Loop-driven conformational transition between the alternative and collapsed form of prethrombin-2: targeted molecular dynamics study.

PubMed

Wu, Sangwook

2017-01-01

Two distinct crystal structures of prethrombin-2, the alternative and collapsed forms, are elucidated by X-ray crystallogrphy. We analyzed the conformational transition from the alternative to the collapsed form employing targeted molecular dynamics (TMD) simulation. Despite small RMSD difference in the two X-ray crystal structures, some hydrophobic residues (W60d, W148, W215, and F227) show a significant difference between the two conformations. TMD simulation shows that the four hydrophobic residues undergo concerted movement from dimer to trimer transition via tetramer state in the conformational change from the alternative to the collapsed form. We reveal that the concerted movement of the four hydrophobic residues is controlled by movement of specific loop regions behind. In this paper, we propose a sequential scenario for the conformational transition from the alternative form to the collapsed form, which is partially supported by the mutant W148A simulation.

Conformational Heterogeneity of Bax Helix 9 Dimer for Apoptotic Pore Formation

NASA Astrophysics Data System (ADS)

Liao, Chenyi; Zhang, Zhi; Kale, Justin; Andrews, David W.; Lin, Jialing; Li, Jianing

2016-07-01

Helix α9 of Bax protein can dimerize in the mitochondrial outer membrane (MOM) and lead to apoptotic pores. However, it remains unclear how different conformations of the dimer contribute to the pore formation on the molecular level. Thus we have investigated various conformational states of the α9 dimer in a MOM model — using computer simulations supplemented with site-specific mutagenesis and crosslinking of the α9 helices. Our data not only confirmed the critical membrane environment for the α9 stability and dimerization, but also revealed the distinct lipid-binding preference of the dimer in different conformational states. In our proposed pathway, a crucial iso-parallel dimer that mediates the conformational transition was discovered computationally and validated experimentally. The corroborating evidence from simulations and experiments suggests that, helix α9 assists Bax activation via the dimer heterogeneity and interactions with specific MOM lipids, which eventually facilitate proteolipidic pore formation in apoptosis regulation.

Molecular structure and conformational preferences of gaseous 1-iodo-1-silacyclohexane

NASA Astrophysics Data System (ADS)

Belyakov, A. V.; Baskakov, A. A.; Berger, R. J. F.; Mitzel, N. W.; Oberhammer, H.; Arnason, I.; Wallevik, S. Ò.

2012-03-01

The molecular structure of the axial and equatorial conformers of 1-iodo-1-silacyclohexane, CH2(CH2CH2)2SiH-I, as well as thermodynamic equilibrium between these species were investigated by means of gas-phase electron diffraction (GED) and quantum chemical calculations up to MP2(full)/SDB-AUG-CC-pVTZ level of theory (MP2). According to electron diffraction data, the vapor of this compound comprises a mixture of conformers with chair conformation and Cs symmetry differing in the axial and equatorial position of the Si-I bond (axial = 73(7) mol%/equatorial = 27(7) mol%) at T = 352 K. This corresponds to a free energy difference of A = -0.59(22) kcal mol-1. The observed gas-phase electron diffraction parameters are in good agreement with those obtained from theory. NBO analysis revealed that axial conformer of 1-iodo-1-silacyclohexane is an example for electrostatic stabilization of a conformer which is unfavorable in terms of steric and conjugation interaction.

Ligand-Induced Modulation of the Free-Energy Landscape of G Protein-Coupled Receptors Explored by Adaptive Biasing Techniques

PubMed Central

Provasi, Davide; Artacho, Marta Camacho; Negri, Ana; Mobarec, Juan Carlos; Filizola, Marta

