loop segments, each eight residues long and taken from a diverse set of 22 protein structures. We find that the simulations generally sampled conformations with low global root-mean-square-deviation (RMSD) for loop backbone coordinates from the known structures, whereas clustering conformations in RMSD space and scoring detected less favorable loop structures. Specifically, the lattice simulations sampled basins that exhibited an average global RMSD of 2.21 +/- 1.42 A, whereas clustering and scoring the loop conformations determined an RMSD of 3.72 +/- 1.91 A. Using CHARMM19/GB to refine the lattice conformations improved the sampling RMSD to 1.57 +/- 0.98 A and detection to 2.58 +/- 1.48 A. We found that further improvement could be gained from extending the upper temperature in the all-atom refinement from 400 to 800 K, where the results typically yield a reduction of approximately 1 A or greater in the RMSD of the detected loop. Overall, CHARMM19 with a simple pairwise GB solvent model is more efficient at sampling low-RMSD loop basins than CHARMM22 with a higher-resolution modified analytical GB model; however, the latter simulation method provides a more accurate description of the all-atom energy surface, yet demands a much greater computational cost. (c) 2007 Wiley Periodicals, Inc.

Metadynamics Simulations Reveal a Na+ Independent Exiting Path of Galactose for the Inward-Facing Conformation of vSGLT

PubMed Central

Bisha, Ina; Rodriguez, Alex; Laio, Alessandro; Magistrato, Alessandra

2014-01-01

Sodium-Galactose Transporter (SGLT) is a secondary active symporter which accumulates sugars into cells by using the electrochemical gradient of Na+ across the membrane. Previous computational studies provided insights into the release process of the two ligands (galactose and sodium ion) into the cytoplasm from the inward-facing conformation of Vibrio parahaemolyticus sodium/galactose transporter (vSGLT). Several aspects of the transport mechanism of this symporter remain to be clarified: (i) a detailed kinetic and thermodynamic characterization of the exit path of the two ligands is still lacking; (ii) contradictory conclusions have been drawn concerning the gating role of Y263; (iii) the role of Na+ in modulating the release path of galactose is not clear. In this work, we use bias-exchange metadynamics simulations to characterize the free energy profile of the galactose and Na+ release processes toward the intracellular side. Surprisingly, we find that the exit of Na+ and galactose is non-concerted as the cooperativity between the two ligands is associated to a transition that is not rate limiting. The dissociation barriers are of the order of 11–12 kcal/mol for both the ion and the substrate, in line with kinetic information concerning this type of transporters. On the basis of these results we propose a branched six-state alternating access mechanism, which may be shared also by other members of the LeuT-fold transporters. PMID:25522004

Linear Response Path Following: A Molecular Dynamics Method To Simulate Global Conformational Changes of Protein upon Ligand Binding.

PubMed

Tamura, Koichi; Hayashi, Shigehiko

2015-07-14

Molecular functions of proteins are often fulfilled by global conformational changes that couple with local events such as the binding of ligand molecules. High molecular complexity of proteins has, however, been an obstacle to obtain an atomistic view of the global conformational transitions, imposing a limitation on the mechanistic understanding of the functional processes. In this study, we developed a new method of molecular dynamics (MD) simulation called the linear response path following (LRPF) to simulate a protein's global conformational changes upon ligand binding. The method introduces a biasing force based on a linear response theory, which determines a local reaction coordinate in the configuration space that represents linear coupling between local events of ligand binding and global conformational changes and thus provides one with fully atomistic models undergoing large conformational changes without knowledge of a target structure. The overall transition process involving nonlinear conformational changes is simulated through iterative cycles consisting of a biased MD simulation with an updated linear response force and a following unbiased MD simulation for relaxation. We applied the method to the simulation of global conformational changes of the yeast calmodulin N-terminal domain and successfully searched out the end conformation. The atomistically detailed trajectories revealed a sequence of molecular events that properly lead to the global conformational changes and identified key steps of local-global coupling that induce the conformational transitions. The LRPF method provides one with a powerful means to model conformational changes of proteins such as motors and transporters where local-global coupling plays a pivotal role in their functional processes.

Replica exchange molecular dynamics simulations provide insight into substrate recognition by small heat shock proteins.

PubMed

Patel, Sunita; Vierling, Elizabeth; Tama, Florence

2014-06-17

The small heat shock proteins (sHSPs) are a virtually ubiquitous and diverse group of molecular chaperones that can bind and protect unfolding proteins from irreversible aggregation. It has been suggested that intrinsic disorder of the N-terminal arm (NTA) of sHSPs is important for substrate recognition. To investigate conformations of the NTA that could recognize substrates we performed replica exchange molecular dynamics simulations. Behavior at normal and stress temperatures of the dimeric building blocks of dodecameric HSPs from wheat (Ta16.9) and pea (Ps18.1) were compared because they display high sequence similarity, but Ps18.1 is more efficient in binding specific substrates. In our simulations, the NTAs of the dimer are flexible and dynamic; however, rather than exhibiting highly extended conformations they retain considerable α-helical character and contacts with the conserved α-crystallin domain (ACD). Network analysis and clustering methods reveal that there are two major conformational forms designated either "open" or "closed" based on the relative position of the two NTAs and their hydrophobic solvent accessible surface area. The equilibrium constant for the closed to open transition is significantly different for Ta16.9 and Ps18.1, with the latter showing more open conformations at elevated temperature correlated with its more effective chaperone activity. In addition, the Ps18.1 NTAs have more hydrophobic solvent accessible surface than those of Ta16.9. NTA hydrophobic patches are comparable in size to the area buried in many protein-protein interactions, which would enable sHSPs to bind early unfolding intermediates. Reduced interactions of the Ps18.1 NTAs with each other and with the ACD contribute to the differences in dynamics and hydrophobic surface area of the two sHSPs. These data support a major role for the conformational equilibrium of the NTA in substrate binding and indicate features of the NTA that contribute to sHSP chaperone efficiency. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Structure-guided design and functional characterization of an artificial red light-regulated guanylate/adenylate cyclase for optogenetic applications.

PubMed

Etzl, Stefan; Lindner, Robert; Nelson, Matthew D; Winkler, Andreas

2018-06-08

Genetically targeting biological systems to control cellular processes with light is the concept of optogenetics. Despite impressive developments in this field, underlying molecular mechanisms of signal transduction of the employed photoreceptor modules are frequently not sufficiently understood to rationally design new optogenetic tools. Here, we investigate the requirements for functional coupling of red light-sensing phytochromes with non-natural enzymatic effectors by creating a series of constructs featuring the Deinococcus radiodurans bacteriophytochrome linked to a Synechocystis guanylate/adenylate cyclase. Incorporating characteristic structural elements important for cyclase regulation in our designs, we identified several red light-regulated fusions with promising properties. We provide details of one light-activated construct with low dark-state activity and high dynamic range that outperforms previous optogenetic tools in vitro and expands our in vivo toolkit, as demonstrated by manipulation of Caenorhabditis elegans locomotor activity. The full-length crystal structure of this phytochrome-linked cyclase revealed molecular details of photoreceptor-effector coupling, highlighting the importance of the regulatory cyclase element. Analysis of conformational dynamics by hydrogen-deuterium exchange in different functional states enriched our understanding of phytochrome signaling and signal integration by effectors. We found that light-induced conformational changes in the phytochrome destabilize the coiled-coil sensor-effector linker, which releases the cyclase regulatory element from an inhibited conformation, increasing cyclase activity of this artificial system. Future designs of optogenetic functionalities may benefit from our work, indicating that rational considerations for the effector improve the rate of success of initial designs to obtain optogenetic tools with superior properties. © 2018 Etzl et al.

Molecular dynamics simulation of β₂-microglobulin in denaturing and stabilizing conditions.

PubMed

Fogolari, Federico; Corazza, Alessandra; Varini, Nicola; Rotter, Matteo; Gumral, Devrim; Codutti, Luca; Rennella, Enrico; Viglino, Paolo; Bellotti, Vittorio; Esposito, Gennaro

2011-03-01

β₂-Microglobulin has been a model system for the study of fibril formation for 20 years. The experimental study of β₂-microglobulin structure, dynamics, and thermodynamics in solution, at atomic detail, along the pathway leading to fibril formation is difficult because the onset of disorder and aggregation prevents signal resolution in Nuclear Magnetic Resonance experiments. Moreover, it is difficult to characterize conformers in exchange equilibrium. To gain insight (at atomic level) on processes for which experimental information is available at molecular or supramolecular level, molecular dynamics simulations have been widely used in the last decade. Here, we use molecular dynamics to address three key aspects of β₂-microglobulin, which are known to be relevant to amyloid formation: (1) 60 ns molecular dynamics simulations of β₂-microglobulin in trifluoroethanol and in conditions mimicking low pH are used to study the behavior of the protein in environmental conditions that are able to trigger amyloid formation; (2) adaptive biasing force molecular dynamics simulation is used to force cis-trans isomerization at Proline 32 and to calculate the relative free energy in the folded and unfolded state. The native-like trans-conformer (known as intermediate 2 and determining the slow phase of refolding), is simulated for 10 ns, detailing the possible link between cis-trans isomerization and conformational disorder; (3) molecular dynamics simulation of highly concentrated doxycycline (a molecule able to suppress fibril formation) in the presence of β₂-microglobulin provides details of the binding modes of the drug and a rationale for its effect. Copyright © 2010 Wiley-Liss, Inc.

Water-mediated electron transfer between protein redox centers.

PubMed

Migliore, Agostino; Corni, Stefano; Felice, Rosa Di; Molinari, Elisa

2007-04-12

Recent experimental and theoretical investigations show that water molecules between or near redox partners can significantly affect their electron-transfer (ET) properties. Here we study the effects of intervening water molecules on the electron self-exchange reaction of azurin (Az), by performing a conformational sampling on the water medium and by using a newly developed ab initio method to calculate transfer integrals between molecular redox sites. We show that the insertion of water molecules at the interface between the copper active sites of Az dimers slightly increases the overall ET rate, while some favorable water conformations can considerably enhance the ET kinetics. These features are traced back to the interplay of two competing factors: the electrostatic interaction between the water and protein subsystems (mainly opposing the ET process for the water arrangements drawn from MD simulations) and the effectiveness of water in mediating ET coupling pathways. Such an interplay provides a physical basis for the found absence of correlation between the electronic couplings derived through ab initio electronic structure calculations and the related quantities obtained through the Empirical Pathways (EP) method. In fact, the latter does not account for electrostatic effects on the transfer integrals. Thus, we conclude that the water-mediated electron tunneling is not controlled by the geometry of a single physical pathway. We discuss the results in terms of the interplay between different ET pathways controlled by the conformational changes of one of the water molecules via its electrostatic influence. Finally, we examine the dynamical effects of the interfacial water and check the validity of the Condon approximation.

Levels of Conformity to Islamic Values and the Process of Identification.

ERIC Educational Resources Information Center

Nassir, Balkis

This study was conducted to measure the conformity levels and the identification process among university women students in an Islamic culture. Identity/conformity tests and costume identity tests were administered to 129 undergraduate female students at King Abdulaziz University in Saudi Arabia. The Photographic Costume Identity Test and the…

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2012-12-11

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Numerical Sensitivity of Trajectories Across Conformational Energy Hypersurfaces from Geometry Optimized Molecular Orbital Calculations: AM1, MNDO, and MINDO/3

DTIC Science & Technology

1988-01-01

relationship studies, it became corn- surfaces are being traversed, the molecule monly believed that compounds with higher h can go along different paths on...1975). AMPAC, and consanguineous programs 15. W. Thiel, Quantum Chem. Prog. Exchange Cata- should be done with the tightest available log, 11, 353

DNA Recombinase Proteins, their Function and Structure in the Active Form, a Computational Study

NASA Technical Reports Server (NTRS)

Carra, Claudio; Cucinotta, Francis A.

2007-01-01

Homologous recombination is a crucial sequence of reactions in all cells for the repair of double strand DNA (dsDNA) breaks. While it was traditionally considered as a means for generating genetic diversity, it is now known to be essential for restart of collapsed replication forks that have met a lesion on the DNA template (Cox et al., 2000). The central stage of this process requires the presence of the DNA recombinase protein, RecA in bacteria, RadA in archaea, or Rad51 in eukaryotes, which leads to an ATP-mediated DNA strand-exchange process. Despite many years of intense study, some aspects of the biochemical mechanism, and structure of the active form of recombinase proteins are not well understood. Our theoretical study is an attempt to shed light on the main structural and mechanistic issues encountered on the RecA of the e-coli, the RecA of the extremely radio resistant Deinococcus Radiodurans (promoting an inverse DNA strand-exchange repair), and the homolog human Rad51. The conformational changes are analyzed for the naked enzymes, and when they are linked to ATP and ADP. The average structures are determined over 2ns time scale of Langevian dynamics using a collision frequency of 1.0 ps(sup -1). The systems are inserted in an octahedron periodic box with a 10 Angstrom buffer of water molecules explicitly described by the TIP3P model. The corresponding binding free energies are calculated in an implicit solvent using the Poisson-Boltzmann solvent accessible surface area, MM-PBSA model. The role of the ATP is not only in stabilizing the interaction RecA-DNA, but its hydrolysis is required to allow the DNA strand-exchange to proceed. Furthermore, we extended our study, using the hybrid QM/MM method, on the mechanism of this chemical process. All the calculations were performed using the commercial code Amber 9.

SAIDE: A Semi-Automated Interface for Hydrogen/Deuterium Exchange Mass Spectrometry.

PubMed

Villar, Maria T; Miller, Danny E; Fenton, Aron W; Artigues, Antonio

2010-01-01

Deuterium/hydrogen exchange in combination with mass spectrometry (DH MS) is a sensitive technique for detection of changes in protein conformation and dynamics. Since temperature, pH and timing control are the key elements for reliable and efficient measurement of hydrogen/deuterium content in proteins and peptides, we have developed a small, semiautomatic interface for deuterium exchange that interfaces the HPLC pumps with a mass spectrometer. This interface is relatively inexpensive to build, and provides efficient temperature and timing control in all stages of enzyme digestion, HPLC separation and mass analysis of the resulting peptides. We have tested this system with a series of standard tryptic peptides reconstituted in a solvent containing increasing concentration of deuterium. Our results demonstrate the use of this interface results in minimal loss of deuterium due to back exchange during HPLC desalting and separation. For peptides reconstituted in a buffer containing 100% deuterium, and assuming that all amide linkages have exchanged hydrogen with deuterium, the maximum loss of deuterium content is only 17% of the label, indicating the loss of only one deuterium molecule per peptide.

SAIDE: A Semi-Automated Interface for Hydrogen/Deuterium Exchange Mass Spectrometry

PubMed Central

Villar, Maria T.; Miller, Danny E.; Fenton, Aron W.; Artigues, Antonio

2011-01-01

Deuterium/hydrogen exchange in combination with mass spectrometry (DH MS) is a sensitive technique for detection of changes in protein conformation and dynamics. Since temperature, pH and timing control are the key elements for reliable and efficient measurement of hydrogen/deuterium content in proteins and peptides, we have developed a small, semiautomatic interface for deuterium exchange that interfaces the HPLC pumps with a mass spectrometer. This interface is relatively inexpensive to build, and provides efficient temperature and timing control in all stages of enzyme digestion, HPLC separation and mass analysis of the resulting peptides. We have tested this system with a series of standard tryptic peptides reconstituted in a solvent containing increasing concentration of deuterium. Our results demonstrate the use of this interface results in minimal loss of deuterium due to back exchange during HPLC desalting and separation. For peptides reconstituted in a buffer containing 100% deuterium, and assuming that all amide linkages have exchanged hydrogen with deuterium, the maximum loss of deuterium content is only 17% of the label, indicating the loss of only one deuterium molecule per peptide. PMID:25309638

Conformational analysis of processivity clamps in solution demonstrates that tertiary structure does not correlate with protein dynamics.

PubMed

Fang, Jing; Nevin, Philip; Kairys, Visvaldas; Venclovas, Česlovas; Engen, John R; Beuning, Penny J

2014-04-08

The relationship between protein sequence, structure, and dynamics has been elusive. Here, we report a comprehensive analysis using an in-solution experimental approach to study how the conservation of tertiary structure correlates with protein dynamics. Hydrogen exchange measurements of eight processivity clamp proteins from different species revealed that, despite highly similar three-dimensional structures, clamp proteins display a wide range of dynamic behavior. Differences were apparent both for structurally similar domains within proteins and for corresponding domains of different proteins. Several of the clamps contained regions that underwent local unfolding with different half-lives. We also observed a conserved pattern of alternating dynamics of the α helices lining the inner pore of the clamps as well as a correlation between dynamics and the number of salt bridges in these α helices. Our observations reveal that tertiary structure and dynamics are not directly correlated and that primary structure plays an important role in dynamics. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Conformal Template and New Perspectives for Quantum Chromodynamics

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

Brodsky, Stanley J.; /SLAC

2007-03-06

Conformal symmetry provides a systematic approximation to QCD in both its perturbative and nonperturbative domains. One can use the AdS/CFT correspondence between Anti-de Sitter space and conformal gauge theories to obtain an analytically tractable approximation to QCD in the regime where the QCD coupling is large and constant. For example, there is an exact correspondence between the fifth-dimensional coordinate of AdS space and a specific impact variable which measures the separation of the quark constituents within the hadron in ordinary space-time. This connection allows one to compute the analytic form of the frame-independent light-front wavefunctions of mesons and baryons, themore » fundamental entities which encode hadron properties and allow the computation of exclusive scattering amplitudes. One can also use conformal symmetry as a template for perturbative QCD predictions where the effects of the nonzero beta function can be systematically included in the scale of the QCD coupling. This leads to fixing of the renormalization scale and commensurate scale relations which relate observables without scale or scheme ambiguity. The results are consistent with the renormalization group and the analytic connection of QCD to Abelian theory at N{sub C} {yields} 0. I also discuss a number of novel phenomenological features of QCD. Initial- and .nal-state interactions from gluon-exchange, normally neglected in the parton model, have a profound effect in QCD hard-scattering reactions, leading to leading-twist single-spin asymmetries, diffractive deep inelastic scattering, di.ractive hard hadronic reactions, the breakdown of the Lam Tung relation in Drell-Yan reactions, and nuclear shadowing and non-universal antishadowing--leading-twist physics not incorporated in the light-front wavefunctions of the target computed in isolation. I also discuss tests of hidden color in nuclear wavefunctions, the use of diffraction to materialize the Fock states of a hadronic projectile and test QCD color transparency, nonperturbative antisymmetric sea quark distributions, anomalous heavy quark e.ects, and the unexpected effects of direct higher-twist processes.« less

Conformation study of HA(306-318) antigenic peptide of the haemagglutinin influenza virus protein

NASA Astrophysics Data System (ADS)

Bertrand, A.; Brito, R. M.; Alix, A. J. P.; Lancelin, J. M.; Carvalho, R. A.; Geraldes, C. F. G. C.; Lakhdar-Ghazal, F.

2006-11-01

Several HLA-DR alleles present the immunodominant HA(306-318) peptide of haemagglutinin of the influenza virus to T cells. NMR data of the peptide in various water solutions exclude any α-helix or turn conformations. Circular dichroism and Fourier transform infrared spectroscopies indicate an estimated β-extended structure in water of 31% and 28%, respectively, with spectra shape similar to the ones observed for β-sheet containing proteins. The H/D amide exchange suggests a stable length-dependent interchain hydrogen-bonding. The partially β-extended conformation of HA(306-318) in solution might be close to the one found in HA(306-318)-HLA-DR1 complex. These results suggest different interconverting extended conformations of HA(306-318), depending on the microenvironment of the solution medium. This flexibility emphasizes the ability of some peptides to fit more easily the binding site of several HLA-DR molecules. Similar results were obtained on the HIV P25(263-277) peptide which has been previously shown to be a good DR1 binder. From a vibrational point of view, infrared Amide I frequencies of secondary structures in peptides were ascertained. As previously demonstrated for proteins in solution, Fourier transform infrared and circular dichroism spectroscopies appear to be valuable tools for conformational properties of peptides. Their use may contribute to the detection of peptide conformation-binding relationship which has to be further tested by biochemical and biological studies.

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

The mechanism of monomer transfer between two structurally distinct PrP oligomers

PubMed Central

Armiento, Aurora; Martin, Davy; Lepejova, Nad’a

2017-01-01

In mammals, Prion pathology refers to a class of infectious neuropathologies whose mechanism is based on the self-perpetuation of structural information stored in the pathological conformer. The characterisation of the PrP folding landscape has revealed the existence of a plethora of pathways conducing to the formation of structurally different assemblies with different biological properties. However, the biochemical interconnection between these diverse assemblies remains unclear. The PrP oligomerisation process leads to the formation of neurotoxic and soluble assemblies called O1 oligomers with a high size heterodispersity. By combining the measurements in time of size distribution and average size with kinetic models and data assimilation, we revealed the existence of at least two structurally distinct sets of assemblies, termed Oa and Ob, forming O1 assemblies. We propose a kinetic model representing the main processes in prion aggregation pathway: polymerisation, depolymerisation, and disintegration. The two groups interact by exchanging monomers through a disintegration process that increases the size of Oa. Our observations suggest that PrP oligomers constitute a highly dynamic population. PMID:28746342

The mechanism of monomer transfer between two structurally distinct PrP oligomers.

PubMed

Armiento, Aurora; Moireau, Philippe; Martin, Davy; Lepejova, Nad'a; Doumic, Marie; Rezaei, Human

2017-01-01

In mammals, Prion pathology refers to a class of infectious neuropathologies whose mechanism is based on the self-perpetuation of structural information stored in the pathological conformer. The characterisation of the PrP folding landscape has revealed the existence of a plethora of pathways conducing to the formation of structurally different assemblies with different biological properties. However, the biochemical interconnection between these diverse assemblies remains unclear. The PrP oligomerisation process leads to the formation of neurotoxic and soluble assemblies called O1 oligomers with a high size heterodispersity. By combining the measurements in time of size distribution and average size with kinetic models and data assimilation, we revealed the existence of at least two structurally distinct sets of assemblies, termed Oa and Ob, forming O1 assemblies. We propose a kinetic model representing the main processes in prion aggregation pathway: polymerisation, depolymerisation, and disintegration. The two groups interact by exchanging monomers through a disintegration process that increases the size of Oa. Our observations suggest that PrP oligomers constitute a highly dynamic population.

Minimal model for the secondary structures and conformational conversions in proteins

NASA Astrophysics Data System (ADS)

Imamura, Hideo

Better understanding of protein folding process can provide physical insights on the function of proteins and makes it possible to benefit from genetic information accumulated so far. Protein folding process normally takes place in less than seconds but even seconds are beyond reach of current computational power for simulations on a system of all-atom detail. Hence, to model and explore protein folding process it is crucial to construct a proper model that can adequately describe the physical process and mechanism for the relevant time scale. We discuss the reduced off-lattice model that can express _-helix and ?-hairpin conformations defined solely by a given sequence in order to investigate a protein folding mechanism of conformations such as a ?-hairpin and also to investigate conformational conversions in proteins. The first two chapters introduce and review essential concepts in protein folding modelling physical interaction in proteins, various simple models, and also review computational methods, in particular, the Metropolis Monte Carlo method, its dynamic interpretation and thermodynamic Monte Carlo algorithms. Chapter 3 describes the minimalist model that represents both _-helix and ?-sheet conformations using simple potentials. The native conformation can be specified by the sequence without particular conformational biases to a reference state. In Chapter 4, the model is used to investigate the folding mechanism of ?-hairpins exhaustively using the dynamic Monte Carlo and a thermodynamic Monte Carlo method an effcient combination of the multicanonical Monte Carlo and the weighted histogram analysis method. We show that the major folding pathways and folding rate depend on the location of a hydrophobic. The conformational conversions between _-helix and ?-sheet conformations are examined in Chapter 5 and 6. First, the conformational conversion due to mutation in a non-hydrophobic system and then the conformational conversion due to mutation with a hydrophobic pair at a different position at various temperatures are examined.