2011-01-01

Extensive experimental information supports the formation of ligand-specific conformations of G protein-coupled receptors (GPCRs) as a possible molecular basis for their functional selectivity for signaling pathways. Taking advantage of the recently published inactive and active crystal structures of GPCRs, we have implemented an all-atom computational strategy that combines different adaptive biasing techniques to identify ligand-specific conformations along pre-determined activation pathways. Using the prototypic GPCR β2-adrenergic receptor as a suitable test case for validation, we show that ligands with different efficacies (either inverse agonists, neutral antagonists, or agonists) modulate the free-energy landscape of the receptor by shifting the conformational equilibrium towards active or inactive conformations depending on their elicited physiological response. Notably, we provide for the first time a quantitative description of the thermodynamics of the receptor in an explicit atomistic environment, which accounts for the receptor basal activity and the stabilization of different active-like states by differently potent agonists. Structural inspection of these metastable states reveals unique conformations of the receptor that may have been difficult to retrieve experimentally. PMID:22022248

In God we trust? Neural measures reveal lower social conformity among non-religious individuals.

PubMed

Thiruchselvam, Ravi; Gopi, Yashoda; Kilekwang, Leonard; Harper, Jessica; Gross, James J

2017-06-01

Even in predominantly religious societies, there are substantial individual differences in religious commitment. Why is this? One possibility is that differences in social conformity (i.e. the tendency to think and behave as others do) underlie inclination towards religiosity. However, the link between religiosity and conformity has not yet been directly examined. In this study, we tested the notion that non-religious individuals show dampened social conformity, using both self-reported and neural (EEG-based ERPs) measures of sensitivity to others' influence. Non-religious vs religious undergraduate subjects completed an experimental task that assessed levels of conformity in a domain unrelated to religion (i.e. in judgments of facial attractiveness). Findings showed that, although both groups yielded to conformity pressures at the self-report level, non-religious individuals did not yield to such pressures in their neural responses. These findings highlight a novel link between religiosity and social conformity, and hold implications for prominent theories about the psychological functions of religion. © The Author (2017). Published by Oxford University Press.

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

Rycyna, R.E.; Alderfer, J.L.

Uridylyl(3'-5')uridine (UpU) is subjected to aqueous acetone photosensitized radiation with sunlamps. These irradiation conditions form only cyclobutane-type photodimers. Purification of a specific configurational photodimer is accomplished by using C-18 reverse-phase high-performance liquid chromatography. Multinuclear NMR analysis is used to analyze photoproduct formation and to determine conformational features of these photodimers. Four photodimers are identified, with the cis-syn isomer predominant. The cis-syn and trans-syn photodimers of UpU exhibit markedly different furanose and exocyclic bond conformations. A comparison of the properties of the cis-syn dimers of UpU with those of dTpdT reveal many similar conformational features but also some that are different.

Identification of Serine Conformers by Matrix-Isolation IR Spectroscopy Aided by Near-Infrared Laser Induced Conformational Change, 2D Correlation Analysis, and Quantum Mechanical Anharmonic Computations

PubMed Central

Najbauer, Eszter E.; Bazsó, Gábor; Apóstolo, Rui; Fausto, Rui; Biczysko, Malgorzata; Barone, Vincenzo; Tarczay, György

2018-01-01

The conformers of α-serine were investigated by matrix-isolation IR spectroscopy combined with NIR laser irradiation. This method, aided by 2D correlation analysis, enabled unambiguously grouping the spectral lines to individual conformers. On the basis of comparison of at least nine experimentally observed vibrational transitions of each conformer with empirically scaled (SQM) and anharmonic (GVPT2) computed IR spectra, 6 conformers were identified. In addition, the presence of at least one more conformer in Ar matrix was proved, and a short-lived conformer with a half-live of (3.7±0.5)·103 s in N2 matrix was generated by NIR irradiation. The analysis of the NIR laser induced conversions revealed that the excitation of the stretching overtone of both the side-chain and the carboxylic OH groups can effectively promote conformational changes, but remarkably different paths were observed for the two kinds of excitations. PMID:26201050

Phospho-selective mechanisms of arrestin conformations and functions revealed by unnatural amino acid incorporation and 19F-NMR

PubMed Central

Yang, Fan; Yu, Xiao; Liu, Chuan; Qu, Chang-Xiu; Gong, Zheng; Liu, Hong-Da; Li, Fa-Hui; Wang, Hong-Mei; He, Dong-Fang; Yi, Fan; Song, Chen; Tian, Chang-Lin; Xiao, Kun-Hong; Wang, Jiang-Yun; Sun, Jin-Peng