Chain Assembly and Disassembly Processes Differently Affect the Conformational Space of Ubiquitin Chains.

PubMed

Kniss, Andreas; Schuetz, Denise; Kazemi, Sina; Pluska, Lukas; Spindler, Philipp E; Rogov, Vladimir V; Husnjak, Koraljka; Dikic, Ivan; Güntert, Peter; Sommer, Thomas; Prisner, Thomas F; Dötsch, Volker

2018-02-06

Ubiquitination is the most versatile posttranslational modification. The information is encoded by linkage type as well as chain length, which are translated by ubiquitin binding domains into specific signaling events. Chain topology determines the conformational space of a ubiquitin chain and adds an additional regulatory layer to this ubiquitin code. In particular, processes that modify chain length will be affected by chain conformations as they require access to the elongation or cleavage sites. We investigated conformational distributions in the context of chain elongation and disassembly using pulsed electron-electron double resonance spectroscopy in combination with molecular modeling. Analysis of the conformational space of diubiquitin revealed conformational selection or remodeling as mechanisms for chain recognition during elongation or hydrolysis, respectively. Chain elongation to tetraubiquitin increases the sampled conformational space, suggesting that a high intrinsic flexibility of K48-linked chains may contribute to efficient proteasomal degradation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural basis for profilin-mediated actin nucleotide exchange

PubMed Central

Porta, Jason C.; Borgstahl, Gloria E.O.

2015-01-01

Actin is a ubiquitous eukaryotic protein that is responsible for cellular scaffolding, motility and division. The ability of actin to form a helical filament is the driving force behind these cellular activities. Formation of a filament is dependent the successful exchange of actin’s ADP for ATP. Mammalian profilin is a small actin binding protein that catalyzes the exchange of nucleotide and facilitates the addition of an actin monomer to a growing filament. Here, crystal structures of profilin:actin have been determined showing an actively exchanging ATP. The structural analysis shows how the binding of profilin to the barbed end of actin causes a rotation of the small domain relative to the large domain. This conformational change is propagated to the ATP site and causes a shift in the nucleotide loops which in turn causes a repositioning of Ca2+ to its canonical position as the cleft closes around ATP. Reversing the solvent exposure of Trp-356 is also involved in cleft closure. In addition, secondary calcium binding sites were identified. PMID:22366544

Predicting Three-Dimensional Conformations of Peptides Constructed of Only Glycine, Alanine, Aspartic Acid, and Valine

NASA Astrophysics Data System (ADS)

Oda, Akifumi; Fukuyoshi, Shuichi

2015-06-01

The GADV hypothesis is a form of the protein world hypothesis, which suggests that life originated from proteins (Lacey et al. 1999; Ikehara 2002; Andras 2006). In the GADV hypothesis, life is thought to have originated from primitive proteins constructed of only glycine, alanine, aspartic acid, and valine ([GADV]-proteins). In this study, the three-dimensional (3D) conformations of randomly generated short [GADV]-peptides were computationally investigated using replica-exchange molecular dynamics (REMD) simulations (Sugita and Okamoto 1999). Because the peptides used in this study consisted of only 20 residues each, they could not form certain 3D structures. However, the conformational tendencies of the peptides were elucidated by analyzing the conformational ensembles generated by REMD simulations. The results indicate that secondary structures can be formed in several randomly generated [GADV]-peptides. A long helical structure was found in one of the hydrophobic peptides, supporting the conjecture of the GADV hypothesis that many peptides aggregated to form peptide multimers with enzymatic activity in the primordial soup. In addition, these results indicate that REMD simulations can be used for the structural investigation of short peptides.

Multiple welding of long fiber epoxy vitrimer composites.

PubMed

Chabert, Erwan; Vial, Jérôme; Cauchois, Jean-Pierre; Mihaluta, Marius; Tournilhac, François

2016-05-25

Vitrimers appear as a new class of polymers that exhibit mechanical strength and are insoluble even at high temperatures, like thermosets, and yet, like thermoplastics, they are heat processable, recyclable and weldable. The question arises whether this welding property is maintained in composite materials made of more than 50 vol% of reinforcing fibers. In this paper, we quantitatively analyze the bond strength of epoxy vitrimer-based composite plates made by resin transfer molding and compare them to their non-vitrimer counterparts made of a standard thermoset epoxy. It is demonstrated that only epoxy vitrimer samples show substantial bond strength and the ability to be repeatedly welded thanks to the exchange reactions, which promote improved surface conformity and chemical bonding between the adherands at the joint interface. This opens the way towards joining composite parts without adhesives nor mechanical fasteners.

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Quantitative Predictions of Binding Free Energy Changes in Drug-Resistant Influenza Neuraminidase

DTIC Science & Technology

2012-08-30

drug resistance to two antiviral drugs, zanamivir and oseltamivir. We augmented molecular dynamics (MD) with Hamiltonian Replica Exchange and...conformations that are virtually identical to WT [10]. Molecular simulations that rigorously model the microscopic structure and thermodynamics PLOS...influenza neuraminidase (NA) that confer drug resistance to two antiviral drugs, zanamivir and oseltamivir. We augmented molecular dynamics (MD) with

Mapping allosteric connections from the receptor to the nucleotide-binding pocket of heterotrimeric G proteins

PubMed Central

Oldham, William M.; Van Eps, Ned; Preininger, Anita M.; Hubbell, Wayne L.; Hamm, Heidi E.

2007-01-01

Heterotrimeric G proteins function as molecular relays that mediate signal transduction from heptahelical receptors in the cell membrane to intracellular effector proteins. Crystallographic studies have demonstrated that guanine nucleotide exchange on the Gα subunit causes specific conformational changes in three key “switch” regions of the protein, which regulate binding to Gβγ subunits, receptors, and effector proteins. In the present study, nitroxide side chains were introduced at sites within the switch I region of Gαi to explore the structure and dynamics of this region throughout the G protein cycle. EPR spectra obtained for each of the Gα(GDP), Gα(GDP)βγ heterotrimer and Gα(GTPγS) conformations are consistent with the local environment observed in the corresponding crystal structures. Binding of the heterotrimer to activated rhodopsin to form the nucleotide-free (empty) complex, for which there is no crystal structure, causes prominent changes relative to the heterotrimer in the structure of switch I and contiguous sequences. The data identify a putative pathway of allosteric changes triggered by receptor binding and, together with previously published data, suggest elements of a mechanism for receptor-catalyzed nucleotide exchange. PMID:17463080

Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes

PubMed Central

Crepin, Thibaut; Shalak, Vyacheslav F.; Yaremchuk, Anna D.; Vlasenko, Dmytro O.; McCarthy, Andrew; Negrutskii, Boris S.; Tukalo, Michail A.; El'skaya, Anna V.

2014-01-01

Eukaryotic elongation factor eEF1A transits between the GTP- and GDP-bound conformations during the ribosomal polypeptide chain elongation. eEF1A*GTP establishes a complex with the aminoacyl-tRNA in the A site of the 80S ribosome. Correct codon–anticodon recognition triggers GTP hydrolysis, with subsequent dissociation of eEF1A*GDP from the ribosome. The structures of both the ‘GTP’- and ‘GDP’-bound conformations of eEF1A are unknown. Thus, the eEF1A-related ribosomal mechanisms were anticipated only by analogy with the bacterial homolog EF-Tu. Here, we report the first crystal structure of the mammalian eEF1A2*GDP complex which indicates major differences in the organization of the nucleotide-binding domain and intramolecular movements of eEF1A compared to EF-Tu. Our results explain the nucleotide exchange mechanism in the mammalian eEF1A and suggest that the first step of eEF1A*GDP dissociation from the 80S ribosome is the rotation of the nucleotide-binding domain observed after GTP hydrolysis. PMID:25326326

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

Free-energy landscape of a hyperstable RNA tetraloop

PubMed Central

Miner, Jacob C.; Chen, Alan A.; García, Angel E.

2016-01-01

We report the characterization of the energy landscape and the folding/unfolding thermodynamics of a hyperstable RNA tetraloop obtained through high-performance molecular dynamics simulations at microsecond timescales. Sampling of the configurational landscape is conducted using temperature replica exchange molecular dynamics over three isochores at high, ambient, and negative pressures to determine the thermodynamic stability and the free-energy landscape of the tetraloop. The simulations reveal reversible folding/unfolding transitions of the tetraloop into the canonical A-RNA conformation and the presence of two alternative configurations, including a left-handed Z-RNA conformation and a compact purine Triplet. Increasing hydrostatic pressure shows a stabilizing effect on the A-RNA conformation and a destabilization of the left-handed Z-RNA. Our results provide a comprehensive description of the folded free-energy landscape of a hyperstable RNA tetraloop and highlight the significant advances of all-atom molecular dynamics in describing the unbiased folding of a simple RNA secondary structure motif. PMID:27233937

Structure of the full-length glucagon class B G protein-coupled receptor

PubMed Central

Zhang, Haonan; Qiao, Anna; Yang, Dehua; Yang, Linlin; Dai, Antao; de Graaf, Chris; Reedtz-Runge, Steffen; Dharmarajan, Venkatasubramanian; Zhang, Hui; Han, Gye Won; Grant, Thomas D.; Sierra, Raymond G.; Weierstall, Uwe; Nelson, Garrett; Liu, Wei; Wu, Yanhong; Ma, Limin; Cai, Xiaoqing; Lin, Guangyao; Wu, Xiaoai; Geng, Zhi; Dong, Yuhui; Song, Gaojie; Griffin, Patrick R.; Lau, Jesper; Cherezov, Vadim; Yang, Huaiyu; Hanson, Michael A.; Stevens, Raymond C.; Zhao, Qiang; Jiang, Hualiang; Wang, Ming-Wei; Wu, Beili

2017-01-01

The human glucagon receptor (GCGR) belongs to the class B G protein-coupled receptor (GPCR) family and plays a key role in glucose homeostasis and the pathophysiology of type 2 diabetes. Here we report the 3.0 Å crystal structure of full-length GCGR containing both extracellular domain (ECD) and transmembrane domain (TMD) in an inactive conformation. The two domains are connected by a 12-residue segment termed the ‘stalk’, which adopts a β-strand conformation, instead of forming an α-helix as observed in the previously solved structure of GCGR-TMD. The first extracellular loop (ECL1) exhibits a β-hairpin conformation and interacts with the stalk to form a compact β-sheet structure. Hydrogen/deuterium exchange, disulfide cross-linking and molecular dynamics studies suggest that the stalk and ECL1 play critical roles in modulating peptide ligand binding and receptor activation. These insights into the full-length GCGR structure deepen our understanding about the signaling mechanisms of class B GPCRs. PMID:28514451

Effect of single-strand break on branch migration and folding dynamics of Holliday junctions.

PubMed

Palets, Dmytro; Lushnikov, Alexander Y; Karymov, Mikhail A; Lyubchenko, Yuri L

2010-09-22

The Holliday junction (HJ), or four-way junction, is a central intermediate state of DNA for homologous genetic recombination and other genetic processes such as replication and repair. Branch migration is the process by which the exchange of homologous DNA regions occurs, and it can be spontaneous or driven by proteins. Unfolding of the HJ is required for branch migration. Our previous single-molecule fluorescence studies led to a model according to which branch migration is a stepwise process consisting of consecutive migration and folding steps. Folding of the HJ in one of the folded conformations terminates the branch migration phase. At the same time, in the unfolded state HJ rapidly migrates over entire homology region of the HJ in one hop. This process can be affected by irregularities in the DNA double helical structure, so mismatches almost terminate a spontaneous branch migration. Single-stranded breaks or nicks are the most ubiquitous defects in the DNA helix; however, to date, their effect on the HJ branch migration has not been studied. In addition, although nicked HJs are specific substrates for a number of enzymes involved in DNA recombination and repair, the role of this substrate specificity remains unclear. Our main goal in this work was to study the effect of nicks on the efficiency of HJ branch migration and the dynamics of the HJ. To accomplish this goal, we applied two single-molecule methods: atomic force microscopy and fluorescence resonance energy transfer. The atomic force microscopy data show that the nick does not prevent branch migration, but it does decrease the probability that the HJ will pass the DNA lesion. The single-molecule fluorescence resonance energy transfer approaches were instrumental in detailing the effects of nicks. These studies reveal a dramatic change of the HJ dynamics. The nick changes the structure and conformational dynamics of the junctions, leading to conformations with geometries that are different from those for the intact HJ. On the basis of these data, we propose a model of branch migration in which the propensity of the junction to unfold decreases the lifetimes of folded states, thereby increasing the frequency of junction fluctuations between the folded states. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Effects of forcefield and sampling method in all-atom simulations of inherently disordered proteins: Application to conformational preferences of human amylin

PubMed Central

Peng, Enxi; Todorova, Nevena

2017-01-01

Although several computational modelling studies have investigated the conformational behaviour of inherently disordered protein (IDP) amylin, discrepancies in identifying its preferred solution conformations still exist between various forcefields and sampling methods used. Human islet amyloid polypeptide has long been a subject of research, both experimentally and theoretically, as the aggregation of this protein is believed to be the lead cause of type-II diabetes. In this work, we present a systematic forcefield assessment using one of the most advanced non-biased sampling techniques, Replica Exchange with Solute Tempering (REST2), by comparing the secondary structure preferences of monomeric amylin in solution. This study also aims to determine the ability of common forcefields to sample a transition of the protein from a helical membrane bound conformation into the disordered solution state of amylin. Our results demonstrated that the CHARMM22* forcefield showed the best ability to sample multiple conformational states inherent for amylin. It is revealed that REST2 yielded results qualitatively consistent with experiments and in quantitative agreement with other sampling methods, however far more computationally efficiently and without any bias. Therefore, combining an unbiased sampling technique such as REST2 with a vigorous forcefield testing could be suggested as an important step in developing an efficient and robust strategy for simulating IDPs. PMID:29023509

Cholesterol dependent conformational exchange of the C-terminal domain of the influenza A M2 protein

PubMed Central

Kim, Sangwoo S.; Upshur, Mary Alice; Saotome, Kei; Sahu, Indra D.; McCarrick, Robert M.; Feix, Jimmy B.; Lorigan, Gary A.; Howard, Kathleen P.

2016-01-01

The C-terminal amphipathic helix of the influenza A M2 protein plays a critical cholesterol dependent role in viral budding. To provide atomic-level detail on the impact cholesterol has on the conformation of M2 protein, we spin-labeled sites right before and within the C-terminal amphipathic helix of the M2 protein. We studied the spin-labeled M2 proteins in membranes both with and without cholesterol. We used a multipronged site-directed spin-label electron paramagnetic resonance (SDSL-EPR) approach and collected data on line shapes, relaxation rates, accessibility of sites to the membrane, and distances between symmetry related sites within the tetrameric protein. We demonstrate that the C-terminal amphipathic helix of M2 populates at least two conformations in POPC/POPG 4:1 bilayers. Furthermore, we show that the conformational state that becomes more populated in the presence of cholesterol is less dynamic, less membrane buried, and more tightly packed than the other state. Cholesterol dependent changes in M2 could be attributed to the changes cholesterol induces in bilayer properties and/or direct binding of cholesterol to the protein. We propose a model consistent with all our experimental data that suggests that the predominant conformation we observe in the presence of cholesterol is relevant for the understanding of viral budding. PMID:26569023

Effects of forcefield and sampling method in all-atom simulations of inherently disordered proteins: Application to conformational preferences of human amylin.

PubMed

Peng, Enxi; Todorova, Nevena; Yarovsky, Irene

2017-01-01

Although several computational modelling studies have investigated the conformational behaviour of inherently disordered protein (IDP) amylin, discrepancies in identifying its preferred solution conformations still exist between various forcefields and sampling methods used. Human islet amyloid polypeptide has long been a subject of research, both experimentally and theoretically, as the aggregation of this protein is believed to be the lead cause of type-II diabetes. In this work, we present a systematic forcefield assessment using one of the most advanced non-biased sampling techniques, Replica Exchange with Solute Tempering (REST2), by comparing the secondary structure preferences of monomeric amylin in solution. This study also aims to determine the ability of common forcefields to sample a transition of the protein from a helical membrane bound conformation into the disordered solution state of amylin. Our results demonstrated that the CHARMM22* forcefield showed the best ability to sample multiple conformational states inherent for amylin. It is revealed that REST2 yielded results qualitatively consistent with experiments and in quantitative agreement with other sampling methods, however far more computationally efficiently and without any bias. Therefore, combining an unbiased sampling technique such as REST2 with a vigorous forcefield testing could be suggested as an important step in developing an efficient and robust strategy for simulating IDPs.

Structural Insights into the Calcium-Mediated Allosteric Transition in the C-Terminal Domain of Calmodulin from Nuclear Magnetic Resonance Measurements.

PubMed

Kukic, Predrag; Lundström, Patrik; Camilloni, Carlo; Evenäs, Johan; Akke, Mikael; Vendruscolo, Michele

2016-01-12

Calmodulin is a two-domain signaling protein that becomes activated upon binding cooperatively two pairs of calcium ions, leading to large-scale conformational changes that expose its binding site. Despite significant advances in understanding the structural biology of calmodulin functions, the mechanistic details of the conformational transition between closed and open states have remained unclear. To investigate this transition, we used a combination of molecular dynamics simulations and nuclear magnetic resonance (NMR) experiments on the Ca(2+)-saturated E140Q C-terminal domain variant. Using chemical shift restraints in replica-averaged metadynamics simulations, we obtained a high-resolution structural ensemble consisting of two conformational states and validated such an ensemble against three independent experimental data sets, namely, interproton nuclear Overhauser enhancements, (15)N order parameters, and chemical shift differences between the exchanging states. Through a detailed analysis of this structural ensemble and of the corresponding statistical weights, we characterized a calcium-mediated conformational transition whereby the coordination of Ca(2+) by just one oxygen of the bidentate ligand E140 triggers a concerted movement of the two EF-hands that exposes the target binding site. This analysis provides atomistic insights into a possible Ca(2+)-mediated activation mechanism of calmodulin that cannot be achieved from static structures alone or from ensemble NMR measurements of the transition between conformations.

Structural and Functional Characterization of a Hole-Hole Homodimer Variant in a "Knob-Into-Hole" Bispecific Antibody.

PubMed

Zhang, Hui-Min; Li, Charlene; Lei, Ming; Lundin, Victor; Lee, Ho Young; Ninonuevo, Milady; Lin, Kevin; Han, Guanghui; Sandoval, Wendy; Lei, Dongsheng; Ren, Gang; Zhang, Jennifer; Liu, Hongbin

2017-12-19

Bispecific antibodies have great potential to be the next-generation biotherapeutics due to their ability to simultaneously recognize two different targets. Compared to conventional monoclonal antibodies, knob-into-hole bispecific antibodies face unique challenges in production and characterization due to the increase in variant possibilities, such as homodimerization in covalent and noncovalent forms. In this study, a storage- and pH-sensitive hydrophobic interaction chromatography (HIC) profile change was observed for the hole-hole homodimer, and the multiple HIC peaks were explored and shown to be conformational isomers. We combined traditional analytical methods with hydrogen/deuterium exchange mass spectrometry (HDX MS), native mass spectrometry, and negative-staining electron microscopy to comprehensively characterize the hole-hole homodimer. HDX MS revealed conformational changes at the resolution of a few amino acids overlapping the C H 2-C H 3 domain interface. Conformational heterogeneity was also assessed by HDX MS isotopic distribution. The hole-hole homodimer was demonstrated to adopt a more homogeneous conformational distribution during storage. This conformational change is likely caused by a lack of C H 3 domain dimerization (due to the three "hole" point mutations), resulting in a unique storage- and pH-dependent conformational destabilization and refolding of the hole-hole homodimer Fc. Compared with the hole-hole homodimer under different storage conditions, the bispecific heterodimer, guided by the knob-into-hole assembly, proved to be a stable conformation with homogeneous distribution, confirming its high quality as a desired therapeutic. Functional studies by antigen binding and neonatal Fc receptor (FcRn) binding correlated very well with the structural characterization. Comprehensive interpretation of the results has provided a better understanding of both the homodimer variant and the bispecific molecule.

A novel lipid transfer protein from the pea Pisum sativum: isolation, recombinant expression, solution structure, antifungal activity, lipid binding, and allergenic properties.

PubMed

Bogdanov, Ivan V; Shenkarev, Zakhar O; Finkina, Ekaterina I; Melnikova, Daria N; Rumynskiy, Eugene I; Arseniev, Alexander S; Ovchinnikova, Tatiana V

2016-04-30

Plant lipid transfer proteins (LTPs) assemble a family of small (7-9 kDa) ubiquitous cationic proteins with an ability to bind and transport lipids as well as participate in various physiological processes including defense against phytopathogens. They also form one of the most clinically relevant classes of plant allergens. Nothing is known to date about correlation between lipid-binding and IgE-binding properties of LTPs. The garden pea Pisum sativum is widely consumed crop and important allergenic specie of the legume family. This work is aimed at isolation of a novel LTP from pea seeds and characterization of its structural, functional, and allergenic properties. Three novel lipid transfer proteins, designated as Ps-LTP1-3, were found in the garden pea Pisum sativum, their cDNA sequences were determined, and mRNA expression levels of all the three proteins were measured at different pea organs. Ps-LTP1 was isolated for the first time from the pea seeds, and its complete amino acid sequence was determined. The protein exhibits antifungal activity and is a membrane-active compound that causes a leakage from artificial liposomes. The protein binds various lipids including bioactive jasmonic acid. Spatial structure of the recombinant uniformly (13)C,(15)N-labelled Ps-LTP1 was solved by heteronuclear NMR spectroscopy. In solution the unliganded protein represents the mixture of two conformers (relative populations ~ 85:15) which are interconnected by exchange process with characteristic time ~ 100 ms. Hydrophobic residues of major conformer form a relatively large internal tunnel-like lipid-binding cavity (van der Waals volume comes up to ~1000 Å(3)). The minor conformer probably corresponds to the protein with the partially collapsed internal cavity. For the first time conformational heterogeneity in solution was shown for an unliganded plant lipid transfer protein. Heat denaturation profile and simulated gastrointestinal digestion assay showed that Ps-LTP1 displayed a high thermal and digestive proteolytic resistance proper for food allergens. The reported structural and immunological findings seem to describe Ps-LTP1 as potential cross-reactive allergen in LTP-sensitized patients, mostly Pru p 3(+) ones. Similarly to allergenic LTPs the potential IgE-binding epitope of Ps-LTP1 is located near the proposed entrance into internal cavity and could be involved in lipid-binding.

On the Solutions of Two-Extended Principal Conformal Toda Theory

NASA Astrophysics Data System (ADS)

Chao, L.; Hou, B. Y.

1994-02-01

The solutions of the two-extended principal conformal Toda theory (2-EPCT theory, also called bosonic superconformal Toda theory) are constructed in two different ways: (1) Leznov-Saveliev algebraic analysis and (2) the associated chiral embedding surface. The first approach gives rise to the general solution in terms of appropriate matrix elements in different fundamental representations of the underlying Lie algebra, whilst the second one leads to a special solution in the form of Wronski determinants and their co-minors, and it gives an explicit geometrical interpretation of the WZNW → 2-EPCT reduction. The key points of both approaches are the chiral vectors derived recently by the authors, which constitute a closed exchange algebra of the theory.

Roles of Bridging Ligand Topology and Conformation in Controlling Exchange Interactions between Paramagnetic Molybdenum Fragments in Dinuclear and Trinuclear Complexes.

PubMed

Ung VÂ, V&acaron;n Ân; Cargill Thompson, Alexander M. W.; Bardwell, David A.; Gatteschi, Dante; Jeffery, John C.; McCleverty, Jon A.; Totti, Federico; Ward, Michael D.

1997-07-30

The magnetic properties of two series of dinuclear complexes, and one trinuclear complex, have been examined as a function of the bridging pathway between the metal centers. The first series of dinuclear complexes is [{Mo(V)(O)(Tp)Cl}(2)(&mgr;-OO)], where "OO" is [1,4-O(C(6)H(4))(n)O](2)(-) (n = 1, 1; n = 2, 3), [4,4'-O(C(6)H(3)-2-Me)(2)O](2)(-) (4), or [1,3-OC(6)H(4)O](2)(-) (2) [Tp = tris(3,5-dimethylpyrazolyl)hydroborate]. The second series of dinuclear complexes is [{Mo(I)(NO)(Tp)Cl}(2)(&mgr;-NN)], where "NN" is 4,4'-bipyridyl (5), 3,3'-dimethyl-4,4'-bipyridine (6), 3,8-phenanthroline (7), or 2,7-diazapyrene (8). The trinuclear complex is [{Mo(V)(O)(Tp)Cl}(3)(1,3,5-C(6)H(3)O(3))] (9), whose crystal structure was determined [9.5CH(2)Cl(2): C(56)H(81)B(3)Cl(13)Mo(3)N(18)O(6); monoclinic, P2(1)/n; a = 13.443, b = 41.46(2), c = 14.314(6) Å; beta = 93.21(3) degrees; V = 7995(5) Å(3); Z = 4; R(1) = 0.106]. In these complexes, the sign and magnitude of the exchange coupling constant J is clearly related to both the topology and the conformation of the bridging ligand [where J is derived from H = -JS(1)().S(2)() for 1-8 and H = -J(S(1)().S(2)() + S(2)().S(3)() + S(1)().S(3)()) for 9]. The values are as follows: 1, -80 cm(-)(1); 2, +9.8 cm(-)(1); 3, -13.2 cm(-)(1); 4, -2.8 cm(-)(1); 5, -33 cm(-)(1); 6, -3.5 cm(-)(1); 7, -35.6 cm(-)(1); 8, -35.0 cm(-)(1); 9, +14.4 cm(-)(1). In particular the following holds: (1) J is negative (antiferromagnetic exchange) across the para-substituted bridges ligands of 1 and 3-8 but positive (ferromagnetic exchange) across the meta-substituted bridging ligands of 2 and 9. (2) J decreases in magnitude dramatically as the bridging ligand conformation changes from planar to twisted (compare 3 and 4, or 6 and 8). These observations are consistent with a spin-polarization mechanism for the exchange interaction, propagated across the pi-system of the bridging ligand by via overlap of bridging ligand p(pi) orbitals with the d(pi) magnetic orbitals of the metals. The EPR spectrum of 9 is characteristic of a quartet species and shows weak Deltam(s) = 2 and Deltam(s) = 3 transitions at one-half and one-third, respectively, of the field strength of the principal Deltam(s) = 1 component.

Application of statistical process control and process capability analysis procedures in orbiter processing activities at the Kennedy Space Center

NASA Technical Reports Server (NTRS)

Safford, Robert R.; Jackson, Andrew E.; Swart, William W.; Barth, Timothy S.

1994-01-01

Successful ground processing at KSC requires that flight hardware and ground support equipment conform to specifications at tens of thousands of checkpoints. Knowledge of conformance is an essential requirement for launch. That knowledge of conformance at every requisite point does not, however, enable identification of past problems with equipment, or potential problem areas. This paper describes how the introduction of Statistical Process Control and Process Capability Analysis identification procedures into existing shuttle processing procedures can enable identification of potential problem areas and candidates for improvements to increase processing performance measures. Results of a case study describing application of the analysis procedures to Thermal Protection System processing are used to illustrate the benefits of the approaches described in the paper.

Peptide:lipid ratio and membrane surface charge determine the mechanism of action of the antimicrobial peptide BP100. Conformational and functional studies.

PubMed

Manzini, Mariana C; Perez, Katia R; Riske, Karin A; Bozelli, José C; Santos, Talita L; da Silva, Marcia A; Saraiva, Greice K V; Politi, Mario J; Valente, Ana P; Almeida, Fábio C L; Chaimovich, Hernan; Rodrigues, Magali A; Bemquerer, Marcelo P; Schreier, Shirley; Cuccovia, Iolanda M

2014-07-01

The cecropin-melittin hybrid antimicrobial peptide BP100 (H-KKLFKKILKYL-NH2) is selective for Gram-negative bacteria, negatively charged membranes, and weakly hemolytic. We studied BP100 conformational and functional properties upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG). CD and NMR spectra showed that upon binding to PG-containing LUVs BP100 acquires α-helical conformation, the helix spanning residues 3-11. Theoretical analyses indicated that the helix is amphipathic and surface-seeking. CD and dynamic light scattering data evinced peptide and/or vesicle aggregation, modulated by peptide:lipid ratio and PG content. BP100 decreased the absolute value of the zeta potential (ζ) of LUVs with low PG contents; for higher PG, binding was analyzed as an ion-exchange process. At high salt, BP100-induced LUVS leakage requires higher peptide concentration, indicating that both electrostatic and hydrophobic interactions contribute to peptide binding. While a gradual release took place at low peptide:lipid ratios, instantaneous loss occurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs confirmed BP100-promoted disruption of negatively charged membranes. The mechanism of action of BP100 is determined by both peptide:lipid ratio and negatively charged lipid content. While gradual release results from membrane perturbation by a small number of peptide molecules giving rise to changes in acyl chain packing, lipid clustering (leading to membrane defects), and/or membrane thinning, membrane disruption results from a sequence of events - large-scale peptide and lipid clustering, giving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism. Copyright © 2014 Elsevier B.V. All rights reserved.

Thermoswitchable Janus Gold Nanoparticles with Stimuli-Responsive Hydrophilic Polymer Brushes.

PubMed

Niu, Xiaoqin; Ran, Fen; Chen, Limei; Lu, Gabriella Jia-En; Hu, Peiguang; Deming, Christopher P; Peng, Yi; Rojas-Andrade, Mauricio D; Chen, Shaowei

2016-05-03

Well-defined thermoswitchable Janus gold nanoparticles with stimuli-responsive hydrophilic polymer brushes were fabricated by combining ligand exchange reactions and the Langmuir technique. Stimuli-responsive polydi(ethylene glycol) methyl ether methacrylate was prepared by addition-fragmentation chain-transfer polymerization. The polymer brushes were then anchored onto the nanoparticle surface by interfacial ligand exchange reactions with hexanethiolate-protected gold nanoparticles, leading to the formation of a hydrophilic (polymer) hemisphere and a hydrophobic (hexanethiolate) one. The resulting Janus nanoparticles showed temperature-switchable wettability, hydrophobicity at high temperatures, and hydrophilicity at low temperatures, due to thermally induced conformational transition of the polymer ligands. The results further highlight the importance of interfacial engineering in the deliberate functionalization of nanoparticle materials.

HDX Workbench: Software for the Analysis of H/D Exchange MS Data

NASA Astrophysics Data System (ADS)

Pascal, Bruce D.; Willis, Scooter; Lauer, Janelle L.; Landgraf, Rachelle R.; West, Graham M.; Marciano, David; Novick, Scott; Goswami, Devrishi; Chalmers, Michael J.; Griffin, Patrick R.

2012-09-01

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is an established method for the interrogation of protein conformation and dynamics. While the data analysis challenge of HDX-MS has been addressed by a number of software packages, new computational tools are needed to keep pace with the improved methods and throughput of this technique. To address these needs, we report an integrated desktop program titled HDX Workbench, which facilitates automation, management, visualization, and statistical cross-comparison of large HDX data sets. Using the software, validated data analysis can be achieved at the rate of generation. The application is available at the project home page http://hdx.florida.scripps.edu.

Tube support grid and spacer therefor

DOEpatents

Ringsmuth, Richard J.; Kaufman, Jay S.

1986-01-01

A tube support grid and spacers therefor provide radially inward preloading of heat exchange tubes to minimize stress upon base welds due to differential thermal expansion. The grid comprises a concentric series of rings and spacers with opposing concave sides for conforming to the tubes and V-shaped ends to provide resilient flexibility. The flexibility aids in assembly and in transmitting seismic vibrations from the tubes to a shroud. The tube support grid may be assembled in place to achieve the desired inwardly radial preloading of the heat exchange tubes. Tab and slot assembly further minimizes stresses in the system. The radii of the grid rings may be preselected to effect the desired radially inward preloading.

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Supramolecular organization of calix[4]pyrrole with a methyl-trialkylammonium anion exchanger leads to remarkable reversal of selectivity for sulfate extraction vs. nitrate

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

Borman, Christopher J.; Custelcean, Radu; Hay, Ben P.

Here, meso-Octamethylcalix[4]pyrrole (C4P) enhances sulfate selectivity in solvent extraction by Aliquat 336N, an effect ascribed to the supramolecular preorganization and thermodynamic stability imparted by insertion of the methyl group of the Aliquat cation into the cup of C4P in its cone conformation.

Free-Energy Profiles of Membrane Insertion of the M2 Transmembrane Peptide from Influenza A Virus

DTIC Science & Technology

2008-12-01

ABSTRACT The insertion of the M2 transmembrane peptide from influenza A virus into a membrane has been studied with molecular - dynamics simulations ...performed replica-exchange molecular - dynamics simulations with umbrella-sampling techniques to characterize the probability distribution and conformation...atomic- detailed molecular dynamics (MD) simulation techniques represent a valuable complementary methodology to inves- tigate membrane-insertion of

NMR Chemical Exchange as a Probe for Ligand-Binding Kinetics in a Theophylline-Binding RNA Aptamer

PubMed Central

Latham, Michael P.; Zimmermann, Grant R.; Pardi, Arthur

2009-01-01

The apparent on- and off-rate constants for theophylline binding to its RNA aptamer in the absence of Mg2+ were determined here by 2D 1H-1H NMR ZZ-exchange spectroscopy. Analysis of the build-up rate of the exchange cross peaks for several base-paired imino protons in the RNA yielded an apparent kon of 600 M-1 s-1. This small apparent kon results from the free RNA existing as a dynamic equilibrium of inactive states rapidly interconverting with a low population of active species. The data here indicate that the RNA aptamer employs a conformational selection mechanism for binding theophylline in the absence of Mg2+. The kinetic data here also explain a very unusual property of this RNA-theophylline system, slow exchange on the NMR chemical shift timescale for a weak-binding complex. To our knowledge, it is unprecedented to have such a weak binding complex (Kd ≈ 3.0 mM at 15 °C) show slow exchange on the NMR chemical shift timescale, but the results clearly demonstrate that slow exchange and weak binding are readily rationalized by a small kon. Comparisons with other ligand-receptor interactions are presented. PMID:19317486

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.

Replica Exchange Gaussian Accelerated Molecular Dynamics: Improved Enhanced Sampling and Free Energy Calculation.

PubMed

Huang, Yu-Ming M; McCammon, J Andrew; Miao, Yinglong

2018-04-10

Through adding a harmonic boost potential to smooth the system potential energy surface, Gaussian accelerated molecular dynamics (GaMD) provides enhanced sampling and free energy calculation of biomolecules without the need of predefined reaction coordinates. This work continues to improve the acceleration power and energy reweighting of the GaMD by combining the GaMD with replica exchange algorithms. Two versions of replica exchange GaMD (rex-GaMD) are presented: force constant rex-GaMD and threshold energy rex-GaMD. During simulations of force constant rex-GaMD, the boost potential can be exchanged between replicas of different harmonic force constants with fixed threshold energy. However, the algorithm of threshold energy rex-GaMD tends to switch the threshold energy between lower and upper bounds for generating different levels of boost potential. Testing simulations on three model systems, including the alanine dipeptide, chignolin, and HIV protease, demonstrate that through continuous exchanges of the boost potential, the rex-GaMD simulations not only enhance the conformational transitions of the systems but also narrow down the distribution width of the applied boost potential for accurate energetic reweighting to recover biomolecular free energy profiles.

Multiscale Kinetic Modeling Reveals an Ensemble of Cl–/H+ Exchange Pathways in ClC-ec1 Antiporter

PubMed Central

2018-01-01

Despite several years of research, the ion exchange mechanisms in chloride/proton antiporters and many other coupled transporters are not yet understood at the molecular level. Here, we present a novel approach to kinetic modeling and apply it to ion exchange in ClC-ec1. Our multiscale kinetic model is developed by (1) calculating the state-to-state rate coefficients with reactive and polarizable molecular dynamics simulations, (2) optimizing these rates in a global kinetic network, and (3) predicting new electrophysiological results. The model shows that the robust Cl:H exchange ratio (2.2:1) can indeed arise from kinetic coupling without large protein conformational changes, indicating a possible facile evolutionary connection to chloride channels. The E148 amino acid residue is shown to couple chloride and proton transport through protonation-dependent blockage of the central anion binding site and an anion-dependent pKa value, which influences proton transport. The results demonstrate how an ensemble of different exchange pathways, as opposed to a single series of transitions, culminates in the macroscopic observables of the antiporter, such as transport rates, chloride/proton stoichiometry, and pH dependence. PMID:29332400

Assessing the Chemical Accuracy of Protein Structures via Peptide Acidity

PubMed Central

Anderson, Janet S.; Hernández, Griselda; LeMaster, David M.

2012-01-01

Although the protein native state is a Boltzmann conformational ensemble, practical applications often require a representative model from the most populated region of that distribution. The acidity of the backbone amides, as reflected in hydrogen exchange rates, is exquisitely sensitive to the surrounding charge and dielectric volume distribution. For each of four proteins, three independently determined X-ray structures of differing crystallographic resolution were used to predict exchange for the static solvent-exposed amide hydrogens. The average correlation coefficients range from 0.74 for ubiquitin to 0.93 for Pyrococcus furiosus rubredoxin, reflecting the larger range of experimental exchange rates exhibited by the latter protein. The exchange prediction errors modestly correlate with the crystallographic resolution. MODELLER 9v6-derived homology models at ~60% sequence identity (36% identity for chymotrypsin inhibitor CI2) yielded correlation coefficients that are ~0.1 smaller than for the cognate X-ray structures. The most recently deposited NOE-based ubiquitin structure and the original NMR structure of CI2 fail to provide statistically significant predictions of hydrogen exchange. However, the more recent RECOORD refinement study of CI2 yielded predictions comparable to the X-ray and homology model-based analyses. PMID:23182463

Surface-Tension Replica-Exchange Molecular Dynamics Method for Enhanced Sampling of Biological Membrane Systems.

PubMed

Mori, Takaharu; Jung, Jaewoon; Sugita, Yuji

2013-12-10

Conformational sampling is fundamentally important for simulating complex biomolecular systems. The generalized-ensemble algorithm, especially the temperature replica-exchange molecular dynamics method (T-REMD), is one of the most powerful methods to explore structures of biomolecules such as proteins, nucleic acids, carbohydrates, and also of lipid membranes. T-REMD simulations have focused on soluble proteins rather than membrane proteins or lipid bilayers, because explicit membranes do not keep their structural integrity at high temperature. Here, we propose a new generalized-ensemble algorithm for membrane systems, which we call the surface-tension REMD method. Each replica is simulated in the NPγT ensemble, and surface tensions in a pair of replicas are exchanged at certain intervals to enhance conformational sampling of the target membrane system. We test the method on two biological membrane systems: a fully hydrated DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine) lipid bilayer and a WALP23-POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) membrane system. During these simulations, a random walk in surface tension space is realized. Large-scale lateral deformation (shrinking and stretching) of the membranes takes place in all of the replicas without collapse of the lipid bilayer structure. There is accelerated lateral diffusion of DPPC lipid molecules compared with conventional MD simulation, and a much wider range of tilt angle of the WALP23 peptide is sampled due to large deformation of the POPC lipid bilayer and through peptide-lipid interactions. Our method could be applicable to a wide variety of biological membrane systems.

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A novel restraint spraying-Conform process for manufacturing hypereutectic Al-Si alloy with enhanced properties

NASA Astrophysics Data System (ADS)

Chen, Y. G.; Yang, H.; Zhang, B. Q.; Liu, Y. L.; Yin, J. C.; Wei, W.; Zhong, Y.

2017-02-01

A novel restraint spraying-Conform (RS-C) process, which directly combines spraying with Conform to process metals in one step, has been proposed. Al-20Si alloy selected as experimental material was successfully fabricated by the RS-C process. The microstructures were dominated with fine and uniform primary silicon phases. The tensile strength and elongation to failure of the Al-20Si alloy were 204 MPa and 7.2% respectively after the RS-C process. The wear resistance of the processed Al-20Si alloy was increased significantly, about 1.7 times over the as-cast ingot. The experimental results indicate that RS-C is a promising near net shape forming technology.

Online hydrogen/deuterium exchange performed in the ion mobility cell of a hybrid mass spectrometer.

PubMed

Nagy, Kornél; Redeuil, Karine; Rezzi, Serge

2009-11-15

The present paper describes the performance of online, gas-phase hydrogen/deuterium exchange implemented in the ion mobility cell of a quadrupole time-of-flight mass spectrometer. Deuterium oxide and deuterated methanol were utilized to create deuterated vapor that is introduced into the ion mobility region of the mass spectrometer. Hydrogen/deuterium exchange occurs spontaneously in the milliseconds time frame without the need of switching the instrument into ion mobility mode. The exchange was studied in case of low molecular weight molecules and proteins. The observed number of exchanged hydrogens was equal to the number of theoretically exchangeable hydrogens for all low molecular weight compounds. This method needs only minimal instrumental modifications, is simple, cheap, environment friendly, compatible with ultraperformance liquid chromatography, and can be implemented on commercially available instruments. It does not compromise choice of liquid chromatographic solvents and accurate mass or parallel-fragmentation (MS(E)) methods. The performance of this method was compared to that of conventional alternatives where the deuterated solvent is introduced into the cone gas of the instrument. Although the degree of exchange was similar between the two methods, the "cone gas method" requires 10 times higher deuterated solvent volumes (50 muL/min) and offers reduced sensitivity in the tandem mass spectrometry (MS/MS) mode. The presented method is suggested as a standard future element of mass spectrometers to aid online structural characterization of unknowns and to study conformational changes of proteins with hydrogen/deuterium exchange.

Conformal doping of topographic silicon structures using a radial line slot antenna plasma source

NASA Astrophysics Data System (ADS)

Ueda, Hirokazu; Ventzek, Peter L. G.; Oka, Masahiro; Horigome, Masahiro; Kobayashi, Yuuki; Sugimoto, Yasuhiro; Nozawa, Toshihisa; Kawakami, Satoru

2014-06-01

Fin extension doping for 10 nm front end of line technology requires ultra-shallow high dose conformal doping. In this paper, we demonstrate a new radial line slot antenna plasma source based doping process that meets these requirements. Critical to reaching true conformality while maintaining fin integrity is that the ion energy be low and controllable, while the dose absorption is self-limited. The saturated dopant later is rendered conformal by concurrent amorphization and dopant containing capping layer deposition followed by stabilization anneal. Dopant segregation assists in driving dopants from the capping layer into the sub silicon surface. Very high resolution transmission electron microscopy-Energy Dispersive X-ray spectroscopy, used to prove true conformality, was achieved. We demonstrate these results using an n-type arsenic based plasma doping process on 10 to 40 nm high aspect ratio fins structures. The results are discussed in terms of the different types of clusters that form during the plasma doping process.

Using the conformity to masculine norms inventory to work with men in a clinical setting.

PubMed

Mahalik, James R; Talmadge, W Tracy; Locke, Benjamin D; Scott, Ryan P J

2005-06-01

Given that gender roles are increasingly viewed as salient in clinical work with men, this article describes a process of exploring masculine gender roles with male clients in therapy by using the Conformity to Masculine Norms Inventory (CMNI). Specifically, this article (a) discusses how men's degree of conformity to masculine norms may be connected to a variety of benefits and costs, (b) describes the CMNI as a tool that can be used to explore men's degree of conformity to masculine norms, (c) describes a process by which to use the CMNI to explore the relevance of men's masculine selves to their presenting concerns, and (d) illustrates the process with a case example. As such, the paper is intended to provide a systematic procedure for clinicians working with men who want to explore the benefits and costs that both conformity, and nonconformity, to specific masculinity norms brings for male clients. Copyright 2005 Wiley Periodicals, Inc.

Reciprocity Outperforms Conformity to Promote Cooperation.

PubMed

Romano, Angelo; Balliet, Daniel

2017-10-01

Evolutionary psychologists have proposed two processes that could give rise to the pervasiveness of human cooperation observed among individuals who are not genetically related: reciprocity and conformity. We tested whether reciprocity outperformed conformity in promoting cooperation, especially when these psychological processes would promote a different cooperative or noncooperative response. To do so, across three studies, we observed participants' cooperation with a partner after learning (a) that their partner had behaved cooperatively (or not) on several previous trials and (b) that their group members had behaved cooperatively (or not) on several previous trials with that same partner. Although we found that people both reciprocate and conform, reciprocity has a stronger influence on cooperation. Moreover, we found that conformity can be partly explained by a concern about one's reputation-a finding that supports a reciprocity framework.

Fibrillar dimer formation of islet amyloid polypeptides

DOE PAGES

Chiu, Chi -cheng; de Pablo, Juan J.

2015-05-08

Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimentalmore » and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.« less

Interaction of packaging motor with the polymerase complex of dsRNA bacteriophage

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

Lisal, Jiri; Kainov, Denis E.; Lam, TuKiet T.

2006-07-20

Many viruses employ molecular motors to package their genomes into preformed empty capsids (procapsids). In dsRNA bacteriophages the packaging motor is a hexameric ATPase P4, which is an integral part of the multisubunit procapsid. Structural and biochemical studies revealed a plausible RNA-translocation mechanism for the isolated hexamer. However, little is known about the structure and regulation of the hexamer within the procapsid. Here we use hydrogen-deuterium exchange and mass spectrometry to delineate the interactions of the P4 hexamer with the bacteriophage phi12 procapsid. P4 associates with the procapsid via its C-terminal face. The interactions also stabilize subunit interfaces within themore » hexamer. The conformation of the virus-bound hexamer is more stable than the hexamer in solution, which is prone to spontaneous ring openings. We propose that the stabilization within the viral capsid increases the packaging processivity and confers selectivity during RNA loading.« less

Structures of glycans bound to receptors from saturation transfer difference (STD) NMR spectroscopy: quantitative analysis by using CORCEMA-ST.