2015-01-01

Specific arrestin conformations are coupled to distinct downstream effectors, which underlie the functions of many G-protein-coupled receptors (GPCRs). Here, using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance (19F-NMR) spectroscopy, we demonstrate that distinct receptor phospho-barcodes are translated to specific β-arrestin-1 conformations and direct selective signalling. With its phosphate-binding concave surface, β-arrestin-1 ‘reads' the message in the receptor phospho-C-tails and distinct phospho-interaction patterns are revealed by 19F-NMR. Whereas all functional phosphopeptides interact with a common phosphate binding site and induce the movements of finger and middle loops, different phospho-interaction patterns induce distinct structural states of β-arrestin-1 that are coupled to distinct arrestin functions. Only clathrin recognizes and stabilizes GRK2-specific β-arrestin-1 conformations. The identified receptor-phospho-selective mechanism for arrestin conformation and the spacing of the multiple phosphate-binding sites in the arrestin enable arrestin to recognize plethora phosphorylation states of numerous GPCRs, contributing to the functional diversity of receptors. PMID:26347956

IR-IR Conformation Specific Spectroscopy of Na+(Glucose) Adducts

NASA Astrophysics Data System (ADS)

Voss, Jonathan M.; Kregel, Steven J.; Fischer, Kaitlyn C.; Garand, Etienne

2018-01-01

We report an IR-IR double resonance study of the structural landscape present in the Na+(glucose) complex. Our experimental approach involves minimal modifications to a typical IR predissociation setup, and can be carried out via ion-dip or isomer-burning methods, providing additional flexibility to suit different experimental needs. In the current study, the single-laser IR predissociation spectrum of Na+(glucose), which clearly indicates contributions from multiple structures, was experimentally disentangled to reveal the presence of three α-conformers and five β-conformers. Comparisons with calculations show that these eight conformations correspond to the lowest energy gas-phase structures with distinctive Na+ coordination. [Figure not available: see fulltext.

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.

Kinetic evidence for interaction of TMPyP4 with two different G-quadruplex conformations of human telomeric DNA.

PubMed

Pérez-Arnaiz, Cristina; Busto, Natalia; Santolaya, Javier; Leal, José M; Barone, Giampaolo; García, Begoña

2018-03-01

Stabilization of G-quadruplex helices by small ligands has attracted growing attention because they inhibit the activity of the enzyme telomerase, which is overexpressed in >80% cancer cells. TMPyP4, one of the most studied G-quadruplex ligands, is used as a model to show that the ligands can exhibit different binding features with different conformations of a human telomeric specific sequence. UV-Vis, FRET melting Assay, Isothermal Titration Calorimetry, Time-resolved Fluorescence lifetime, T-Jump and Molecular Dynamics. TMPyP4 yields two different complexes with two Tel22 telomeric conformations in the presence of Na + or K + . T-Jump kinetic experiments show that the rates of formation and dissociation of these complexes in the ms time scale differ by one order of magnitude. MD simulations reveal that, in K + buffer, "hybrid 1" conformation yields kinetic constants on interaction with TMPyP4 one order lower than "hybrid 2". The binding involves π-π stacking with external loop bases. For the first time we show that for a particular buffer TMPyP4 interacts in a kinetically different way with the two Tel22 conformations even if the complexes formed are thermodynamically indistinguishable. G-quadruplexes, endowed with technological applications and potential impact on regulation mechanisms, define a new research field. The possibility of building different conformations from same sequence is a complex issue that confers G-quadruplexes very interesting features. The obtaining of reliable kinetic data constitutes an efficient tool to determine reaction mechanisms between conformations and small molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

Electrostatics effects on Ca(2+) binding and conformational changes in EF-hand domains: Functional implications for EF-hand proteins.