PubMed

Enríquez-Navas, Pedro M; Guzzi, Cinzia; Muñoz-García, Juan C; Nieto, Pedro M; Angulo, Jesús

2015-01-01

Glycan-receptor interactions are of fundamental relevance for a large number of biological processes, and their kinetics properties (medium/weak binding affinities) make them appropriated to be studied by ligand observed NMR techniques, among which saturation transfer difference (STD) NMR spectroscopy has been shown to be a very robust and powerful approach. The quantitative analysis of the results from a STD NMR study of a glycan-receptor interaction is essential to be able to translate the resulting spectral intensities into a 3D molecular model of the complex. This chapter describes how to carry out such a quantitative analysis by means of the Complete Relaxation and Conformational Exchange Matrix Approach for STD NMR (CORCEMA-ST), in general terms, and an example of a previous work on an antibody-glycan interaction is also shown.

Fibrillar dimer formation of islet amyloid polypeptides

NASA Astrophysics Data System (ADS)

Chiu, Chi-cheng; de Pablo, Juan J.

2015-09-01

Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 - 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 - 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

Molecular Characteristics and Biological Functions of Surface-Active and Surfactant Proteins.

PubMed

Sunde, Margaret; Pham, Chi L L; Kwan, Ann H

2017-06-20

Many critical biological processes take place at hydrophobic:hydrophilic interfaces, and a wide range of organisms produce surface-active proteins and peptides that reduce surface and interfacial tension and mediate growth and development at these boundaries. Microorganisms produce both small lipid-associated peptides and amphipathic proteins that allow growth across water:air boundaries, attachment to surfaces, predation, and improved bioavailability of hydrophobic substrates. Higher-order organisms produce surface-active proteins with a wide variety of functions, including the provision of protective foam environments for vulnerable reproductive stages, evaporative cooling, and gas exchange across airway membranes. In general, the biological functions supported by these diverse polypeptides require them to have an amphipathic nature, and this is achieved by a diverse range of molecular structures, with some proteins undergoing significant conformational change or intermolecular association to generate the structures that are surface active.

Benchmarking all-atom simulations using hydrogen exchange

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

Skinner, John J.; Yu, Wookyung; Gichana, Elizabeth K.

We are now able to fold small proteins reversibly to their native structures [Lindorff-Larsen K, Piana S, Dror RO, Shaw DE (2011) Science 334(6055):517–520] using long-time molecular dynamics (MD) simulations. Our results indicate that modern force fields can reproduce the energy surface near the native structure. In this paper, to test how well the force fields recapitulate the other regions of the energy surface, MD trajectories for a variant of protein G are compared with data from site-resolved hydrogen exchange (HX) and other biophysical measurements. Because HX monitors the breaking of individual H-bonds, this experimental technique identifies the stability andmore » H-bond content of excited states, thus enabling quantitative comparison with the simulations. Contrary to experimental findings of a cooperative, all-or-none unfolding process, the simulated denatured state ensemble, on average, is highly collapsed with some transient or persistent native 2° structure. The MD trajectories of this protein G variant and other small proteins exhibit excessive intramolecular H-bonding even for the most expanded conformations, suggesting that the force fields require improvements in describing H-bonding and backbone hydration. Finally and moreover, these comparisons provide a general protocol for validating the ability of simulations to accurately capture rare structural fluctuations.« less

Spectroscopic studies of bacteriorhodopsin fragments dissolved in organic solution.

PubMed Central

Torres, J; Padrós, E

1995-01-01

Fourier transform infrared and UV fourth-derivative spectroscopies were used to study the secondary structure of bacteriorhodopsin and its chymotryptic and one of the sodium borohydride fragments dissolved in chloroform-methanol (1:1, v/v), 0.1 M LiClO4. The C1 fragment (helices C, D, E, F, and G) showed an alpha-helical content of about 53%, whereas C2 (helices A and B) had about 60%, and B2 (helices F and G) about 65% alpha-helix. The infrared main band indicated differences in alpha-helical properties between these fragments. These techniques were also used to obtain information on the interactions among helices. According to the results obtained from the hydrogen/deuterium exchange kinetics, about 40% of the amide protons of C2 are particularly protected against exchange, whereas for the C1 fragment this process is unexpectedly fast. UV fourth-derivative spectra of these samples were used to obtain information about the environment of Trp side chains. The results showed that the Trp residues of C2 are more shielded from the solvent than those of C1 or B2. The results of this work indicate that the specific interactions existing between the transmembrane segments induce different types of helical conformations in native bacteriorhodopsin. PMID:7612847

Adsorption of bovine serum albumin on nano and bulk oxide particles in deionized water.

PubMed

Song, Lei; Yang, Kun; Jiang, Wei; Du, Peng; Xing, Baoshan

2012-06-01

In this work, the influence of particle size and surface functional groups on the adsorption behavior of bovine serum albumin (BSA) by three types of oxide nanoparticles (NPs), TiO(2) (50±5 nm), SiO(2) (30±5 nm), and Al(2)O(3) (150±5 nm for α type and 60±5 nm for γ type) was investigated in deionized water, in order to explore their interaction mechanisms without competitive influence of other ions. Bulkparticles (BPs) were also used for comparison with NPs. BSA adsorption maxima on oxide particles were controlled by the surface area and hydrogen content, while adsorption process was primarily induced by electrostatic interaction, hydrophobic interaction and ligand exchange between BSA and oxide surfaces. With the increase of hydrogen content, the BSA adsorption mechanism switched from mainly hydrophobic interaction to hydrogen bonding and ligand exchange. Calculations, based on surface area and BSA size, suggested that a multilayer of BSA covered on α-Al(2)O(3), and single layer on the other oxide particle surfaces. BPs led to greater conformational change of BSA molecules after the adsorption on the surfaces of oxide particles though NPs adsorbed more BSA than BPs. Copyright © 2012 Elsevier B.V. All rights reserved.

Benchmarking all-atom simulations using hydrogen exchange

DOE PAGES

Skinner, John J.; Yu, Wookyung; Gichana, Elizabeth K.; ...

2014-10-27

We are now able to fold small proteins reversibly to their native structures [Lindorff-Larsen K, Piana S, Dror RO, Shaw DE (2011) Science 334(6055):517–520] using long-time molecular dynamics (MD) simulations. Our results indicate that modern force fields can reproduce the energy surface near the native structure. In this paper, to test how well the force fields recapitulate the other regions of the energy surface, MD trajectories for a variant of protein G are compared with data from site-resolved hydrogen exchange (HX) and other biophysical measurements. Because HX monitors the breaking of individual H-bonds, this experimental technique identifies the stability andmore » H-bond content of excited states, thus enabling quantitative comparison with the simulations. Contrary to experimental findings of a cooperative, all-or-none unfolding process, the simulated denatured state ensemble, on average, is highly collapsed with some transient or persistent native 2° structure. The MD trajectories of this protein G variant and other small proteins exhibit excessive intramolecular H-bonding even for the most expanded conformations, suggesting that the force fields require improvements in describing H-bonding and backbone hydration. Finally and moreover, these comparisons provide a general protocol for validating the ability of simulations to accurately capture rare structural fluctuations.« less

Atropisomerism about Aryl-C(sp(3)) Bonds: Conformational Behavior of Substituted Phenylcyclohexanes in Solution.

PubMed

Flos, Manon; Lameiras, Pedro; Denhez, Clément; Mirand, Catherine; Berber, Hatice

2016-03-18

A catalytic hydrogenation of cannabidiol derivatives known as phenylcyclohexenes was used to prepare epimeric (1R,1S) and/or rotameric (M,P) phenylcyclohexanes. The reaction is diastereoselective, in favor of the 1S epimer, when large groups are attached to the phenyl ring. For each epimer, variable-temperature NMR experiments, including EXSY spectroscopy and DFT calculations, were used to determine the activation energies of the conformational exchange arising from the restricted rotation about the aryl-C(sp(3)) bond that led to two unequally populated rotamers. The conformational preference arises essentially from steric interactions between substituents vicinal to the pivot bond. The conformers of epimers (1S)-2e,f show high rotational barriers of up to 92 kJ mol(-1), unlike those of (1R)-2e,f and with much lower barriers of ∼72 kJ mol(-1). The height of the barriers not only depends on the substituents at the axis of chirality but also is influenced by the position of a methyl group on the monoterpene ring. The feature most favorable to high rotational barriers is when the methyl at C1 lies equatorially. This additional substituent effect, highlighted for the first time, seems fundamental to allowing atropisomerism in hindered ortho-substituted phenylcyclohexanes.

Quantum chemical calculation (electronic and topologic) and experimental (FT-IR, FT-Raman and UV) analysis of isonicotinic acid N-oxide

NASA Astrophysics Data System (ADS)

Karaca, Caglar; Atac, Ahmet; Karabacak, Mehmet

2015-04-01

In this work, the molecular conformation, vibrational and electronic analysis of isonicotinic acid N-oxide (iso-NANO) were presented in the ground state using experimental techniques (FT-IR, FT-Raman and UV) and density functional theory (DFT) employing B3LYP exchange correlation with the 6-311++G(d,p) basis set. The geometry optimization and energies associated possible two conformers (Rot-I and Rot-II) were computed. The vibrational spectra were calculated and fundamental vibrations were assigned on the basis of the total energy distribution (TED) of the vibrational modes, calculated with scaled quantum mechanics (SQM) method and PQS program. The obtained structures were analyzed with the Atoms in Molecules (AIMs) methodology. The computational results diagnose the most stable conformer of iso-NANO as the Rot-I form. Total density of state (TDOS) and partial density of state (PDOS) and also overlap population density of state (OPDOS) diagrams analysis for the most stable conformer (Rot-I) were calculated using the same method. Thermodynamic properties (heat capacity, entropy and enthalpy) of the title compound at different temperatures were calculated. As a result, the optimized geometry and calculated spectroscopic data show a good agreement with the experimental results.

Interpretation of IR and Raman spectra of dopamine neurotransmitter and effect of hydrogen bond in HCl

NASA Astrophysics Data System (ADS)

Yadav, T.; Mukherjee, V.

2018-05-01

The potential energy scanning with respect to the different dihedral angles were performed to search possible numbers of dopamine (neutral) conformers and further, fifteen conformers of dopamine were identified on the basis of energy minima. Vibrational frequencies were calculated for all the conformers of dopamine. Density functional theory was employed to carry out all the computations. The exchange correlation functional B3LYP and the basis set 6-31++G(d,p) were included in DFT calculation. The FTIR and FT-Raman spectra of dopamine hydrochloride were also recorded in the spectral region 400-4000 cm-1 and 50-4000 cm-1 respectively. The normal coordinate analysis was also performed to scale DFT calculated force constants and to calculate potential energy distributions. The detailed vibrational spectral analysis and the assignments of the bands, done on the best-fit basis comparison of the experimentally obtained and theoretically calculated IR and Raman spectra, match quite well indicating DFT calculations as very accurate source of normal mode assignments. The interaction of the most stable conformer of dopamine with HCl was also studied to know the effect of hydrogen bond on its geometry and dynamics. The stability of the dopamine in isolated and protonated forms arising from hyperconjugative interactions was also analyzed by natural bond orbital analysis.

Insights on Na(+) binding and conformational dynamics in multidrug and toxic compound extrusion transporter NorM.

PubMed

Song, Jianing; Ji, Changge; Zhang, John Z H

2014-02-01

MATE (multidrug and toxic compound extrusion) transporter proteins mediate metabolite transport in plants and multidrug resistance in bacteria and mammals. MATE transporter NorM from Vibrio cholerae is an antiporter that is driven by Na+ gradient to extrude the substrates. To understand the molecular mechanism of Na+-substrate exchange, molecular dynamics simulation was performed to study conformational changes of both wild-type and mutant NorM with and without cation bindings. Our results show that NorM is able to bind two Na(+) ions simultaneously, one to each of the carboxylic groups of E255 and D371 in the binding pocket. Furthermore, this di-Na(+) binding state is likely more efficient for conformational changes of NorM_VC toward the inward-facing conformation than single-Na(+) binding state. The observation of two Na(+) binding sites of NorM_VC is consistent with the previous study that two sites for ion binding (denoted as Na1/Na2 sites) are found in the transporter LeuT and BetP, another two secondary transporters. Taken together, our findings shed light on the structure rearrangements of NorM on Na(+) binding and enrich our knowledge of the transport mechanism of secondary transporters. Copyright © 2013 Wiley Periodicals, Inc.

Active-State Model of a Dopamine D2 Receptor - Gαi Complex Stabilized by Aripiprazole-Type Partial Agonists

PubMed Central

Kling, Ralf C.; Tschammer, Nuska; Lanig, Harald; Clark, Timothy; Gmeiner, Peter

2014-01-01

Partial agonists exhibit a submaximal capacity to enhance the coupling of one receptor to an intracellular binding partner. Although a multitude of studies have reported different ligand-specific conformations for a given receptor, little is known about the mechanism by which different receptor conformations are connected to the capacity to activate the coupling to G-proteins. We have now performed molecular-dynamics simulations employing our recently described active-state homology model of the dopamine D2 receptor-Gαi protein-complex coupled to the partial agonists aripiprazole and FAUC350, in order to understand the structural determinants of partial agonism better. We have compared our findings with our model of the D2R-Gαi-complex in the presence of the full agonist dopamine. The two partial agonists are capable of inducing different conformations of important structural motifs, including the extracellular loop regions, the binding pocket and, in particular, intracellular G-protein-binding domains. As G-protein-coupling to certain intracellular epitopes of the receptor is considered the key step of allosterically triggered nucleotide-exchange, it is tempting to assume that impaired coupling between the receptor and the G-protein caused by distinct ligand-specific conformations is a major determinant of partial agonist efficacy. PMID:24932547

Pauling and Corey's alpha-pleated sheet structure may define the prefibrillar amyloidogenic intermediate in amyloid disease.

PubMed

Armen, Roger S; DeMarco, Mari L; Alonso, Darwin O V; Daggett, Valerie

2004-08-10

Transthyretin, beta(2)-microglobulin, lysozyme, and the prion protein are four of the best-characterized proteins implicated in amyloid disease. Upon partial acid denaturation, these proteins undergo conformational change into an amyloidogenic intermediate that can self-assemble into amyloid fibrils. Many experiments have shown that pH-mediated changes in structure are required for the formation of the amyloidogeneic intermediate, but it has proved impossible to characterize these conformational changes at high resolution using experimental means. To probe these conformational changes at atomic resolution, we have performed molecular dynamics simulations of these proteins at neutral and low pH. In low-pH simulations of all four proteins, we observe the formation of alpha-pleated sheet secondary structure, which was first proposed by L. Pauling and R. B. Corey [(1951) Proc. Natl. Acad. Sci. USA 37, 251-256]. In all beta-sheet proteins, transthyretin and beta(2)-microglobulin, alpha-pleated sheet structure formed over the strands that are highly protected in hydrogen-exchange experiments probing amyloidogenic conditions. In lysozyme and the prion protein, alpha-sheets formed in the specific regions of the protein implicated in the amyloidogenic conversion. We propose that the formation of alpha-pleated sheet structure may be a common conformational transition in amyloidosis.

Pauling and Corey's α-pleated sheet structure may define the prefibrillar amyloidogenic intermediate in amyloid disease

PubMed Central

Armen, Roger S.; DeMarco, Mari L.; Alonso, Darwin O. V.; Daggett, Valerie

2004-01-01

Transthyretin, β2-microglobulin, lysozyme, and the prion protein are four of the best-characterized proteins implicated in amyloid disease. Upon partial acid denaturation, these proteins undergo conformational change into an amyloidogenic intermediate that can self-assemble into amyloid fibrils. Many experiments have shown that pH-mediated changes in structure are required for the formation of the amyloidogeneic intermediate, but it has proved impossible to characterize these conformational changes at high resolution using experimental means. To probe these conformational changes at atomic resolution, we have performed molecular dynamics simulations of these proteins at neutral and low pH. In low-pH simulations of all four proteins, we observe the formation of α-pleated sheet secondary structure, which was first proposed by L. Pauling and R. B. Corey [(1951) Proc. Natl. Acad. Sci. USA 37, 251–256]. In all β-sheet proteins, transthyretin and β2-microglobulin, α-pleated sheet structure formed over the strands that are highly protected in hydrogen-exchange experiments probing amyloidogenic conditions. In lysozyme and the prion protein, α-sheets formed in the specific regions of the protein implicated in the amyloidogenic conversion. We propose that the formation of α-pleated sheet structure may be a common conformational transition in amyloidosis. PMID:15280548

Effective Application of Bicelles for Conformational Analysis of G Protein-Coupled Receptors by Hydrogen/Deuterium Exchange Mass Spectrometry

NASA Astrophysics Data System (ADS)

Duc, Nguyen Minh; Du, Yang; Thorsen, Thor S.; Lee, Su Youn; Zhang, Cheng; Kato, Hideaki; Kobilka, Brian K.; Chung, Ka Young

2015-05-01

G protein-coupled receptors (GPCRs) have important roles in physiology and pathology, and 40% of drugs currently on the market target GPCRs for the treatment of various diseases. Because of their therapeutic importance, the structural mechanism of GPCR signaling is of great interest in the field of drug discovery. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for analyzing ligand binding sites, the protein-protein interaction interface, and conformational changes of proteins. However, its application to GPCRs has been limited for various reasons, including the hydrophobic nature of GPCRs and the use of detergents in their preparation. In the present study, we tested the application of bicelles as a means of solubilizing GPCRs for HDX-MS studies. GPCRs (e.g., β2-adrenergic receptor [β2AR], μ-opioid receptor, and protease-activated receptor 1) solubilized in bicelles produced better sequence coverage (greater than 90%) than GPCRs solubilized in n-dodecyl-β-D-maltopyranoside (DDM), suggesting that bicelles are a more effective method of solubilization for HDX-MS studies. The HDX-MS profile of β2AR in bicelles showed that transmembrane domains (TMs) undergo lower deuterium uptake than intracellular or extracellular regions, which is consistent with the fact that the TMs are highly ordered and embedded in bicelles. The overall HDX-MS profiles of β2AR solubilized in bicelles and in DDM were similar except for intracellular loop 3. Interestingly, we detected EX1 kinetics, an important phenomenon in protein dynamics, at the C-terminus of TM6 in β2AR. In conclusion, we suggest the application of bicelles as a useful method for solubilizing GPCRs for conformational analysis by HDX-MS.

Replica Exchange Molecular Dynamics Study of Dimerization in Prion Protein: Multiple Modes of Interaction and Stabilization.

PubMed

Chamachi, Neharika G; Chakrabarty, Suman

2016-08-04

The pathological forms of prions are known to be a result of misfolding, oligomerization, and aggregation of the cellular prion. While the mechanism of misfolding and aggregation in prions has been widely studied using both experimental and computational tools, the structural and energetic characterization of the dimer form have not garnered as much attention. On one hand dimerization can be the first step toward a nucleation-like pathway to aggregation, whereas on the other hand it may also increase the conformational stability preventing self-aggregation. In this work, we have used extensive all-atom replica exchange molecular dynamics simulations of both monomer and dimer forms of a mouse prion protein to understand the structural, dynamic, and thermodynamic stability of dimeric prion as compared to the monomeric form. We show that prion proteins can dimerize spontaneously being stabilized by hydrophobic interactions as well as intermolecular hydrogen bonding and salt bridge formation. We have computed the conformational free energy landscapes for both monomer and dimer forms to compare the thermodynamic stability and misfolding pathways. We observe large conformational heterogeneity among the various modes of interactions between the monomers and the strong intermolecular interactions may lead to as high as 20% β-content. The hydrophobic regions in helix-2, surrounding coil regions, terminal regions along with the natively present β-sheet region appear to actively participate in prion-prion intermolecular interactions. Dimerization seems to considerably suppress the inherent dynamic instability observed in monomeric prions, particularly because the regions of structural frustration constitute the dimer interface. Further, we demonstrate an interesting reversible coupling between the Q160-G131 interaction (which leads to inhibition of β-sheet extension) and the G131-V161 H-bond formation.

Conformational flexibility of arginine-82 as source for the heterogeneous and pH-dependent kinetics of the primary proton transfer step in the bacteriorhodopsin photocycle: An electrostatic model

NASA Astrophysics Data System (ADS)

Scharnagl, Christina; Fischer, Sighart F.

1996-11-01

We use equilibrium thermodynamic concepts to relate protein conformational and protonation substates and their pH-dependent population to kinetic schemes for the rise of the M intermediate in the photocycle of bacteriorhodopsin. Conformational flexibility of arginine R82 is described by a two-state model. The analysis accounts for the electrostatic coupling between its orientation and hydrogen ion titration and presents a structural basis for the linkage between the protonation states of the primary proton acceptor, aspartate D85, and the extracellular release group, glutamate E204. We find that the charge state of D85 is a significant determinant for the orientation of R82. The molecular model predicts the following: the primary proton transfer to D85 can be described by a kinetic scheme with two heterogeneous substates. They control the event with different activation parameters due to the reorientation of R82 away from the chromophore binding site. Their population depends on the external pH and the proton exchange equilibrium between the membrane buried residues and the bulk aqueous solvent. Proton transfer in the physiologic pH range is strongly activated and followed by the reorientation of R82 which shifts the equilibrium toward complete transfer. In the alkaline pH region a different mechanism operates, which involves the increased population of a substate with already reoriented R82 as a consequence of the deprotonation of E204, leading to accelerated proton transfer. Assuming full proton exchange equilibrium with the bulk water on the millisecond time scale leads to an increased population of substates which are non-productive for proton transfer.

Solution and Gas-Phase H/D Exchange of Protein-Small-Molecule Complexes: Cex and Its Inhibitors

NASA Astrophysics Data System (ADS)

Kang, Yang; Terrier, Peran; Ding, Chuanfan; Douglas, D. J.

2012-01-01

The properties of noncovalent complexes of the enzyme exo-1,4-β-D-glycanase ("Cex") with three aza-sugar inhibitors, deoxynojirimycin (X2DNJ), isofagomine lactam (X2IL), and isofagomine (X2IF), have been studied with solution and gas-phase hydrogen deuterium exchange (H/Dx) and measurements of collision cross sections of gas-phase ions. In solution, complexes have lower H/Dx levels than free Cex because binding the inhibitors blocks some sites from H/Dx and reduces fluctuations of the protein. In mass spectra of complexes, abundant Cex ions are seen, which mostly are formed by dissociation of complexes in the ion sampling interface. Both complex ions and Cex ions formed from a solution containing complexes have lower cross sections than Cex ions from a solution of Cex alone. This suggests the Cex ions formed by dissociation "remember" their solution conformations. For a given charge, ions of the complexes have greater gas-phase H/Dx levels than ions of Cex. Unlike cross sections, H/Dx levels of the complexes do not correlate with the relative gas-phase binding strengths measured by MS/MS. Cex ions from solutions with or without inhibitors, which have different cross sections, show the same H/Dx level after 15 s, indicating the ions may fold or unfold on the seconds time scale of the H/Dx experiment. Thus, cross sections show that complexes have more compact conformations than free protein ions on the time scale of ca. 1 ms. The gas-phase H/Dx measurements show that at least some complexes retain different conformations from the Cex ions on a time scale of seconds.