PubMed

Ababou, Abdessamad; Zaleska, Mariola

2015-12-01

Mutations of Gln41 and Lys75 with nonpolar residues in the N-terminal domain of calmodulin (N-Cam) revealed the importance of solvation energetics in conformational change of Ca(2+) sensor EF-hand domains. While in general these domains have polar residues at these corresponding positions yet the extent of their conformational response to Ca(2+) binding and their Ca(2+) binding affinity can be different from N-Cam. Consequently, here we address the charge state of the polar residues at these positions. The results show that the charge state of these polar residues can affect substantially the conformational change and the Ca(2+) binding affinity of our N-Cam variants. Since all the variants kept their conformational activity in the presence of Ca(2+) suggests that the differences observed among them mainly originate from the difference in their molecular dynamics. Hence we propose that the molecular dynamics of Ca(2+) sensor EF-hand domains is a key factor in the multifunctional aspect of EF-hand proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

From a structural average to the conformational ensemble of a DNA bulge

PubMed Central

Shi, Xuesong; Beauchamp, Kyle A.; Harbury, Pehr B.; Herschlag, Daniel

2014-01-01

Direct experimental measurements of conformational ensembles are critical for understanding macromolecular function, but traditional biophysical methods do not directly report the solution ensemble of a macromolecule. Small-angle X-ray scattering interferometry has the potential to overcome this limitation by providing the instantaneous distance distribution between pairs of gold-nanocrystal probes conjugated to a macromolecule in solution. Our X-ray interferometry experiments reveal an increasing bend angle of DNA duplexes with bulges of one, three, and five adenosine residues, consistent with previous FRET measurements, and further reveal an increasingly broad conformational ensemble with increasing bulge length. The distance distributions for the AAA bulge duplex (3A-DNA) with six different Au-Au pairs provide strong evidence against a simple elastic model in which fluctuations occur about a single conformational state. Instead, the measured distance distributions suggest a 3A-DNA ensemble with multiple conformational states predominantly across a region of conformational space with bend angles between 24 and 85 degrees and characteristic bend directions and helical twists and displacements. Additional X-ray interferometry experiments revealed perturbations to the ensemble from changes in ionic conditions and the bulge sequence, effects that can be understood in terms of electrostatic and stacking contributions to the ensemble and that demonstrate the sensitivity of X-ray interferometry. Combining X-ray interferometry ensemble data with molecular dynamics simulations gave atomic-level models of representative conformational states and of the molecular interactions that may shape the ensemble, and fluorescence measurements with 2-aminopurine-substituted 3A-DNA provided initial tests of these atomistic models. More generally, X-ray interferometry will provide powerful benchmarks for testing and developing computational methods. PMID:24706812

Uncoupling of Protease trans-Cleavage and Helicase Activities in Pestivirus NS3

PubMed Central

Zheng, Fengwei; Lu, Guoliang; Li, Ling

2017-01-01

ABSTRACT The nonstructural protein NS3 from the Flaviviridae family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å2 intramolecular protease-helicase interface comprising three clusters of interactions, representing a “closed” global conformation related to the NS3-NS4A cis-cleavage event. Although this conformation is incompatible with protease trans-cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo. Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein. IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis-cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different conformational states. PMID:28835495

The conformational dynamics of the mitochondrial Hsp70 chaperone.

PubMed

Mapa, Koyeli; Sikor, Martin; Kudryavtsev, Volodymyr; Waegemann, Karin; Kalinin, Stanislav; Seidel, Claus A M; Neupert, Walter; Lamb, Don C; Mokranjac, Dejana

2010-04-09

Heat shock proteins 70 (Hsp70) represent a ubiquitous and conserved family of molecular chaperones involved in a plethora of cellular processes. The dynamics of their ATP hydrolysis-driven and cochaperone-regulated conformational cycle are poorly understood. We used fluorescence spectroscopy to analyze, in real time and at single-molecule resolution, the effects of nucleotides and cochaperones on the conformation of Ssc1, a mitochondrial member of the family. We report that the conformation of its ADP state is unexpectedly heterogeneous, in contrast to a uniform ATP state. Substrates are actively involved in determining the conformation of Ssc1. The J protein Mdj1 does not interact transiently with the chaperone, as generally believed, but rather is released slowly upon ATP hydrolysis. Analysis of the major bacterial Hsp70 revealed important differences between highly homologous members of the family, possibly explaining tuning of Hsp70 chaperones to meet specific functions in different organisms and cellular compartments. 2010 Elsevier Inc. All rights reserved.