NMR resolved multiple anesthetic binding sites in the TM domains of the α4β2 nAChR

PubMed Central

Bondarenko, Vasyl; Mowrey, David; Liu, Lu Tian; Xu, Yan; Tang, Pei

2012-01-01

The α4β2 nicotinic acetylcholine receptor (nAChR) has significant roles in nervous system function and disease. It is also a molecular target of general anesthetics. Anesthetics inhibit the α4β2 nAChR at clinically relevant concentrations, but their binding sites in α4β2 remain unclear. The recently determined NMR structures of the α4β2 nAChR transmembrane (TM) domains provide valuable frameworks for identifying the binding sites. In this study, we performed solution NMR experiments on the α4β2 TM domains in the absence and presence of halothane and ketamine. Both anesthetics were found in an intra-subunit cavity near the extracellular end of the 2 transmembrane helices, homologous to a common anesthetic binding site observed in X-ray structures of anesthetic-bound GLIC (Nury, et. al. 2011). Halothane, but not ketamine, was also found in cavities adjacent to the common anesthetic site at the interface of α4 and β2. In addition, both anesthetics bound to cavities near the ion selectivity filter at the intracellular end of the TM domains. Anesthetic binding induced profound changes in protein conformational exchanges. A number of residues, close to or remote from the binding sites, showed resonance signal splitting from single to double peaks, signifying that anesthetics decreased conformation exchange rates. It was also evident that anesthetics shifted population of two conformations. Altogether, the study comprehensively resolved anesthetic binding sites in the α4β2 nAChR. Furthermore, the study provided compelling experimental evidence of anesthetic-induced changes in protein dynamics, especially near regions of the hydrophobic gate and ion selectivity filter that directly regulate channel functions. PMID:23000369

NMR resolved multiple anesthetic binding sites in the TM domains of the α4β2 nAChR.

PubMed

Bondarenko, Vasyl; Mowrey, David; Liu, Lu Tian; Xu, Yan; Tang, Pei

2013-02-01

The α4β2 nicotinic acetylcholine receptor (nAChR) has significant roles in nervous system function and disease. It is also a molecular target of general anesthetics. Anesthetics inhibit the α4β2 nAChR at clinically relevant concentrations, but their binding sites in α4β2 remain unclear. The recently determined NMR structures of the α4β2 nAChR transmembrane (TM) domains provide valuable frameworks for identifying the binding sites. In this study, we performed solution NMR experiments on the α4β2 TM domains in the absence and presence of halothane and ketamine. Both anesthetics were found in an intra-subunit cavity near the extracellular end of the β2 transmembrane helices, homologous to a common anesthetic binding site observed in X-ray structures of anesthetic-bound GLIC (Nury et al., [32]). Halothane, but not ketamine, was also found in cavities adjacent to the common anesthetic site at the interface of α4 and β2. In addition, both anesthetics bound to cavities near the ion selectivity filter at the intracellular end of the TM domains. Anesthetic binding induced profound changes in protein conformational exchanges. A number of residues, close to or remote from the binding sites, showed resonance signal splitting from single to double peaks, signifying that anesthetics decreased conformation exchange rates. It was also evident that anesthetics shifted population of two conformations. Altogether, the study comprehensively resolved anesthetic binding sites in the α4β2 nAChR. Furthermore, the study provided compelling experimental evidence of anesthetic-induced changes in protein dynamics, especially near regions of the hydrophobic gate and ion selectivity filter that directly regulate channel functions. Copyright © 2012 Elsevier B.V. All rights reserved.

Interaction of influenza virus polymerase with viral RNA in the 'corkscrew' conformation.

PubMed

Flick, R; Hobom, G

1999-10-01

The influenza virus RNA (vRNA) promoter structure is known to consist of the 5'- and 3'-terminal sequences of the RNA, within very narrow boundaries of 16 and 15 nucleotides, respectively. A complete set of single nucleotide substitutions led to the previously proposed model of a binary hooked or 'corkscrew' conformation for the vRNA promoter when it interacts with the viral polymerase. This functional structure is confirmed here with a complete set of complementary double substitutions, of both the regular A:U and G:C type and also the G:U type of base-pair exchanges. The proposed structure consists of a six base-pair RNA rod in the distal element in conjunction with two stem-loop structures of two short-range base-pairs (positions 2-9; 3-8). These support an exposed tetranucleotide loop within each branch of the proximal element, in an overall oblique organization due to a central unpaired A residue at position 10 in the 5' sequence. Long-range base-pairing between the entire 5' and 3' branches, as required for an unmodified 'panhandle' model, has been excluded for the proximal element, while it is known to represent the mode of interaction within the distal element. A large number of short-range base-pair exchanges in the proximal element constitute promoter-up mutations, which show activities several times above that of the wild-type in reporter gene assays. The unique overall conformation and rather few invariant nucleotides appear to be the core elements in vRNA recognition by polymerase and also in viral ribonucleoprotein packaging, to allow discrimination against the background of other RNA molecules in the cell.

Effects of 3d-4f magnetic exchange interactions on the dynamics of the magnetization of Dy(III)-M(II)-Dy(III) trinuclear clusters.

PubMed

Pointillart, Fabrice; Bernot, Kevin; Sessoli, Roberta; Gatteschi, Dante

2007-01-01

[{Dy(hfac)(3)}(2){Fe(bpca)(2)}] x CHCl(3) ([Dy(2)Fe]) and [{Dy(hfac)(3)}(2){Ni(bpca)(2)}]CHCl(3) ([Dy(2)Ni]) (in which hfac(-)=1,1,1,5,5,5-hexafluoroacetylacetonate and bpca(-)=bis(2-pyridylcarbonyl)amine anion) were synthesized and characterized. Single-crystal X-ray diffraction shows that [Dy(2)Fe] and [Dy(2)Ni] are linear trinuclear complexes. Static magnetic susceptibility measurements reveal a weak ferromagnetic exchange interaction between Ni(II) and Dy(III) ions in [Dy(2)Ni], whereas the use of the diamagnetic Fe(II) ion leads to the absence of magnetic exchange interaction in [Dy(2)Fe]. Dynamic susceptibility measurements show a thermally activated behavior with the energy barrier of 9.7 and 4.9 K for the [Dy(2)Fe] and [Dy(2)Ni] complexes, respectively. A surprising negative effect of the ferromagnetic exchange interaction has been found and has been attributed to the structural conformation of these trinuclear complexes.

Sampling enhancement for the quantum mechanical potential based molecular dynamics simulations: a general algorithm and its extension for free energy calculation on rugged energy surface.

PubMed

Li, Hongzhi; Yang, Wei

2007-03-21

An approach is developed in the replica exchange framework to enhance conformational sampling for the quantum mechanical (QM) potential based molecular dynamics simulations. Importantly, with our enhanced sampling treatment, a decent convergence for electronic structure self-consistent-field calculation is robustly guaranteed, which is made possible in our replica exchange design by avoiding direct structure exchanges between the QM-related replicas and the activated (scaled by low scaling parameters or treated with high "effective temperatures") molecular mechanical (MM) replicas. Although the present approach represents one of the early efforts in the enhanced sampling developments specifically for quantum mechanical potentials, the QM-based simulations treated with the present technique can possess the similar sampling efficiency to the MM based simulations treated with the Hamiltonian replica exchange method (HREM). In the present paper, by combining this sampling method with one of our recent developments (the dual-topology alchemical HREM approach), we also introduce a method for the sampling enhanced QM-based free energy calculations.

Integration of biochemical sensors into wearable biomaterial platforms

NASA Astrophysics Data System (ADS)

Jandhyala, Sidhartha; Walper, Scott A.; Cargill, Allison A.; Ozual, Abigail; Daniele, Michael A.

2016-05-01

With rapidly inflating healthcare costs, a limited supply of physicians and an alarming surge in lifestyle diseases, radical changes must be made to improve preventative medicine and ensure a sustainable healthcare system. A compelling solution is to equip the population with wearable health monitors to continuously record representative and actionable physiological data. Herein, we present a preliminary design and evaluation of a biochemical sensor node enabled by a substrate comprised of a nanocellulose thin-film that conforms to the skin and carries a printed sensor element. The nanocellulose layer ensures conformal and biocompatible contact with the skin, while a printed layer provides a high surface-area electrode. While the recognition/transduction element can be exchanged for many different sensing motifs, we utilize the general structure of an electrochemical glucose sensor.

An arbitrary-shaped acoustic cloak with merits beyond the internal and external cloaks

NASA Astrophysics Data System (ADS)

Li, Baolei; Li, Tinghua; Wu, Jun; Hui, Ming; Yuan, Gang; Zhu, Yongsheng

2017-01-01

Based on transformation acoustics, an arbitrary-shaped acoustic cloak capable of functioning as an information exchange-enabling internal cloak and a movement-allowing external cloak is presented. The general expressions of material parameters for the acoustic cloaks with arbitrarily conformal or non-conformal boundaries are derived, and then the performances of developed cloaks are validated by full-wave simulations. Finally, the different characteristics of the linear and nonlinear transformations-based cloaks are compared and analyzed. The proposed cloak could lead to wider applications beyond that of normal cloaks, since it effectively compensates the insufficiencies of traditional internal and external cloaks. Besides, this work also provides a new method to design bifunctional device and suggests an alternative way to make a large object invisible.

Utilizing tagged paramagnetic shift reagents to monitor protein dynamics by NMR.

PubMed

Ye, Libin; Van Eps, Ned; Li, Xiang; Ernst, Oliver P; Prosser, R Scott

2017-11-01

Calmodulin is a ubiquitous calcium sensor protein, known to serve as a critical interaction hub with a wide range of signaling partners. While the holo form of calmodulin (CaM-4Ca 2+ ) has a well-defined ground state structure, it has been shown to undergo exchange, on a millisecond timescale, to a conformation resembling that of the peptide bound state. Tagged paramagnetic relaxation agents have been previously used to identify long-range dipolar interactions through relaxation effects on nuclear spins of interest. In the case of calmodulin, this lead to the determination of the relative orientation of the N- and C-terminal domains and the presence of a weakly populated peptide bound like state. Here, we make use of pseudocontact shifts from a tagged paramagnetic shift reagent which allows us to define minor states both in 13 C and 15 N NMR spectra and through 13 C- and 15 N-edited 1 H-CPMG relaxation dispersion measurements. This is validated by pulsed EPR (DEER) spectroscopy which reveals an ensemble consisting of a compact peptide-bound like conformer, an intermediate peptide-bound like conformer, and a (dumbbell-like) extended ground state conformer of CaM-4Ca 2+ , where addition of the MLCK peptide increases the population of the peptide-bound conformers. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

Bioactive Conformations of Two Seminal Delta Opioid Receptor Penta-peptides Inferred from Free-Energy Profiles

PubMed Central

Scarabelli, Guido; Provasi, Davide; Negri, Ana; Filizola, Marta

2013-01-01

Delta-opioid (DOP) receptors are members of the G protein-coupled receptor (GPCR) sub-family of opioid receptors, and are evolutionarily related, with homology exceeding 70%, to cognate mu-opioid (MOP), kappa-opioid (KOP), and nociceptin opioid (NOP) receptors. DOP receptors are considered attractive drug targets for pain management because agonists at these receptors are reported to exhibit strong antinociceptive activity with relatively few side effects. Among the most potent analgesics targeting the DOP receptor are the linear and cyclic enkephalin analogs known as DADLE (Tyr-D-Ala-GlyPhe-D-Leu) and DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen), respectively. Several computational and experimental studies have been carried out over the years to characterize the conformational profile of these penta-peptides with the ultimate goal of designing potent peptidomimetic agonists for the DOP receptor. The computational studies published to date, however, have investigated only a limited range of timescales and used over-simplified representations of the solvent environment. We provide here a thorough exploration of the conformational space of DADLE and DPDPE in an explicit solvent, using microsecond-scale molecular dynamics and bias-exchange metadynamics simulations. Free-energy profiles derived from these simulations point to a small number of DADLE and DPDPE conformational minima in solution, which are separated by relatively small energy barriers. Candidate bioactive forms of these peptides are selected from identified common spatial arrangements of key pharmacophoric points within all sampled conformations. PMID:23564013

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

Comprehensive Peptide Ion Structure Studies Using Ion Mobility Techniques: Part 1. An Advanced Protocol for Molecular Dynamics Simulations and Collision Cross-Section Calculation.

PubMed

Ghassabi Kondalaji, Samaneh; Khakinejad, Mahdiar; Tafreshian, Amirmahdi; J Valentine, Stephen

2017-05-01

Collision cross-section (CCS) measurements with a linear drift tube have been utilized to study the gas-phase conformers of a model peptide (acetyl-PAAAAKAAAAKAAAAKAAAAK). Extensive molecular dynamics (MD) simulations have been conducted to derive an advanced protocol for the generation of a comprehensive pool of in-silico structures; both higher energy and more thermodynamically stable structures are included to provide an unbiased sampling of conformational space. MD simulations at 300 K are applied to the in-silico structures to more accurately describe the gas-phase transport properties of the ion conformers including their dynamics. Different methods used previously for trajectory method (TM) CCS calculation employing the Mobcal software [1] are evaluated. A new method for accurate CCS calculation is proposed based on clustering and data mining techniques. CCS values are calculated for all in-silico structures, and those with matching CCS values are chosen as candidate structures. With this approach, more than 300 candidate structures with significant structural variation are produced; although no final gas-phase structure is proposed here, in a second installment of this work, gas-phase hydrogen deuterium exchange data will be utilized as a second criterion to select among these structures as well as to propose relative populations for these ion conformers. Here the need to increase conformer diversity and accurate CCS calculation is demonstrated and the advanced methods are discussed. Graphical Abstract ᅟ.

Comprehensive Peptide Ion Structure Studies Using Ion Mobility Techniques: Part 1. An Advanced Protocol for Molecular Dynamics Simulations and Collision Cross-Section Calculation

NASA Astrophysics Data System (ADS)

Ghassabi Kondalaji, Samaneh; Khakinejad, Mahdiar; Tafreshian, Amirmahdi; J. Valentine, Stephen

2017-05-01

Collision cross-section (CCS) measurements with a linear drift tube have been utilized to study the gas-phase conformers of a model peptide (acetyl-PAAAAKAAAAKAAAAKAAAAK). Extensive molecular dynamics (MD) simulations have been conducted to derive an advanced protocol for the generation of a comprehensive pool of in-silico structures; both higher energy and more thermodynamically stable structures are included to provide an unbiased sampling of conformational space. MD simulations at 300 K are applied to the in-silico structures to more accurately describe the gas-phase transport properties of the ion conformers including their dynamics. Different methods used previously for trajectory method (TM) CCS calculation employing the Mobcal software [1] are evaluated. A new method for accurate CCS calculation is proposed based on clustering and data mining techniques. CCS values are calculated for all in-silico structures, and those with matching CCS values are chosen as candidate structures. With this approach, more than 300 candidate structures with significant structural variation are produced; although no final gas-phase structure is proposed here, in a second installment of this work, gas-phase hydrogen deuterium exchange data will be utilized as a second criterion to select among these structures as well as to propose relative populations for these ion conformers. Here the need to increase conformer diversity and accurate CCS calculation is demonstrated and the advanced methods are discussed.

Atomistic simulations and network-based modeling of the Hsp90-Cdc37 chaperone binding with Cdk4 client protein: A mechanism of chaperoning kinase clients by exploiting weak spots of intrinsically dynamic kinase domains

PubMed Central

Czemeres, Josh; Buse, Kurt

2017-01-01

A fundamental role of the Hsp90 and Cdc37 chaperones in mediating conformational development and activation of diverse protein kinase clients is essential in signal transduction. There has been increasing evidence that the Hsp90-Cdc37 system executes its chaperoning duties by recognizing conformational instability of kinase clients and modulating their folding landscapes. The recent cryo-electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex has provided a framework for dissecting regulatory principles underlying differentiation and recruitment of protein kinase clients to the chaperone machinery. In this work, we have combined atomistic simulations with protein stability and network-based rigidity decomposition analyses to characterize dynamic factors underlying allosteric mechanism of the chaperone-kinase cycle and identify regulatory hotspots that control client recognition. Through comprehensive characterization of conformational dynamics and systematic identification of stabilization centers in the unbound and client- bound Hsp90 forms, we have simulated key stages of the allosteric mechanism, in which Hsp90 binding can induce instability and partial unfolding of Cdk4 client. Conformational landscapes of the Hsp90 and Cdk4 structures suggested that client binding can trigger coordinated dynamic changes and induce global rigidification of the Hsp90 inter-domain regions that is coupled with a concomitant increase in conformational flexibility of the kinase client. This process is allosteric in nature and can involve reciprocal dynamic exchanges that exert global effect on stability of the Hsp90 dimer, while promoting client instability. The network-based rigidity analysis and emulation of thermal unfolding of the Cdk4-cyclin D complex and Hsp90-Cdc37-Cdk4 complex revealed weak spots of kinase instability that are present in the native Cdk4 structure and are targeted by the chaperone during client recruitment. Our findings suggested that this mechanism may be exploited by the Hsp90-Cdc37 chaperone to recruit and protect intrinsically dynamic kinase clients from degradation. The results of this investigation are discussed and interpreted in the context of diverse experimental data, offering new insights into mechanisms of chaperone regulation and binding. PMID:29267381

Molecular dynamics study of the phosphorylation effect on the conformational states of the C-terminal domain of RNA polymerase II.

PubMed

Yonezawa, Yasushige

2014-05-01

The carboxyl-terminal domain (CTD) of RNA polymerase II in eukaryotes regulates mRNA processing processes by recruiting various regulation factors. A main function of the CTD relies on the heptad consensus sequence (YSPTSPS). The CTD dynamically changes its conformational state to recognize and bind different regulation factors. The dynamical conformation changes are caused by modifications, mainly phosphorylation and dephosphorylation, to the serine residues. In this study, we investigate the conformational states of the unit consensus CTD peptide with various phosphorylation patterns of the serine residues by extended ensemble simulations. The results show that the CTD without phosphorylation has a flexible disordered structure distributed between twisted and extended states, but phosphorylation tends to reduce the conformational space. It was found that phosphorylation induces a β-turn around the phosphorylated serine residue and the cis conformation of the proline residue significantly inhibits the β-turn formation. The β-turn should contribute to specific CTD binding of the different regulation factors by changing the conformation propensity combined with induced fit.

15 CFR 287.2 - Definitions. 1

Code of Federal Regulations, 2010 CFR

2010-01-01

... management of product, process or service quality and environmental performance. Sampling means the selection..., process, service, or person's qualifications conforms to specified requirements. Conformity assessment.... Requirements for products, services, systems, and organizations are those defined by law or regulation or by an...

Differential hydrogen/deuterium exchange mass spectrometry analysis of protein–ligand interactions

PubMed Central

Chalmers, Michael J; Busby, Scott A; Pascal, Bruce D; West, Graham M; Griffin, Patrick R

2011-01-01

Functional regulation of ligand-activated receptors is driven by alterations in the conformational dynamics of the protein upon ligand binding. Differential hydrogen/deuterium exchange (HDX) coupled with mass spectrometry has emerged as a rapid and sensitive approach for characterization of perturbations in conformational dynamics of proteins following ligand binding. While this technique is sensitive to detecting ligand interactions and alterations in receptor dynamics, it also can provide important mechanistic insights into ligand regulation. For example, HDX has been used to determine a novel mechanism of ligand activation of the nuclear receptor peroxisome proliferator activated receptor-γ, perform detailed analyses of binding modes of ligands within the ligand-binding pocket of two estrogen receptor isoforms, providing insight into selectivity, and helped classify different types of estrogen receptor-α ligands by correlating their pharmacology with the way they interact with the receptor based solely on hierarchical clustering of receptor HDX signatures. Beyond small-molecule–receptor interactions, this technique has also been applied to study protein–protein complexes, such as mapping antibody–antigen interactions. In this article, we summarize the current state of the differential HDX approaches and the future outlook. We summarize how HDX analysis of protein–ligand interactions has had an impact on biology and drug discovery. PMID:21329427

W-band PELDOR with 1 kW microwave power: molecular geometry, flexibility and exchange coupling.

PubMed

Reginsson, Gunnar W; Hunter, Robert I; Cruickshank, Paul A S; Bolton, David R; Sigurdsson, Snorri Th; Smith, Graham M; Schiemann, Olav

2012-03-01

A technique that is increasingly being used to determine the structure and conformational flexibility of biomacromolecules is Pulsed Electron-Electron Double Resonance (PELDOR or DEER), an Electron Paramagnetic Resonance (EPR) based technique. At X-band frequencies (9.5 GHz), PELDOR is capable of precisely measuring distances in the range of 1.5-8 nm between paramagnetic centres but the orientation selectivity is weak. In contrast, working at higher frequencies increases the orientation selection but usually at the expense of decreased microwave power and PELDOR modulation depth. Here it is shown that a home-built high-power pulsed W-band EPR spectrometer (HiPER) with a large instantaneous bandwidth enables one to achieve PELDOR data with a high degree of orientation selectivity and large modulation depths. We demonstrate a measurement methodology that gives a set of PELDOR time traces that yield highly constrained data sets. Simulating the resulting time traces provides a deeper insight into the conformational flexibility and exchange coupling of three bisnitroxide model systems. These measurements provide strong evidence that W-band PELDOR may prove to be an accurate and quantitative tool in assessing the relative orientations of nitroxide spin labels and to correlate those orientations to the underlying biological structure and dynamics. Copyright © 2012 Elsevier Inc. All rights reserved.

XRD, TEM, and thermal analysis of Arizona Ca-montmorillonites modified with didodecyldimethylammonium bromide.

PubMed

Sun, Zhiming; Park, Yuri; Zheng, Shuilin; Ayoko, Godwin A; Frost, Ray L

2013-10-15

An Arizona SAz-2 calcium montmorillonite was modified by a typical dialkyl cationic surfactant (didodecyldimethylammonium bromide, abbreviated to DDDMA) through direct ion exchange. The obtained organoclays were characterized by X-ray diffraction (XRD), high-resolution transmission electron microscopy (HR-TEM), high-resolution thermogravimetric analysis (HR-TG), and infrared emission spectroscopy (IES). The intercalation of surfactants greatly increased the basal spacing of the interlayers and the conformation arrangement of the loaded surfactant were assessed based on the XRD and TEM measurements. This work shows that the dialkyl surfactant can be directly intercalated into the montmorillonite without first undergoing Na(+) exchange. Moreover, the thermal stability of organoclays and the different arrangements of the surfactant molecules intercalated in the SAz-2 Ca-montmorillonite were determined by a combination of TG and IES techniques. The detailed conformational ordering of different intercalated surfactants under different conditions was also studied. The surfactant molecule DDDMA has proved to be thermally stable even at 400°C which indicates that the prepared organoclay is stable to significantly high temperatures. This study offers new insights into the structure and thermal stabilities of SAz-2 Ca-montmorillonite modified with DDDMA. The experimental results also confirm the potential applications of organic SAz-2 Ca-montmorillonites as adsorbents and polymer-clay nanocomposites. Copyright © 2013 Elsevier Inc. All rights reserved.

Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes.

PubMed

Crepin, Thibaut; Shalak, Vyacheslav F; Yaremchuk, Anna D; Vlasenko, Dmytro O; McCarthy, Andrew; Negrutskii, Boris S; Tukalo, Michail A; El'skaya, Anna V

2014-11-10

Eukaryotic elongation factor eEF1A transits between the GTP- and GDP-bound conformations during the ribosomal polypeptide chain elongation. eEF1A*GTP establishes a complex with the aminoacyl-tRNA in the A site of the 80S ribosome. Correct codon-anticodon recognition triggers GTP hydrolysis, with subsequent dissociation of eEF1A*GDP from the ribosome. The structures of both the 'GTP'- and 'GDP'-bound conformations of eEF1A are unknown. Thus, the eEF1A-related ribosomal mechanisms were anticipated only by analogy with the bacterial homolog EF-Tu. Here, we report the first crystal structure of the mammalian eEF1A2*GDP complex which indicates major differences in the organization of the nucleotide-binding domain and intramolecular movements of eEF1A compared to EF-Tu. Our results explain the nucleotide exchange mechanism in the mammalian eEF1A and suggest that the first step of eEF1A*GDP dissociation from the 80S ribosome is the rotation of the nucleotide-binding domain observed after GTP hydrolysis. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Noncoded amino acids in protein engineering: Structure-activity relationship studies of hirudin-thrombin interaction.

PubMed

De Filippis, Vincenzo; Acquasaliente, Laura; Pontarollo, Giulia; Peterle, Daniele

2018-01-01

The advent of recombinant DNA technology allowed to site-specifically insert, delete, or mutate almost any amino acid in a given protein, significantly improving our knowledge of protein structure, stability, and function. Nevertheless, a quantitative description of the physical and chemical basis that makes a polypeptide chain to efficiently fold into a stable and functionally active conformation is still elusive. This mainly originates from the fact that nature combined, in a yet unknown manner, different properties (i.e., hydrophobicity, conformational propensity, polarizability, and hydrogen bonding capability) into the 20 standard natural amino acids, thus making difficult, if not impossible, to univocally relate the change in protein stability or function to the alteration of physicochemical properties caused by amino acid exchange(s). In this view, incorporation of noncoded amino acids with tailored side chains, allowing to finely tune the structure at a protein site, would facilitate to dissect the effects of a given mutation in terms of one or a few physicochemical properties, thus much expanding the scope of physical organic chemistry in the study of proteins. In this review, relevant applications from our laboratory will be presented on the use of noncoded amino acids in structure-activity relationships studies of hirudin binding to thrombin. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

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.

Self-regenerating column chromatography

DOEpatents

Park, Woo K.

1995-05-30

The present invention provides a process for treating both cations and anions by using a self-regenerating, multi-ionic exchange resin column system which requires no separate regeneration steps. The process involves alternating ion-exchange chromatography for cations and anions in a multi-ionic exchange column packed with a mixture of cation and anion exchange resins. The multi-ionic mixed-charge resin column works as a multi-function column, capable of independently processing either cationic or anionic exchange, or simultaneously processing both cationic and anionic exchanges. The major advantage offered by the alternating multi-function ion exchange process is the self-regeneration of the resins.

Reaction Mechanisms of the Atomic Layer Deposition of Tin Oxide Thin Films Using Tributyltin Ethoxide and Ozone.

PubMed

Nanayakkara, Charith E; Liu, Guo; Vega, Abraham; Dezelah, Charles L; Kanjolia, Ravindra K; Chabal, Yves J

2017-06-20

Uniform and conformal deposition of tin oxide thin films is important for several applications in electronics, gas sensing, and transparent conducting electrodes. Thermal atomic layer deposition (ALD) is often best suited for these applications, but its implementation requires a mechanistic understanding of the initial nucleation and subsequent ALD processes. To this end, in situ FTIR and ex situ XPS have been used to explore the ALD of tin oxide films using tributyltin ethoxide and ozone on an OH-terminated, SiO 2 -passivated Si(111) substrate. Direct chemisorption of tributyltin ethoxide on surface OH groups and clear evidence that subsequent ligand exchange are obtained, providing mechanistic insight. Upon ozone pulse, the butyl groups react with ozone, forming surface carbonate and formate. The subsequent tributyltin ethoxide pulse removes the carbonate and formate features with the appearance of the bands for CH stretching and bending modes of the precursor butyl ligands. This ligand-exchange behavior is repeated for subsequent cycles, as is characteristic of ALD processes, and is clearly observed for deposition temperatures of 200 and 300 °C. On the basis of the in situ vibrational data, a reaction mechanism for the ALD process of tributyltin ethoxide and ozone is presented, whereby ligands are fully eliminated. Complementary ex situ XPS depth profiles confirm that the bulk of the films is carbon-free, that is, formate and carbonate are not incorporated into the film during the deposition process, and that good-quality SnO x films are produced. Furthermore, the process was scaled up in a cross-flow reactor at 225 °C, which allowed the determination of the growth rate (0.62 Å/cycle) and confirmed a self-limiting ALD growth at 225 and 268 °C. An analysis of the temperature-dependence data reveals that growth rate increases linearly between 200 and 300 °C.

Conformational Transitions and Convergence of Absolute Binding Free Energy Calculations

PubMed Central

Lapelosa, Mauro; Gallicchio, Emilio; Levy, Ronald M.

2011-01-01

The Binding Energy Distribution Analysis Method (BEDAM) is employed to compute the standard binding free energies of a series of ligands to a FK506 binding protein (FKBP12) with implicit solvation. Binding free energy estimates are in reasonably good agreement with experimental affinities. The conformations of the complexes identified by the simulations are in good agreement with crystallographic data, which was not used to restrain ligand orientations. The BEDAM method is based on λ -hopping Hamiltonian parallel Replica Exchange (HREM) molecular dynamics conformational sampling, the OPLS-AA/AGBNP2 effective potential, and multi-state free energy estimators (MBAR). Achieving converged and accurate results depends on all of these elements of the calculation. Convergence of the binding free energy is tied to the level of convergence of binding energy distributions at critical intermediate states where bound and unbound states are at equilibrium, and where the rate of binding/unbinding conformational transitions is maximal. This finding mirrors similar observations in the context of order/disorder transitions as for example in protein folding. Insights concerning the physical mechanism of ligand binding and unbinding are obtained. Convergence for the largest FK506 ligand is achieved only after imposing strict conformational restraints, which however require accurate prior structural knowledge of the structure of the complex. The analytical AGBNP2 model is found to underestimate the magnitude of the hydrophobic driving force towards binding in these systems characterized by loosely packed protein-ligand binding interfaces. Rescoring of the binding energies using a numerical surface area model corrects this deficiency. This study illustrates the complex interplay between energy models, exploration of conformational space, and free energy estimators needed to obtain robust estimates from binding free energy calculations. PMID:22368530

The pH-dependent tertiary structure of a designed helix-loop-helix dimer.

PubMed

Dolphin, G T; Baltzer, L

1997-01-01

De novo designed helix-loop-helix motifs can fold into well-defined tertiary structures if residues or groups of residues are incorporated at the helix-helix boundary to form helix-recognition sites that restrict the conformational degrees of freedom of the helical segments. Understanding the relationship between structure and function of conformational constraints therefore forms the basis for the engineering of non-natural proteins. This paper describes the design of an interhelical HisH+-Asp- hydrogen-bonded ion pair and the conformational stability of the folded helix-loop-helix motif. GTD-C, a polypeptide with 43 amino acid residues, has been designed to fold into a hairpin helix-loop-helix motif that can dimerise to form a four-helix bundle. The folded motif is in slow conformational exchange on the NMR timescale and has a well-dispersed 1H NMR spectrum, a narrow temperature interval for thermal denaturation and a near-UV CD spectrum with some fine structure. The conformational stability is pH dependent with an optimum that corresponds to the pH for maximum formation of a hydrogen-bonded ion pair between HisH17+ in helix I and Asp27- in helix II. The formation of an interhelical salt bridge is strongly suggested by the pH dependence of a number of spectroscopic probes to generate a well-defined tertiary structure in a designed helix-loop-helix motif. The thermodynamic stability of the folded motif is not increased by the formation of the salt bridge, but neighbouring conformations are destabilised. The use of this novel design principle in combination with hydrophobic interactions that provide sufficient binding energy in the folded structure should be of general use in de novo design of native-like proteins.

Effect of Inactivating Mutations on Peptide Conformational Ensembles: The Plant Polypeptide Hormone Systemin.

PubMed

Chowdhury, Saikat Dutta; Sarkar, Aditya K; Lahiri, Ansuman

2016-07-25

As part of their basal immune mechanism against insect/herbivore attacks, plants have evolved systemic response mechanisms. Such a systemic wound response in tomato was found to involve an 18 amino acid polypeptide called systemin, the first polypeptide hormone to be discovered in plants. Systematic alanine scanning and deletion studies showed differential modulation in its activity, particularly a major loss of function due to alanine substitution at positions 13 and 17 and less extentive loss of function due to substitution at position 12. We have studied the conformational ensembles of wild-type systemin along with its 17 variants by carrying out a total of 5.76 μs of replica-exchange molecular dynamics simulation in an implicit solvent environment. In our simulations, wild-type systemin showed a lack of α-helical and β-sheet structures, in conformity with earlier circular dichroism and NMR data. On the other hand, two regions containing diproline segments showed a tendency to adopt polyproline II structures. Examination of conformational ensembles of the 17 variants revealed a change in the population distributions, suggesting a less flexible structure for alanine substitutions at positions 12 and 13 but not for position 17. Combined with the experimental observations that positions 1-14 of systemin are important for the formation of the peptide-receptor complex, this leads to the hypothesis that loss of conformational flexibility may play a role in the loss of activity of systemin due to the P12A and P13A substitutions, while T17A deactivation probably occurs for a different reason, most likely the loss of the threonine phosphorylation site. We also indicate possible structural reasons why the substitution of the prolines at positions 12 and 13 leads to a loss of conformational freedom in the peptide.

Tadpole-like Conformations of Huntingtin Exon 1 Are Characterized by Conformational Heterogeneity that Persists regardless of Polyglutamine Length.

PubMed

Newcombe, Estella A; Ruff, Kiersten M; Sethi, Ashish; Ormsby, Angelique R; Ramdzan, Yasmin M; Fox, Archa; Purcell, Anthony W; Gooley, Paul R; Pappu, Rohit V; Hatters, Danny M

2018-05-11

Soluble huntingtin exon 1 (Httex1) with expanded polyglutamine (polyQ) engenders neurotoxicity in Huntington's disease. To uncover the physical basis of this toxicity, we performed structural studies of soluble Httex1 for wild-type and mutant polyQ lengths. Nuclear magnetic resonance experiments show evidence for conformational rigidity across the polyQ region. In contrast, hydrogen-deuterium exchange shows absence of backbone amide protection, suggesting negligible persistence of hydrogen bonds. The seemingly conflicting results are explained by all-atom simulations, which show that Httex1 adopts tadpole-like structures with a globular head encompassing the N-terminal amphipathic and polyQ regions and the tail encompassing the C-terminal proline-rich region. The surface area of the globular domain increases monotonically with polyQ length. This stimulates sharp increases in gain-of-function interactions in cells for expanded polyQ, and one of these interactions is with the stress-granule protein Fus. Our results highlight plausible connections between Httex1 structure and routes to neurotoxicity. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Phosphorylation Interferes with Maturation of Amyloid-β Fibrillar Structure in the N Terminus.

PubMed

Rezaei-Ghaleh, Nasrollah; Kumar, Sathish; Walter, Jochen; Zweckstetter, Markus

2016-07-29

Neurodegeneration is characterized by the ubiquitous presence of modifications in protein deposits. Despite their potential significance in the initiation and progression of neurodegenerative diseases, the effects of posttranslational modifications on the molecular properties of protein aggregates are largely unknown. Here, we study the Alzheimer disease-related amyloid-β (Aβ) peptide and investigate how phosphorylation at serine 8 affects the structure of Aβ aggregates. Serine 8 is shown to be located in a region of high conformational flexibility in monomeric Aβ, which upon phosphorylation undergoes changes in local conformational dynamics. Using hydrogen-deuterium exchange NMR and fluorescence quenching techniques, we demonstrate that Aβ phosphorylation at serine 8 causes structural changes in the N-terminal region of Aβ aggregates in favor of less compact conformations. Structural changes induced by serine 8 phosphorylation can provide a mechanistic link between phosphorylation and other biological events that involve the N-terminal region of Aβ aggregates. Our data therefore support an important role of posttranslational modifications in the structural polymorphism of amyloid aggregates and their modulatory effect on neurodegeneration. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Purification, crystallization and preliminary crystallographic analysis of the SH2 domain of IL-2-inducible T-cell kinase.

PubMed

Joseph, Raji E; Ginder, Nathaniel D; Hoy, Julie A; Nix, Jay C; Honzatko, Richard B; Andreotti, Amy H

2011-02-01

Proline is a unique amino acid owing to the relatively small energy difference between the cis and trans conformations of its peptide bond. The X-Pro imide bond readily undergoes cis-trans isomerization in the context of short peptides as well as some proteins. However, the direct detection of cis-trans proline isomerization in folded proteins is technically challenging. NMR spectroscopy is well suited to the direct detection of proline isomerization in folded proteins. It is less clear how well X-ray crystallography can reveal this conformational exchange event in folded proteins. Conformational heterogeneity owing to cis-trans proline isomerization in the Src homology 2 (SH2) domain of the IL-2-inducible T-cell kinase (ITK) has been extensively characterized by NMR. Using the ITK SH2 domain as a test system, an attempt was made to determine whether proline isomerization could be detected in a crystal structure of the ITK SH2 domain. As a first step towards this goal, the purification, crystallization and preliminary characterization of the ITK SH2 domain are described.

Implementing an electronic medication overview in Belgium.

PubMed

Storms, Hannelore; Marquet, Kristel; Nelissen, Katherine; Hulshagen, Leen; Lenie, Jan; Remmen, Roy; Claes, Neree

2014-12-16

An accurate medication overview is essential to reduce medication errors. Therefore, it is essential to keep the medication overview up-to-date and to exchange healthcare information between healthcare professionals and patients. Digitally shared information yields possibilities to improve communication. However, implementing a digitally shared medication overview is challenging. This articles describes the development process of a secured, electronic platform designed for exchanging medication information as executed in a pilot study in Belgium, called "Vitalink". The goal of "Vitalink" is to improve the exchange of medication information between professionals working in healthcare and patients in order to achieve a more efficient cooperation and better quality of care. Healthcare professionals of primary and secondary health care and patients of four Belgian regions participated in the project. In each region project groups coordinated implementation and reported back to the steering committee supervising the pilot study. The electronic medication overview was developed based on consensus in the project groups. The steering committee agreed to establish secured and authorized access through the use of electronic identity documents (eID) and a secured, eHealth-platform conform prior governmental regulations regarding privacy and security of healthcare information. A successful implementation of an electronic medication overview strongly depends on the accessibility and usability of the tool for healthcare professionals. Coordinating teams of the project groups concluded, based on their own observations and on problems reported to them, that secured and quick access to medical data needed to be pursued. According to their observations, the identification process using the eHealth platform, crucial to ensure secured data, was very time consuming. Secondly, software packages should meet the needs of their users, thus be adapted to daily activities of healthcare professionals. Moreover, software should be easy to install and run properly. The project would have benefited from a cost analysis executed by the national bodies prior to implementation.

Process and Formulation Effects on Protein Structure in Lyophilized Solids using Mass Spectrometric Methods

PubMed Central

Iyer, Lavanya K.; Sacha, Gregory A.; Moorthy, Balakrishnan S.; Nail, Steven L.; Topp, Elizabeth M.

2016-01-01

Myoglobin (Mb) was lyophilized in the absence (Mb-A) and presence (Mb-B) of sucrose in a pilot-scale lyophilizer with or without controlled ice nucleation. Cake morphology was characterized using scanning electron microscopy (SEM) and changes in protein structure were monitored using solid-state Fourier-transform infrared spectroscopy (ssFTIR), solid-state hydrogen-deuterium exchange-mass spectrometry (ssHDX-MS) and solid-state photolytic labeling-mass spectrometry (ssPL-MS). The results showed greater variability in nucleation temperature and irregular cake structure for formulations lyophilized without controlled nucleation. Controlled nucleation resulted in nucleation at ~ −5 °C and uniform cake structure. Formulations containing sucrose showed better retention of protein structure by all measures than formulations without sucrose. Samples lyophilized with and without controlled nucleation were similar by most measures of protein structure. However, ssPL-MS showed the greatest pLeu incorporation and more labeled regions for Mb-B lyophilized with controlled nucleation. The data support the use of ssHDX-MS and ssPL-MS to study formulation and process-induced conformational changes in lyophilized proteins. PMID:27044943

Poly(terphenylene) Anion Exchange Membranes: The Effect of Backbone Structure on Morphology and Membrane Property

DOE PAGES

Lee, Woo-Hyung; Park, Eun Joo; Han, Junyoung; ...

2017-05-05

A new design concept for ion-conducting polymers in anion exchange membranes (AEMs) fuel cells is proposed based on structural studies and conformational analysis of polymers and their effect on the properties of AEMs. Thermally, chemically, and mechanically stable terphenyl-based polymers with pendant quaternary ammonium alkyl groups were synthesized to investigate the effect of varying the arrangement of the polymer backbone and cation-tethered alkyl chains. The results demonstrate that the microstructure and morphology of these polymeric membranes significantly influence ion conductivity and fuel cell performance. Finally, the results of this study provide new insights that will guide the molecular design ofmore » polymer electrolyte materials to improve fuel cell performance.« less

Different conformational dynamics of β-arrestin1 and β-arrestin2 analyzed by hydrogen/deuterium exchange mass spectrometry

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

Yun, Youngjoo; Kim, Dong Kyun; Seo, Min-Duk

2015-01-30

Highlights: • The conformational dynamics of β-arrestin1 or β-arrestin2 were analyzed by HDX-MS. • β-Strands II through IV were more dynamic in β-arrestin2 than in β-arrestin1. • The middle loop was less dynamic in β-arrestin2 than in β-arrestin1. • Upon pre-activation by the R169E mutation, β-arrestins became more dynamic. • Pre-activation affected a wider region of β-arrestin1 compared to β-arrestin2. - Abstract: Arrestins have important roles in G protein-coupled receptor (GPCR) signaling including desensitization of GPCRs and G protein-independent signaling. There have been four arrestins identified: arrestin1, arrestin2 (e.g. β-arrestin1), arrestin3 (e.g. β-arrestin2), and arrestin4. β-Arrestin1 and β-arrestin2 are ubiquitouslymore » expressed and regulate a broad range of GPCRs, while arrestin1 and arrestin4 are expressed in the visual system. Although the functions of β-arrestin1 and β-arrestin2 widely overlap, β-arrestin2 has broader receptor selectivity, and a few studies have suggested that β-arrestin1 and β-arrestin2 have distinct cellular functions. Here, we compared the conformational dynamics of β-arrestin1 and β-arrestin2 by hydrogen/deuterium exchange mass spectrometry (HDX-MS). We also used the R169E mutant as a pre-activation model system. HDX-MS data revealed that β-strands II through IV were more dynamic in β-arrestin2 in the basal state, while the middle loop was more dynamic in β-arrestin1. With pre-activation, both β-arrestin1 and β-arrestin2 became more flexible, but broader regions of β-arrestin1 became flexible compared to β-arrestin2. The conformational differences between β-arrestin1 and β-arrestin2 in both the basal and pre-activated states might determine their different receptor selectivities and different cellular functions.« less

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.

Computational infrared and two-dimensional infrared photon echo spectroscopy of both wild-type and double mutant myoglobin-CO proteins.

PubMed

Choi, Jun-Ho; Kwak, Kyung-Won; Cho, Minhaeng

2013-12-12

The CO stretching mode of both wild-type and double mutant ( T67R / S92D ) MbCO (carbonmonoxymyoglobin) proteins is an ideal infrared (IR) probe for studying the local electrostatic environment inside the myoglobin heme pocket. Recently, to elucidate the conformational switching dynamics between two distinguishable states, extensive IR absorption, IR pump-probe, and two-dimensional (2D) IR spectroscopic studies for various mutant MbCO's have been performed by the Fayer group. They showed that the 2D IR spectroscopy of the double mutant, which has a peroxidase enzyme activity, reveals a rapid chemical exchange between two distinct states, whereas that of the wild-type does not. Despite the fact that a few simulation studies on these systems were already performed and reported, such complicated experimental results have not been fully reproduced nor described in terms of conformational state-to-state transition processes. Here, we first develop a distributed vibrational solvatochromic charge model for describing the CO stretch frequency shift reflecting local electric potential changes. Then, by carrying out molecular dynamic simulations of the two MbCO's and examining their CO frequency trajectories, it becomes possible to identify a proper reaction coordinate consisting of His64 imidazole ring rotation and its distance to the CO ligand. From the 2D surfaces of the resulting potential of mean forces, the spectroscopically distinguished A1 and A3 states of the wild-type as well as two more substates of the double mutant are identified and their vibrational frequencies and distributions are separately examined. Our simulated IR absorption and 2D IR spectra of the two MbCO's are directly compared with the previous experimental results reported by the Fayer group. The chemical exchange rate constants extracted from the two-state kinetic analyses of the simulated 2D IR spectra are in excellent agreement with the experimental values. On the basis of the quantitative agreement between the simulated spectra and experimental ones, we further examine the conformational differences in the heme pockets of the two proteins and show that the double mutation, T67R / S92D , suppresses the A1 population, restricts the imidazole ring rotation, and increases hydrogen-bond strength between the imidazole Nε-H and the oxygen atom of the CO ligand. It is believed that such delicate change of distal His64 imidazole ring dynamics induced by the double mutation may be responsible for its enhanced peroxidase catalytic activity as compared to the wild-type myoglobin.

Predicting protein aggregation during storage in lyophilized solids using solid state amide hydrogen/deuterium exchange with mass spectrometric analysis (ssHDX-MS).