IR-IR Conformation Specific Spectroscopy of Na +(Glucose) Adducts

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

Voss, Jonathan M.; Kregel, Steven J.; Fischer, Kaitlyn C.

Here in this paper we report an IR-IR double resonance study of the structural landscape present in the Na +(glucose) complex. Our experimental approach involves minimal modifications to a typical IR predissociation setup, and can be carried out via ion-dip or isomer-burning methods, providing additional flexibility to suit different experimental needs. In the current study, the single-laser IR predissociation spectrum of Na +(glucose), which clearly indicates contributions from multiple structures, was experimentally disentangled to reveal the presence of three α-conformers and five β-conformers. Comparisons with calculations show that these eight conformations correspond to the lowest energy gas-phase structures with distinctivemore » Na+ coordination.« less

IR-IR Conformation Specific Spectroscopy of Na +(Glucose) Adducts

DOE PAGES

Voss, Jonathan M.; Kregel, Steven J.; Fischer, Kaitlyn C.; ...

2017-09-27

Here in this paper we report an IR-IR double resonance study of the structural landscape present in the Na +(glucose) complex. Our experimental approach involves minimal modifications to a typical IR predissociation setup, and can be carried out via ion-dip or isomer-burning methods, providing additional flexibility to suit different experimental needs. In the current study, the single-laser IR predissociation spectrum of Na +(glucose), which clearly indicates contributions from multiple structures, was experimentally disentangled to reveal the presence of three α-conformers and five β-conformers. Comparisons with calculations show that these eight conformations correspond to the lowest energy gas-phase structures with distinctivemore » Na+ coordination.« less

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

Analysis of the structure and dynamics of human serum albumin.

PubMed

Guizado, T R Cuya

2014-10-01

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

Conformational Transition Pathways in Signaling and Enzyme Catalysis Explored by Computational Methods

NASA Astrophysics Data System (ADS)

Pachov, Dimitar V.

Biomolecules are dynamic in nature and visit a number of states while performing their biological function. However, understanding how they interconvert between functional substates is a challenging task. In this thesis, we employ enhanced computational strategies to reveal in atomistic resolution transition states and molecular mechanism along conformational pathways of the signaling protein Nitrogen Regulatory Protein C (NtrC) and the enzyme Adenylate Kinase (Adk). Targeted Molecular Dynamics (TMD) simulations and NMR experiments have previously found the active/inactive interconversion of NtrC is stabilized by non-native transient contacts. To find where along the conformational pathway they lie and probe the existence of multiple intermediates, a beyond 8mus-extensive mapping of the conformational landscape was performed by a multitude of straightforward MD simulations relaxed from the biased TMD pathway. A number of metastable states stabilized by local interactions was found to underline the conformational pathway of NtrC. Two spontaneous transitions of the last stage of the active-to-inactive conversion were identified and used in path sampling procedures to generate an ensemble of truly dynamic reactive pathways. The transition state ensemble (TSE) and mechanistic descriptors of this transition were revealed in atomic detail and verified by committor analysis. By analyzing how pressure affects the dynamics and function of two homologous Adk proteins - the P.Profundum Adk surviving at 700atm pressure in the deep sea, and the E. coli Adk that lives at ambient pressures - we indirectly obtained atomic information about the TSE of the large-amplitude rate-limiting conformational opening of the Adk lids. Guided by NMR experiments showing significantly decreased activation volumes of the piezophile compared to its mesophilic counterpart, TMD simulations revealed the formation of an extended hydrogen-bonded water network in the transition state of the piezophile that can explain the experimentally measured activation volume differences. The transition state of the conformational change was proposed to lie close to the closed state. Additionally, a number of descriptors were used to characterize the free energy landscape of the mesophile. It was found that the features of landscape are highly sensitive to the binding of different ligands, their protonation states and the presence of magnesium.

To conform or not to conform: spontaneous conformity diminishes the sensitivity to monetary outcomes.

PubMed

Yu, Rongjun; Sun, Sai

2013-01-01

When people have different opinions in a group, they often adjust their own attitudes and behaviors to match the group opinion, known as social conformity. The affiliation account of normative conformity states that people conform to norms in order to 'fit in', whereas the accuracy account of informative conformity posits that the motive to learn from others produces herding. Here, we test another possibility that following the crowd reduces the experienced negative emotion when the group decision turns out to be a bad one. Using event related potential (ERP) combined with a novel group gambling task, we found that participants were more likely to choose the option that was predominately chosen by other players in previous trials, although there was little explicit normative pressure at the decision stage and group choices were not informative. When individuals' choices were different from others, the feedback related negativity (FRN), an ERP component sensitive to losses and errors, was enhanced, suggesting that being independent is aversive. At the outcome stage, the losses minus wins FRN effect was significantly reduced following conformity choices than following independent choices. Analyses of the P300 revealed similar patterns both in the response and outcome period. Our study suggests that social conformity serves as an emotional buffer that protects individuals from experiencing strong negative emotion when the outcomes are bad.

Structural Characterization of Proline-rich Tyrosine Kinase 2 (PYK2) Reveals a Unique (DFG-out) Conformation and Enables Inhibitor Design

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

Han, Seungil; Mistry, Anil; Chang, Jeanne S.

Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, non-receptor tyrosine kinase implicated in multiple signaling pathways. It is a negative regulator of osteogenesis and considered a viable drug target for osteoporosis treatment. The high-resolution structures of the human PYK2 kinase domain with different inhibitor complexes establish the conventional bilobal kinase architecture and show the conformational variability of the DFG loop. The basis for the lack of selectivity for the classical kinase inhibitor, PF-431396, within the FAK family is explained by our structural analyses. Importantly, the novel DFG-out conformation with two diarylurea inhibitors (BIRB796, PF-4618433) reveals a distinct subclass of non-receptormore » tyrosine kinases identifiable by the gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. This is the first example of a leucine residue in the hinge loop that blocks the ATP binding site in the DFG-out conformation. Our structural, biophysical, and pharmacological studies suggest that the unique features of the DFG motif, including Leu-504 hinge-loop variability, can be exploited for the development of selective protein kinase inhibitors.« less

Unique Aspects of the Structure and Dynamics of Elementary Iβ Cellulose Microfibrils Revealed by Computational Simulations1[OPEN

PubMed Central

Oehme, Daniel P.; Downton, Matthew T.; Doblin, Monika S.; Wagner, John; Gidley, Michael J.; Bacic, Antony

2015-01-01

The question of how many chains an elementary cellulose microfibril contains is critical to understanding the molecular mechanism(s) of cellulose biosynthesis and regulation. Given the hexagonal nature of the cellulose synthase rosette, it is assumed that the number of chains must be a multiple of six. We present molecular dynamics simulations on three different models of Iβ cellulose microfibrils, 18, 24, and 36 chains, to investigate their structure and dynamics in a hydrated environment. The 36-chain model stays in a conformational space that is very similar to the initial crystalline phase, while the 18- and 24-chain models sample a conformational space different from the crystalline structure yet similar to conformations observed in recent high-temperature molecular dynamics simulations. Major differences in the conformations sampled between the different models result from changes to the tilt of chains in different layers, specifically a second stage of tilt, increased rotation about the O2-C2 dihedral, and a greater sampling of non-TG exocyclic conformations, particularly the GG conformation in center layers and GT conformation in solvent-exposed exocyclic groups. With a reinterpretation of nuclear magnetic resonance data, specifically for contributions made to the C6 peak, data from the simulations suggest that the 18- and 24-chain structures are more viable models for an elementary cellulose microfibril, which also correlates with recent scattering and diffraction experimental data. These data inform biochemical and molecular studies that must explain how a six-particle cellulose synthase complex rosette synthesizes microfibrils likely comprised of either 18 or 24 chains. PMID:25786828

Paths to equilibrium in non-conformal collisions

NASA Astrophysics Data System (ADS)