PubMed

Moorthy, Balakrishnan S; Schultz, Steven G; Kim, Sherry G; Topp, Elizabeth M

2014-06-02

Solid state amide hydrogen/deuterium exchange with mass spectrometric analysis (ssHDX-MS) was used to assess the conformation of myoglobin (Mb) in lyophilized formulations, and the results correlated with the extent of aggregation during storage. Mb was colyophilized with sucrose (1:1 or 1:8 w/w), mannitol (1:1 w/w), or NaCl (1:1 w/w) or in the absence of excipients. Immediately after lyophilization, samples of each formulation were analyzed by ssHDX-MS and Fourier transform infrared spectroscopy (FTIR) to assess Mb conformation, and by dynamic light scattering (DLS) and size exclusion chromatography (SEC) to determine the extent of aggregation. The remaining samples were then placed on stability at 25 °C and 60% RH or 40 °C and 75% RH for up to 1 year, withdrawn at intervals, and analyzed for aggregate content by SEC and DLS. In ssHDX-MS of samples immediately after lyophilization (t = 0), Mb was less deuterated in solids containing sucrose (1:1 and 1:8 w/w) than in those containing mannitol (1:1 w/w), NaCl (1:1 w/w), or Mb alone. Deuterium uptake kinetics and peptide mass envelopes also indicated greater Mb structural perturbation in mannitol, NaCl, or Mb-alone samples at t = 0. The extent of deuterium incorporation and kinetic parameters related to rapidly and slowly exchanging amide pools (Nfast, Nslow), measured at t = 0, were highly correlated with the extent of aggregation on storage as measured by SEC. In contrast, the extent of aggregation was weakly correlated with FTIR band intensity and peak position measured at t = 0. The results support the use of ssHDX-MS as a formulation screening tool in developing lyophilized protein drug products.

A two-dimensional 1H-NMR study of the dam methylase site: comparison between the hemimethylated GATC sequence, its unmethylated analogue and a hemimethylated CATG sequence. The sequence dependence of methylation upon base-pair lifetimes.

PubMed

Fazakerley, G V; Quignard, E; Teoule, R; Guy, A; Guschlbauer, W

1987-09-15

We report two-dimensional NOE (NOESY) spectra on the sequence d(GCGATCATGG).d(CCATGATCGC) which contains the unmethylated dam site. As expected the DNA adopts a B-form conformation but appears to be distorted at the TG step of the second strand. This distorsion, probably bending, is not seen on the opposite strand. When the first strand is methylated on adenine in the GATC or CATG sequence the NOESY spectra indicate little or no change in the conformation. However the single strand-duplex exchange is slowed down to the slow-exchange region on a proton NMR time scale. We have assigned the exchangeable imino and cytidine amino resonances of the three duplexes. From the imino linewidths as a function of temperature, we observe that the unmethylated and the hemimethylated Gm6ATC duplexes melt normally from the ends. However, this is not so for the hemimethylated Cm6ATG duplex which, apart from the terminal base pairs, melts cooperatively and at higher temperature. In spectra recorded in H2O a second duplex is observed, for the Gm6ATC sequence, which we have not been able to identify. It is however unlikely to be a hairpin structure. Ultraviolet-melting curves also indicate the presence of two transitions for this duplex. The effect of methylation upon base-pair lifetimes has been studied by comparing the above three duplexes. Little effect is observed upon methylation in the GATC sequence but a drastic increase in the lifetimes of all base pairs is observed upon methylation in the CATG sequence.

Water on BN doped benzene: A hard test for exchange-correlation functionals and the impact of exact exchange on weak binding

DOE PAGES

Al-Hamdani, Yasmine S.; Alfè, Dario; von Lilienfeld, O. Anatole; ...

2014-10-22

Density functional theory (DFT) studies of weakly interacting complexes have recently focused on the importance of van der Waals dispersion forces, whereas the role of exchange has received far less attention. Here, by exploiting the subtle binding between water and a boron and nitrogen doped benzene derivative (1,2-azaborine) we show how exact exchange can alter the binding conformation within a complex. Benchmark values have been calculated for three orientations of the water monomer on 1,2-azaborine from explicitly correlated quantum chemical methods, and we have also used diffusion quantum Monte Carlo. For a host of popular DFT exchange-correlation functionals we showmore » that the lack of exact exchange leads to the wrong lowest energy orientation of water on 1,2-azaborine. As such, we suggest that a high proportion of exact exchange and the associated improvement in the electronic structure could be needed for the accurate prediction of physisorption sites on doped surfaces and in complex organic molecules. Meanwhile to predict correct absolute interaction energies an accurate description of exchange needs to be augmented by dispersion inclusive functionals, and certain non-local van der Waals functionals (optB88- and optB86b-vdW) perform very well for absolute interaction energies. Through a comparison with water on benzene and borazine (B₃N₃H₆) we show that these results could have implications for the interaction of water with doped graphene surfaces, and suggest a possible way of tuning the interaction energy.« less

7 CFR 22.305 - Conformance with OMB Circular No. A-95.

Code of Federal Regulations, 2010 CFR

2010-01-01

... jurisdictional rural development planning process must conform to the review requirements expressed in OMB... 7 Agriculture 1 2010-01-01 2010-01-01 false Conformance with OMB Circular No. A-95. 22.305 Section 22.305 Agriculture Office of the Secretary of Agriculture RURAL DEVELOPMENT COORDINATION Roles and...

49 CFR 178.39 - Specification 3BN seamless nickel cylinders.

Code of Federal Regulations, 2013 CFR

2013-10-01

... manufactured using equipment and processes adequate to ensure that each cylinder produced conforms to the.... A reasonably smooth and uniform surface finish is required. Cylinders closed in by spinning process... plugs, etc.) for those openings. Threads conforming to the following are required on openings: (1...

49 CFR 178.39 - Specification 3BN seamless nickel cylinders.

Code of Federal Regulations, 2014 CFR

2014-10-01

... manufactured using equipment and processes adequate to ensure that each cylinder produced conforms to the.... A reasonably smooth and uniform surface finish is required. Cylinders closed in by spinning process... plugs, etc.) for those openings. Threads conforming to the following are required on openings: (1...

49 CFR 178.39 - Specification 3BN seamless nickel cylinders.

Code of Federal Regulations, 2012 CFR

2012-10-01

... manufactured using equipment and processes adequate to ensure that each cylinder produced conforms to the.... A reasonably smooth and uniform surface finish is required. Cylinders closed in by spinning process... plugs, etc.) for those openings. Threads conforming to the following are required on openings: (1...

DICOM router: an open source toolbox for communication and correction of DICOM objects.

PubMed

Hackländer, Thomas; Kleber, Klaus; Martin, Jens; Mertens, Heinrich

2005-03-01

Today, the exchange of medical images and clinical information is well defined by the digital imaging and communications in medicine (DICOM) and Health Level Seven (ie, HL7) standards. The interoperability among information systems is specified by the integration profiles of IHE (Integrating the Healthcare Enterprise). However, older imaging modalities frequently do not correctly support these interfaces and integration profiles, and some use cases are not yet specified by IHE. Therefore, corrections of DICOM objects are necessary to establish conformity. The aim of this project was to develop a toolbox that can automatically perform these recurrent corrections of the DICOM objects. The toolbox is composed of three main components: 1) a receiver to receive DICOM objects, 2) a processing pipeline to correct each object, and 3) one or more senders to forward each corrected object to predefined addressees. The toolbox is implemented under Java as an open source project. The processing pipeline is realized by means of plug ins. One of the plug ins can be programmed by the user via an external eXtensible Stylesheet Language (ie, XSL) file. Using this plug in, DICOM objects can also be converted into eXtensible Markup Language (ie, XML) documents or other data formats. DICOM storage services, DICOM CD-ROMs, and the local file system are defined as input and output channel. The toolbox is used clinically for different application areas. These are the automatic correction of DICOM objects from non-IHE-conforming modalities, the import of DICOM CD-ROMs into the picture archiving and communication system and the pseudo naming of DICOM images. The toolbox has been accepted by users in a clinical setting. Because of the open programming interfaces, the functionality can easily be adapted to future applications.

Resolving the Magnetic Asymmetry of the Inner Space in Self-assembled Dimeric Capsules Based on Tetraurea-calix[4]pyrrole Components.

PubMed

Espelt, Mónica; Aragay, Gemma; Ballester, Pablo

2015-01-01

The encapsulation of N,N, N',N'-tetramethyl-1,5-pentanediamine-N,N'-dioxide 2 in a non-chiral capsular assembly formed by dimerization of tetraurea-calix[4]pyrrole 1a produced the observation of the N-methyl groups of the encapsulated guest as two separated singlets resonating highly upfield in the (1)H NMR spectrum. In order to clarify the origin of the observed signal splitting we assembled and studied a series of structurally related dimeric capsules. We used the tetraurea-calix[4]pyrrole 1a , the enantiomerically pure tetraurea-calix[4] pyrrole R-1b and the tetraurea-bisloop calix[4]pyrrole 1c as components of the produced assemblies. The (1)H NMR spectra of the assembled encapsulation complexes with bis-N-oxide 2 evidenced diverse splitting patterns of the N-methyl groups. In addition, 2D EXSY/ROESY NMR experiments revealed the existence of chemical exchange processes involving the separated methyl signals of the encapsulated guest. The capsular assemblies were mainly stabilized by a belt of eight head-to-tail hydrogen-bonded urea groups. The interconversion between the two senses of rotation of the unidirectionally oriented urea groups was slow on the (1)H NMR timescale. These characteristics determined the appearance of a new asymmetry element (supramolecular conformational chirality) in the assemblies that accounted for some of the magnetic asymmetries featured by the capsule's inner space. The racemization of the supramolecular chirality element was fast on the EXSY timescale and produced the chemical exchange processes detected for the encapsulation complexes.

Understanding the connection between conformational changes of peptides and equilibrium thermal fluctuations.

PubMed

Soler, Miguel A; Zúñiga, José; Requena, Alberto; Bastida, Adolfo

2017-02-01

Despite the increasing evidence that conformational transitions in peptides and proteins are driven by specific vibrational energy pathways along the molecule, the current experimental techniques of analysis do as yet not allow to study these biophysical processes in terms of anisotropic energy flows. Computational methods offer a complementary approach to obtain a more detailed understanding of the vibrational and conformational dynamics of these systems. Accordingly, in this work we investigate jointly the vibrational energy distribution and the conformational dynamics of trialanine peptide in water solution at room temperature by applying the Instantaneous Normal Mode analysis to the results derived from equilibrium molecular dynamics simulations. It is shown that conformational changes in trialanine are triggered by the vibrational energy accumulated in the low-frequency modes of the molecule, and that excitation is caused exclusively by thermal fluctuations of the solute-solvent system, thus excluding the possibility of an intramolecular vibrational energy redistribution process.

Molecular Dynamics Simulations of Folding and Insertion of the Ebola Virus Fusion Peptide into a Membrane Bilayer

DTIC Science & Technology

2008-07-01

Molecular Dynamics Simulations of Folding and Insertion of the Ebola Virus Fusion Peptide into a Membrane Bilayer Mark A. Olson1, In...presents replica-exchange molecular dynamics simulations of the folding and insertion of a 16- residue Ebola virus fusion peptide into a membrane...separate calculated structures into conformational basins. 2.1 Simulation models Molecular dynamics simulations were performed using the all-atom

Neural Basis of Two Kinds of Social Influence: Obedience and Conformity

PubMed Central

Xie, Ying; Chen, Mingliang; Lai, Hongxia; Zhang, Wuke; Zhao, Zhen; Anwar, Ch. Mahmood

2016-01-01

Event-related potentials (ERPs) were used in this study to explore the neural mechanism of obedience and conformity on the model of online book purchasing. Participants were asked to decide as quickly as possible whether to buy a book based on limited information including its title, keywords and number of positive and negative reviews. Obedience was induced by forcing participants to buy books which received mostly negative reviews. In contrast, conformity was aroused by majority influence (caused by positive and negative comments). P3 and N2, two kinds of ERP components related to social cognitive process, were measured and recorded with electroencephalogram (EEG) test. The results show that compared with conformity decisions, obedience decisions induced greater cognitive conflicts. In ERP measurements, greater amplitudes of N2 component were observed in the context of obedience. However, consistency level did not make a difference on P3 peak latency for both conformity and obedience. This shows that classification process is implicit in both conformity and obedience decision-making. In addition, for both conformity and obedience decisions, augmented P3 was observed when the reviews consistency (either negative or positive) was higher. PMID:26941632

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

Four-Way Kidney Exchange Transplant With Desensitization Increases Access to Living-Donor Kidney Transplant: First Report From India.

PubMed

Kute, Vivek B; Patel, Himanshu V; Shah, Pankaj R; Modi, Pranjal R; Shah, Veena R; Kasat, Govind S; Patil, Mayur V; Patel, Jaydeep C; Kumar, Deepak P; Trivedi, Hargovind L

2017-09-26

This study reports our experience of the first 4-way kidney exchange transplant combined with desensitization in India, which allows increased access to living-donor kidney transplant for sensitized patients. Four-way kidney exchange transplant procedures were approved by the ethics committee of our institution and the Organ Transplantation Authorization Committee of state governments of India (as per the Transplantation of Human Organs Act of India). The protocols conformed to Declaration of Istanbul principles and the ethical guidelines of the 1975 Helsinki Declaration. Written informed consent was obtained from patients, donors, and their guardians. In April 2016, our transplant team completed simultaneous 4-way kidney exchange transplant procedures without any medical (rejection and infections) or surgical complications. Reasons for being included for kidney exchange transplant were ABO incom-patible (2 recipients) and sensitization (2 recipients). All 4 recipients had stable graft function with no proteinuria and donor-specific antibody at 11-month follow-up on standard triple immunosup-pression. Patient and graft survival rates were both 100%. To the best of our knowledge, this is the first single-center report of 4-way kidney exchange transplant combined with desensitization from India. This procedure has the potential to expand living-donor kidney transplant in disadvantaged groups (eg, sensitized patients). Recipients who are hard to match due to high panel reactive antibody and difficult to desensitize due to strong donor-specific antibodies can receive a transplant with a combination of kidney exchange and desensitization. Our study suggests that 4-way kidney exchange transplant can be performed in developing countries (India) similar to that shown in programs in developed countries with team work, kidney exchange registry, and counseling.

Replica Exchange Improves Sampling in Low-Resolution Docking Stage of RosettaDock

PubMed Central

Zhang, Zhe; Lange, Oliver F.

2013-01-01

Many protein-protein docking protocols are based on a shotgun approach, in which thousands of independent random-start trajectories minimize the rigid-body degrees of freedom. Another strategy is enumerative sampling as used in ZDOCK. Here, we introduce an alternative strategy, ReplicaDock, using a small number of long trajectories of temperature replica exchange. We compare replica exchange sampling as low-resolution stage of RosettaDock with RosettaDock's original shotgun sampling as well as with ZDOCK. A benchmark of 30 complexes starting from structures of the unbound binding partners shows improved performance for ReplicaDock and ZDOCK when compared to shotgun sampling at equal or less computational expense. ReplicaDock and ZDOCK consistently reach lower energies and generate significantly more near-native conformations than shotgun sampling. Accordingly, they both improve typical metrics of prediction quality of complex structures after refinement. Additionally, the refined ReplicaDock ensembles reach significantly lower interface energies and many previously hidden features of the docking energy landscape become visible when ReplicaDock is applied. PMID:24009670

21 CFR 129.1 - Current good manufacturing practice.

Code of Federal Regulations, 2011 CFR

2011-04-01

... drinking water are in conformance with or are operated or administered in conformity with good manufacturing practice to assure that bottled drinking water is safe and that it has been processed, bottled...) FOOD FOR HUMAN CONSUMPTION PROCESSING AND BOTTLING OF BOTTLED DRINKING WATER General Provisions § 129.1...

Improving Group Processes in Transdisciplinary Case Studies for Sustainability Learning

ERIC Educational Resources Information Center

Hansmann, Ralf; Crott, Helmut W.; Mieg, Harald A.; Scholz, Roland W.

2009-01-01

Purpose: Deficient group processes such as conformity pressure can lead to inadequate group decisions with negative social, economic, or environmental consequences. The study aims to investigate how a group technique (called INFO) improves students' handling of conformity pressure and their collective judgments in the context of a…

Temperature: Human Regulating, Ants Conforming

ERIC Educational Resources Information Center

Clopton, Joe R.

2007-01-01

Biological processes speed up as temperature rises. Procedures for demonstrating this with ants traveling on trails, and data gathered by students on the Argentine ant ("Linepithema humile") are presented. The concepts of temperature regulation and conformity are detailed with a focus on the processes rather than on terms that label the organisms.

A flexible docking scheme to explore the binding selectivity of PDZ domains.

PubMed

Gerek, Z Nevin; Ozkan, S Banu

2010-05-01

Modeling of protein binding site flexibility in molecular docking is still a challenging problem due to the large conformational space that needs sampling. Here, we propose a flexible receptor docking scheme: A dihedral restrained replica exchange molecular dynamics (REMD), where we incorporate the normal modes obtained by the Elastic Network Model (ENM) as dihedral restraints to speed up the search towards correct binding site conformations. To our knowledge, this is the first approach that uses ENM modes to bias REMD simulations towards binding induced fluctuations in docking studies. In our docking scheme, we first obtain the deformed structures of the unbound protein as initial conformations by moving along the binding fluctuation mode, and perform REMD using the ENM modes as dihedral restraints. Then, we generate an ensemble of multiple receptor conformations (MRCs) by clustering the lowest replica trajectory. Using ROSETTALIGAND, we dock ligands to the clustered conformations to predict the binding pose and affinity. We apply this method to postsynaptic density-95/Dlg/ZO-1 (PDZ) domains; whose dynamics govern their binding specificity. Our approach produces the lowest energy bound complexes with an average ligand root mean square deviation of 0.36 A. We further test our method on (i) homologs and (ii) mutant structures of PDZ where mutations alter the binding selectivity. In both cases, our approach succeeds to predict the correct pose and the affinity of binding peptides. Overall, with this approach, we generate an ensemble of MRCs that leads to predict the binding poses and specificities of a protein complex accurately.

A flexible docking scheme to explore the binding selectivity of PDZ domains

PubMed Central

Gerek, Z Nevin; Ozkan, S Banu

2010-01-01

Modeling of protein binding site flexibility in molecular docking is still a challenging problem due to the large conformational space that needs sampling. Here, we propose a flexible receptor docking scheme: A dihedral restrained replica exchange molecular dynamics (REMD), where we incorporate the normal modes obtained by the Elastic Network Model (ENM) as dihedral restraints to speed up the search towards correct binding site conformations. To our knowledge, this is the first approach that uses ENM modes to bias REMD simulations towards binding induced fluctuations in docking studies. In our docking scheme, we first obtain the deformed structures of the unbound protein as initial conformations by moving along the binding fluctuation mode, and perform REMD using the ENM modes as dihedral restraints. Then, we generate an ensemble of multiple receptor conformations (MRCs) by clustering the lowest replica trajectory. Using RosettaLigand, we dock ligands to the clustered conformations to predict the binding pose and affinity. We apply this method to postsynaptic density-95/Dlg/ZO-1 (PDZ) domains; whose dynamics govern their binding specificity. Our approach produces the lowest energy bound complexes with an average ligand root mean square deviation of 0.36 Å. We further test our method on (i) homologs and (ii) mutant structures of PDZ where mutations alter the binding selectivity. In both cases, our approach succeeds to predict the correct pose and the affinity of binding peptides. Overall, with this approach, we generate an ensemble of MRCs that leads to predict the binding poses and specificities of a protein complex accurately. PMID:20196074

Packing interface energetics in different crystal forms of the λ Cro dimer.

PubMed

Ahlstrom, Logan S; Miyashita, Osamu

2014-07-01

Variation among crystal structures of the λ Cro dimer highlights conformational flexibility. The structures range from a wild type closed to a mutant fully open conformation, but it is unclear if each represents a stable solution state or if one may be the result of crystal packing. Here we use molecular dynamics (MD) simulation to investigate the energetics of crystal packing interfaces and the influence of site-directed mutagenesis on them in order to examine the effect of crystal packing on wild type and mutant Cro dimer conformation. Replica exchange MD of mutant Cro in solution shows that the observed conformational differences between the wild type and mutant protein are not the direct consequence of mutation. Instead, simulation of Cro in different crystal environments reveals that mutation affects the stability of crystal forms. Molecular Mechanics Poisson-Boltzmann Surface Area binding energy calculations reveal the detailed energetics of packing interfaces. Packing interfaces can have diverse properties in strength, energetic components, and some are stronger than the biological dimer interface. Further analysis shows that mutation can strengthen packing interfaces by as much as ∼5 kcal/mol in either crystal environment. Thus, in the case of Cro, mutation provides an additional energetic contribution during crystal formation that may stabilize a fully open higher energy state. Moreover, the effect of mutation in the lattice can extend to packing interfaces not involving mutation sites. Our results provide insight into possible models for the effect of crystallization on Cro conformational dynamics and emphasize careful consideration of protein crystal structures. © 2013 Wiley Periodicals, Inc.

Packing Interface Energetics in Different Crystal Forms of the λ Cro Dimer

PubMed Central

Ahlstrom, Logan S.; Miyashita, Osamu

2014-01-01

Variation among crystal structures of the λ Cro dimer highlights conformational flexibility. The structures range from a wild type closed to a mutant fully open conformation, but it is unclear if each represents a stable solution state or if one may be the result of crystal packing. Here we use molecular dynamics (MD) simulation to investigate the energetics of crystal packing interfaces and the influence of site-directed mutagenesis on them, in order to examine the effect of crystal packing on wild type and mutant Cro dimer conformation. Replica exchange MD of mutant Cro in solution shows that the observed conformational differences between the wild type and mutant protein are not the direct consequence of mutation. Instead, simulation of Cro in different crystal environments reveals that mutation affects the stability of crystal forms. Molecular Mechanics Poisson-Boltzmann Surface Area binding energy calculations reveal the detailed energetics of packing interfaces. Packing interfaces can have diverse properties in strength, energetic components, and some are stronger than the biological dimer interface. Further analysis shows that mutation can strengthen packing interfaces by as much as ~5 kcal/mol in either crystal environment. Thus, in the case of Cro, mutation provides an additional energetic contribution during crystal formation that may stabilize a fully open higher energy state. Moreover, the effect of mutation in the lattice can extend to packing interfaces not involving mutation sites. Our results provide insight into possible models for the effect of crystallization on Cro conformational dynamics and emphasize careful consideration of protein crystal structures. PMID:24218107

The Conformation and Aggregation of Proline-Rich Surfactant-Like Peptides.

PubMed

Hamley, Ian W; Castelletto, Valeria; Dehsorkhi, Ashkan; Torras, Juan; Aleman, Carlos; Portnaya, Irina; Danino, Dganit

2018-02-15

The secondary structure of proline-rich surfactant-like peptides is examined for the first time and is found to be influenced by charged end groups in peptides P 6 K, P 6 E, and KP 6 E and an equimolar mixture of P 6 K and P 6 E. The peptides exhibit a conformational transition from unordered to polyproline II (PPII) above a critical concentration, detected from circular dichroism (CD) measurements and unexpectedly from fluorescence dye probe measurements. Isothermal titration calorimetry (ITC) measurements provided the Gibbs energies of hydration of P 6 K and P 6 E, which correspond essentially to the hydration energies of the terminal charged residues. A detailed analysis of peptide conformation for these peptides was performed using density functional theory calculations, and this was used as a basis for hybrid quantum mechanics/molecular mechanics molecular dynamics (QM/MM MD) simulations. Quantum mechanics simulations in implicit water show both peptides (and their 1:1 mixture) exhibit PPII conformations. However, hybrid QM/MM MD simulations suggest that some deviations from this conformation are present for P 6 K and P 6 E in peptide bonds close to the charged residue, whereas in the 1:1 mixture a PPII structure is observed. Finally, aggregation of the peptides was investigated using replica exchange molecular dynamics simulations. These reveal a tendency for the average aggregate size (as measured by the radius of gyration) to increase with increasing temperature, which is especially marked for P 6 K, although the fraction of the most populated clusters is larger for P 6 E.

Multiple Replica Repulsion Technique for Efficient Conformational Sampling of Biological Systems

PubMed Central

Malevanets, Anatoly; Wodak, Shoshana J.

2011-01-01

Here, we propose a technique for sampling complex molecular systems with many degrees of freedom. The technique, termed “multiple replica repulsion” (MRR), does not suffer from poor scaling with the number of degrees of freedom associated with common replica exchange procedures and does not require sampling at high temperatures. The algorithm involves creation of multiple copies (replicas) of the system, which interact with one another through a repulsive potential that can be applied to the system as a whole or to portions of it. The proposed scheme prevents oversampling of the most populated states and provides accurate descriptions of conformational perturbations typically associated with sampling ground-state energy wells. The performance of MRR is illustrated for three systems of increasing complexity. A two-dimensional toy potential surface is used to probe the sampling efficiency as a function of key parameters of the procedure. MRR simulations of the Met-enkephalin pentapeptide, and the 76-residue protein ubiquitin, performed in presence of explicit water molecules and totaling 32 ns each, investigate the ability of MRR to characterize the conformational landscape of the peptide, and the protein native basin, respectively. Results obtained for the enkephalin peptide reflect more closely the extensive conformational flexibility of this peptide than previously reported simulations. Those obtained for ubiquitin show that conformational ensembles sampled by MRR largely encompass structural fluctuations relevant to biological recognition, which occur on the microsecond timescale, or are observed in crystal structures of ubiquitin complexes with other proteins. MRR thus emerges as a very promising simple and versatile technique for modeling the structural plasticity of complex biological systems. PMID:21843487

Analyzing slowly exchanging protein conformations by ion mobility mass spectrometry: study of the dynamic equilibrium of prolyl oligopeptidase.

PubMed

López, Abraham; Vilaseca, Marta; Madurga, Sergio; Varese, Monica; Tarragó, Teresa; Giralt, Ernest

2016-07-01

Ion mobility mass spectrometry (IMMS) is a biophysical technique that allows the separation of isobaric species on the basis of their size and shape. The high separation capacity, sensitivity and relatively fast time scale measurements confer IMMS great potential for the study of proteins in slow (µs-ms) conformational equilibrium in solution. However, the use of this technique for examining dynamic proteins is still not generalized. One of the major limitations is the instability of protein ions in the gas phase, which raises the question as to what extent the structures detected reflect those in solution. Here, we addressed this issue by analyzing the conformational landscape of prolyl oligopeptidase (POP) - a model of a large dynamic enzyme in the µs-ms range - by native IMMS and compared the results obtained in the gas phase with those obtained in solution. In order to interpret the experimental results, we used theoretical simulations. In addition, the stability of POP gaseous ions was explored by charge reduction and collision-induced unfolding experiments. Our experiments disclosed two species of POP in the gas phase, which correlated well with the open and closed conformations in equilibrium in solution; moreover, a gas-phase collapsed form of POP was also detected. Therefore, our findings not only support the potential of IMMS for the study of multiple co-existing conformations of large proteins in slow dynamic equilibrium in solution but also stress the need for careful data analysis to avoid artifacts. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Fully transparent conformal organic thin-film transistor array and its application as LED front driving.

PubMed

Cui, Nan; Ren, Hang; Tang, Qingxin; Zhao, Xiaoli; Tong, Yanhong; Hu, Wenping; Liu, Yichun

2018-02-22

A fully transparent conformal organic thin-film field-effect transistor array is demonstrated based on a photolithography-compatible ultrathin metallic grid gate electrode and a solution-processed C 8 -BTBT film. The resulting organic field-effect transistor array exhibits a high optical transparency of >80% over the visible spectrum, mobility up to 2 cm 2 V -1 s -1 , on/off ratio of 10 5 -10 6 , switching current of >0.1 mA, and excellent light stability. The transparent conformal transistor array is demonstrated to adhere well to flat and curved LEDs as front driving. These results present promising applications of the solution-processed wide-bandgap organic semiconductor thin films in future large-scale transparent conformal active-matrix displays.

Rates and equilibrium constants of the ligand-induced conformational transition of an HCN ion channel protein domain determined by DEER spectroscopy.

PubMed

Collauto, Alberto; DeBerg, Hannah A; Kaufmann, Royi; Zagotta, William N; Stoll, Stefan; Goldfarb, Daniella

2017-06-14

Ligand binding can induce significant conformational changes in proteins. The mechanism of this process couples equilibria associated with the ligand binding event and the conformational change. Here we show that by combining the application of W-band double electron-electron resonance (DEER) spectroscopy with microfluidic rapid freeze quench (μRFQ) it is possible to resolve these processes and obtain both equilibrium constants and reaction rates. We studied the conformational transition of the nitroxide labeled, isolated carboxy-terminal cyclic-nucleotide binding domain (CNBD) of the HCN2 ion channel upon binding of the ligand 3',5'-cyclic adenosine monophosphate (cAMP). Using model-based global analysis, the time-resolved data of the μRFQ DEER experiments directly provide fractional populations of the open and closed conformations as a function of time. We modeled the ligand-induced conformational change in the protein using a four-state model: apo/open (AO), apo/closed (AC), bound/open (BO), bound/closed (BC). These species interconvert according to AC + L ⇌ AO + L ⇌ BO ⇌ BC. By analyzing the concentration dependence of the relative contributions of the closed and open conformations at equilibrium, we estimated the equilibrium constants for the two conformational equilibria and the open-state ligand dissociation constant. Analysis of the time-resolved μRFQ DEER data gave estimates for the intrinsic rates of ligand binding and unbinding as well as the rates of the conformational change. This demonstrates that DEER can quantitatively resolve both the thermodynamics and the kinetics of ligand binding and the associated conformational change.

Chimpanzees (Pan troglodytes) flexibly adjust their behaviour in order to maximize payoffs, not to conform to majorities.

PubMed

Van Leeuwen, Edwin J C; Cronin, Katherine A; Schütte, Sebastian; Call, Josep; Haun, Daniel B M

2013-01-01

Chimpanzees have been shown to be adept learners, both individually and socially. Yet, sometimes their conservative nature seems to hamper the flexible adoption of superior alternatives, even to the extent that they persist in using entirely ineffective strategies. In this study, we investigated chimpanzees' behavioural flexibility in two different conditions under which social animals have been predicted to abandon personal preferences and adopt alternative strategies: i) under influence of majority demonstrations (i.e. conformity), and ii) in the presence of superior reward contingencies (i.e. maximizing payoffs). Unlike previous nonhuman primate studies, this study disentangled the concept of conformity from the tendency to maintain one's first-learned strategy. Studying captive (n=16) and semi-wild (n=12) chimpanzees in two complementary exchange paradigms, we found that chimpanzees did not abandon their behaviour in order to match the majority, but instead remained faithful to their first-learned strategy (Study 1a and 1b). However, the chimpanzees' fidelity to their first-learned strategy was overridden by an experimental upgrade of the profitability of the alternative strategy (Study 2). We interpret our observations in terms of chimpanzees' relative weighing of behavioural options as a function of situation-specific trade-offs. More specifically, contrary to previous findings, chimpanzees in our study abandoned their familiar behaviour to maximize payoffs, but not to conform to a majority.

The Mediation of Mothers’ Self-Fulfilling Effects on Their Children’s Alcohol Use: Self-Verification, Informational Conformity and Modeling Processes

PubMed Central

Madon, Stephanie; Guyll, Max; Buller, Ashley A.; Scherr, Kyle C.; Willard, Jennifer; Spoth, Richard

2010-01-01

This research examined whether self-fulfilling prophecy effects are mediated by self-verification, informational conformity, and modeling processes. The authors examined these mediational processes across multiple time frames with longitudinal data obtained from two samples of mother – child dyads (N1 = 487; N2 = 287). Children’s alcohol use was the outcome variable. The results provided consistent support for the mediational process of self-verification. In both samples and across several years of adolescence, there was a significant indirect effect of mothers’ beliefs on children’s alcohol use through children’s self-assessed likelihood of drinking alcohol in the future. Comparatively less support was found for informational conformity and modeling processes as mediators of mothers’ self-fulfilling effects. The potential for self-fulfilling prophecies to produce long lasting changes in targets’ behavior via self-verification processes are discussed. PMID:18665708

The mediation of mothers' self-fulfilling effects on their children's alcohol use: self-verification, informational conformity, and modeling processes.

PubMed

Madon, Stephanie; Guyll, Max; Buller, Ashley A; Scherr, Kyle C; Willard, Jennifer; Spoth, Richard

2008-08-01

This research examined whether self-fulfilling prophecy effects are mediated by self-verification, informational conformity, and modeling processes. The authors examined these mediational processes across multiple time frames with longitudinal data obtained from two samples of mother-child dyads (N-sub-1 = 486; N-sub-2 = 287), with children's alcohol use as the outcome variable. The results provided consistent support for the mediational process of self-verification. In both samples and across several years of adolescence, there was a significant indirect effect of mothers' beliefs on children's alcohol use through children's self-assessed likelihood of drinking alcohol in the future. Comparatively less support was found for informational conformity and modeling processes as mediators of mothers' self-fulfilling effects. The potential for self-fulfilling prophecies to produce long-lasting changes in targets' behavior via self-verification processes are discussed. (c) 2008 APA, all rights reserved

Conformational Transition Pathway in the Activation Process of Allosteric Glucokinase

PubMed Central

Shi, Ting; Zhao, Yaxue; Chen, Yingyi; Li, Xiaobai; Liu, Xinyi; Huang, Zhimin; Zhang, Jian

2013-01-01

Glucokinase (GK) is a glycolytic enzyme that plays an important role in regulating blood glucose level, thus acting as a potentially attractive target for drug discovery in the treatment of diabetes of the young type 2 and persistent hyperinsulinemic hypoglycemia of infancy. To characterize the activation mechanism of GK from the super-open state (inactive state) to the closed state (active state), a series of conventional molecular dynamics (MD) and targeted MD (TMD) simulations were performed on this enzyme. Conventional MD simulation showed a specific conformational ensemble of GK when the enzyme is inactive. Seven TMD simulations depicted a reliably conformational transition pathway of GK from the inactive state to the active state, and the components important to the conformational change of GK were identified by analyzing the detailed structures of the TMD trajectories. In combination with the inactivation process, our findings showed that the whole conformational pathway for the activation-inactivation-activation of GK is a one-direction circulation, and the active state is less stable than the inactive state in the circulation. Additionally, glucose was demonstrated to gradually modulate its binding pose with the help of residues in the large domain and connecting region of GK during the activation process. Furthermore, the obtained energy barriers were used to explain the preexisting equilibrium and the slow binding kinetic process of the substrate by GK. The simulated results are in accordance with the recent findings from the mutagenesis experiments and kinetic analyses. Our observations reveal a complicated conformational process in the allosteric protein, resulting in new knowledge about the delicate mechanisms for allosteric biological macromolecules that will be useful in drug design for targeting allosteric proteins. PMID:23409066

Insights into the conformational switching mechanism of the human vascular endothelial growth factor receptor type 2 kinase domain.

PubMed

Chioccioli, Matteo; Marsili, Simone; Bonaccini, Claudia; Procacci, Piero; Gratteri, Paola

2012-02-27

Human vascular endothelial growth factor receptor type 2 (h-VEFGR2) is a receptor tyrosine kinase involved in the angiogenesis process and regarded as an interesting target for the design of anticancer drugs. Its activation/inactivation mechanism is related to conformational changes in its cytoplasmatic kinase domain, involving first among all the αC-helix in N-lobe and the A-loop in C-lobe. Affinity of inhibitors for the active or inactive kinase form could dictate the open or closed conformation of the A-loop, thus making the different conformations of the kinase domain receptor (KDR) domain different drug targets in drug discovery. In this view, a detailed knowledge of the conformational landscape of KDR domain is of central relevance to rationalize the efficiency and selectivity of kinase inhibitors. Here, molecular dynamics simulations were used to gain insight into the conformational switching activity of the KDR domain and to identify intermediate conformations between the two limiting active and inactive conformations. Specific energy barriers have been selectively removed to induce, and hence highlight at the atomistic level, the regulation mechanism of the A-loop opening. The proposed strategy allowed to repeatedly observe the escape of the KDR domain from the DFG-out free energy basin and to identify rare intermediate conformations between the DFG-out and the DFG-in structures to be employed in a structure-based drug discovery process.

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.

Understanding the kinetic mechanism of RNA single base pair formation

PubMed Central

Xu, Xiaojun; Yu, Tao; Chen, Shi-Jie

2016-01-01

RNA functions are intrinsically tied to folding kinetics. The most elementary step in RNA folding is the closing and opening of a base pair. Understanding this elementary rate process is the basis for RNA folding kinetics studies. Previous studies mostly focused on the unfolding of base pairs. Here, based on a hybrid approach, we investigate the folding process at level of single base pairing/stacking. The study, which integrates molecular dynamics simulation, kinetic Monte Carlo simulation, and master equation methods, uncovers two alternative dominant pathways: Starting from the unfolded state, the nucleotide backbone first folds to the native conformation, followed by subsequent adjustment of the base conformation. During the base conformational rearrangement, the backbone either retains the native conformation or switches to nonnative conformations in order to lower the kinetic barrier for base rearrangement. The method enables quantification of kinetic partitioning among the different pathways. Moreover, the simulation reveals several intriguing ion binding/dissociation signatures for the conformational changes. Our approach may be useful for developing a base pair opening/closing rate model. PMID:26699466

Conformational analysis by intersection: CONAN.

PubMed

Smellie, Andrew; Stanton, Robert; Henne, Randy; Teig, Steve

2003-01-15

As high throughput techniques in chemical synthesis and screening improve, more demands are placed on computer assisted design and virtual screening. Many of these computational methods require one or more three-dimensional conformations for molecules, creating a demand for a conformational analysis tool that can rapidly and robustly cover the low-energy conformational spaces of small molecules. A new algorithm of intersection is presented here, which quickly generates (on average <0.5 seconds/stereoisomer) a complete description of the low energy conformational space of a small molecule. The molecule is first decomposed into nonoverlapping nodes N (usually rings) and overlapping paths P with conformations (N and P) generated in an offline process. In a second step the node and path data are combined to form distinct conformers of the molecule. Finally, heuristics are applied after intersection to generate a small representative collection of conformations that span the conformational space. In a study of approximately 97,000 randomly selected molecules from the MDDR, results are presented that explore these conformations and their ability to cover low-energy conformational space. Copyright 2002 Wiley Periodicals, Inc. J Comput Chem 24: 10-20, 2003

Stretchable Heater Using Ligand-Exchanged Silver Nanowire Nanocomposite for Wearable Articular Thermotherapy.

PubMed

Choi, Suji; Park, Jinkyung; Hyun, Wonji; Kim, Jangwon; Kim, Jaemin; Lee, Young Bum; Song, Changyeong; Hwang, Hye Jin; Kim, Ji Hoon; Hyeon, Taeghwan; Kim, Dae-Hyeong

2015-06-23

Thermal therapy is one of the most popular physiotherapies and it is particularly useful for treating joint injuries. Conventional devices adapted for thermal therapy including heat packs and wraps have often caused discomfort to their wearers because of their rigidity and heavy weight. In our study, we developed a soft, thin, and stretchable heater by using a nanocomposite of silver nanowires and a thermoplastic elastomer. A ligand exchange reaction enabled the formation of a highly conductive and homogeneous nanocomposite. By patterning the nanocomposite with serpentine-mesh structures, conformal lamination of devices on curvilinear joints and effective heat transfer even during motion were achieved. The combination of homogeneous conductive elastomer, stretchable design, and a custom-designed electronic band created a novel wearable system for long-term, continuous articular thermotherapy.

Gauge boson exchange in AdS d+1

NASA Astrophysics Data System (ADS)

D'Hoker, Eric; Freedman, Daniel Z.

1999-04-01

We study the amplitude for exchange of massless gauge bosons between pairs of massive scalar fields in anti-de Sitter space. In the AdS/CFT correspondence this amplitude describes the contribution of conserved flavor symmetry currents to 4-point functions of scalar operators in the boundary conformal theory. A concise, covariant, Y2K compatible derivation of the gauge boson propagator in AdS d + 1 is given. Techniques are developed to calculate the two bulk integrals over AdS space leading to explicit expressions or convenient, simple integral representations for the amplitude. The amplitude contains leading power and sub-leading logarithmic singularities in the gauge boson channel and leading logarithms in the crossed channel. The new methods of this paper are expected to have other applications in the study of the Maldacena conjecture.

Hawking from Catalan

DOE PAGES

Fitzpatrick, A. Liam; Kaplan, Jared; Walters, Matthew T.; ...

2016-05-12

The Virasoro algebra determines all ‘graviton’ matrix elements in AdS 3/CFT 2. We study the explicit exchange of any number of Virasoro gravitons between heavy and light CFT 2 operators at large central charge. These graviton exchanges can be written in terms of new on-shell tree diagrams, organized in a perturbative expansion in h H/c, the heavy operator dimension divided by the central charge. The Virasoro vacuum conformal block, which is the sum of all the tree diagrams, obeys a differential recursion relation generalizing that of the Catalan numbers. Here, we use this recursion relation to sum the on-shell diagramsmore » to all orders, computing the Virasoro vacuum block. Extrapolating to large h H/c determines the Hawking temperature of a BTZ black hole in dual AdS 3 theories.« less

Hawking from Catalan

NASA Astrophysics Data System (ADS)

Fitzpatrick, A. Liam; Kaplan, Jared; Walters, Matthew T.; Wang, Junpu

2016-05-01

The Virasoro algebra determines all `graviton' matrix elements in AdS3/CFT2. We study the explicit exchange of any number of Virasoro gravitons between heavy and light CFT2 operators at large central charge. These graviton exchanges can be written in terms of new on-shell tree diagrams, organized in a perturbative expansion in h H /c, the heavy operator dimension divided by the central charge. The Virasoro vacuum conformal block, which is the sum of all the tree diagrams, obeys a differential recursion relation generalizing that of the Catalan numbers. We use this recursion relation to sum the on-shell diagrams to all orders, computing the Virasoro vacuum block. Extrapolating to large h H /c determines the Hawking temperature of a BTZ black hole in dual AdS3 theories.

Conformational changes in matrix-isolated 6-methoxyindole: Effects of the thermal and infrared light excitations

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

Lopes Jesus, A. J.; CQC, Faculty of Pharmacy, University of Coimbra, 3004-295 Coimbra; Reva, I., E-mail: reva@qui.uc.pt

2016-03-28

Conformational changes induced thermally or upon infrared excitation of matrix-isolated 6-methoxyindole were investigated. Narrowband near-infrared excitation of the first overtone of the N–H stretching vibration of each one of the two identified conformers is found to induce a selective large-scale conversion of the pumped conformer into the other one. This easily controllable bidirectional process consists in the intramolecular reorientation of the methoxy group and allowed a full assignment of the infrared spectra of the two conformers. Matrices with different conformational compositions prepared by narrow-band irradiations were subsequently used to investigate the effects of both thermal and broadband infrared excitations onmore » the conformational mixtures. Particular attention is given to the influence of the matrix medium (Ar vs. Xe) and conformational effects of exposition of the sample to the spectrometer light source during the measurements.« less

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

Cirac, J. Ignacio; Sierra, German; Instituto de Fisica Teorica, UAM-CSIC, Madrid

We generalize the matrix product states method using the chiral vertex operators of conformal field theory and apply it to study the ground states of the XXZ spin chain, the J{sub 1}-J{sub 2} model and random Heisenberg models. We compute the overlap with the exact wave functions, spin-spin correlators, and the Renyi entropy, showing that critical systems can be described by this method. For rotational invariant ansatzs we construct an inhomogenous extension of the Haldane-Shastry model with long-range exchange interactions.

Activation helix orientation of the estrogen receptor is mediated by receptor dimerization: evidence from molecular dynamics simulations.

PubMed

Fratev, Filip

2015-05-28

In recent years, the nuclear receptors (NR) dynamics have been studied extensively by various approaches. However, the transition path of helix 12 (H12) to an agonist or an antagonist conformation and the exchange pathway between these states is not clear yet. A number of accelerated molecular dynamics (aMD) runs were performed on both an ERα monomer and a homodimer with a total length of 2.2 μs. We have been able to sample reasonably well the H12 conformational landscape to reproduce precisely both the agonist and the antagonist conformations, starting from an unfolded position, and to describe the transition path between them, even in the presence of an agonist ligand. These conformations were the most prevalent, suggesting that the extended H12 state is not likely to exist and that the natural ERα H12 position might exist in both the agonist and antagonist states. Remarkably, the H12 transition occurs and is regulated only in a dimer form and the proper agonist or antagonist H12 conformation can be achieved solely in one of the dimer subunits. These results clearly demonstrate that clusters of the two well-known H12 states exist by themselves in the protein free energy landscape, i.e. they are not constituted directly by the ligands, and dimerization favors the switch between them. Conversely, in a monomer, no transitions have been observed. Thus, the dimer formation helps the constitution of populations of discrete H12 conformational states and reshapes the conformational landscape. Further analyses have shown that these observations can be explained by specific interface and long range protein-protein interactions, resulting in conformational fluctuations in helices 5 and 11. Based on these results, a new ERα activation/deactivation mechanism and a sequence of binding events during receptor activity modulation have been suggested according to which ligands control the H12 conformation via alterations of the inter-dimer interactions. These findings agree with the HDX and fluorescence experiments and provide an explanation on a structural basis of these data, demonstrating that the dynamics of H12 are not altered greatly upon ligand binding and large fluctuations at the end of H11 are present.

Peptide Modulation of Class I Major Histocompatibility Complex Protein Molecular Flexibility and the Implications for Immune Recognition*

PubMed Central

Hawse, William F.; Gloor, Brian E.; Ayres, Cory M.; Kho, Kevin; Nuter, Elizabeth; Baker, Brian M.

2013-01-01

T cells use the αβ T cell receptor (TCR) to recognize antigenic peptides presented by class I major histocompatibility complex proteins (pMHCs) on the surfaces of antigen-presenting cells. Flexibility in both TCRs and peptides plays an important role in antigen recognition and discrimination. Less clear is the role of flexibility in the MHC protein; although recent observations have indicated that mobility in the MHC can impact TCR recognition in a peptide-dependent fashion, the extent of this behavior is unknown. Here, using hydrogen/deuterium exchange, fluorescence anisotropy, and structural analyses, we show that the flexibility of the peptide binding groove of the class I MHC protein HLA-A*0201 varies significantly with different peptides. The variations extend throughout the binding groove, impacting regions contacted by TCRs as well as other activating and inhibitory receptors of the immune system. Our results are consistent with statistical mechanical models of protein structure and dynamics, in which the binding of different peptides alters the populations and exchange kinetics of substates in the MHC conformational ensemble. Altered MHC flexibility will influence receptor engagement, impacting conformational adaptations, entropic penalties associated with receptor recognition, and the populations of binding-competent states. Our results highlight a previously unrecognized aspect of the “altered self” mechanism of immune recognition and have implications for specificity, cross-reactivity, and antigenicity in cellular immunity. PMID:23836912