Attems, Maximilian; Bea, Yago; Casalderrey-Solana, Jorge; Mateos, David; Santos-Oliván, Daniel; Sopuerta, Carlos F.; Triana, Miquel; Zilhão, Miguel

2018-03-01

Ever since fast hydrodynamization has been observed in heavy ion collisions the understanding of the hot early out-of-equilibrium stage of such collisions has been a topic of intense research. We use the gauge/gravity duality to model the creation of a strongly coupled Quark-Gluon plasma in a non-conformal gauge theory. This numerical relativity study is the first non-conformal holographic simulation of a heavy ion collision and reveals the existence of new relaxation channels due to the presence of non-vanishing bulk viscosity. We study shock wave collisions at different energies in gauge theories with different degrees of non-conformality and compare three relaxation times which can occur in different orderings: the hydrodynamization time (when hydrodynamics becomes applicable), the EoSization time (when the average pressure approaches its equilibrium value) and the condensate relaxation time (when the expectation value of a scalar operator approaches its equilibrium value). We find that these processes can occur in several different orderings. In particular, the condensate can remain far from equilibrium even long after the plasma has hydrodynamized and EoSized.

Non-rigid molecule of copper(II) diiminate Cu[CF3C(NH)C(F)C(NH)CF3]2, its conformational polymorphism in crystal and structure in solutions (Raman, UV-vis and quantum chemistry study)

NASA Astrophysics Data System (ADS)

Bukalov, Sergey S.; Aysin, Rinat R.; Leites, Larissa A.; Kurykin, Mikhail A.; Khrustalev, Victor N.

2015-10-01

Calculation of potential energy surface (PES) of isolated molecule of copper(II) diiminate Cu[CF3С(NH)C(F)C(NH)CF3]2 (1) resulted a double-well curve with the minima corresponding to equivalent screwed conformations. The low barrier leads to molecular non-rigidity which seems to be the reason of conformational polymorphism in crystals, reported in [1]. For one of newly found polymorphs, the X-ray structure was determined. The differences in the Raman and UV-vis spectra between differently colored species and their solutions were revealed, they are determined by different geometries of Cu(II) coordination polyhedron and different systems of intermolecular interactions in crystals. Transformations of the polymorphs under thermal, mechanical and photo exposures were studied.

Two conformational states of the membrane-associated Bacillus thuringiensis Cry4Ba {delta}-endotoxin complex revealed by electron crystallography: Implications for toxin-pore formation

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

Ounjai, Puey; Laboratory of Molecular Biophysics and Structural Biochemistry, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom 73170; Unger, Vinzenz M.

The insecticidal nature of Cry {delta}-endotoxins produced by Bacillus thuringiensis is generally believed to be caused by their ability to form lytic pores in the midgut cell membrane of susceptible insect larvae. Here we have analyzed membrane-associated structures of the 65-kDa dipteran-active Cry4Ba toxin by electron crystallography. The membrane-associated toxin complex was crystallized in the presence of DMPC via detergent dialysis. Depending upon the charge of the adsorbed surface, 2D crystals of the oligomeric toxin complex have been captured in two distinct conformations. The projection maps of those crystals have been generated at 17 A resolution. Both complexes appeared tomore » be trimeric; as in one crystal form, its projection structure revealed a symmetrical pinwheel-like shape with virtually no depression in the middle of the complex. The other form revealed a propeller-like conformation displaying an obvious hole in the center region, presumably representing the toxin-induced pore. These crystallographic data thus demonstrate for the first time that the 65-kDa activated Cry4Ba toxin in association with lipid membranes could exist in at least two different trimeric conformations, conceivably implying the closed and open states of the pore.« less

Exploration of the conformational landscape in pregnane X receptor reveals a new binding pocket

PubMed Central

Chandran, Aneesh

2016-01-01

Abstract Ligand‐regulated pregnane X receptor (PXR), a member of the nuclear receptor superfamily, plays a central role in xenobiotic metabolism. Despite its critical role in drug metabolism, PXR activation can lead to adverse drug‐drug interactions and early stage metabolism of drugs. Activated PXR can induce cancer drug resistance and enhance the onset of malignancy. Since promiscuity in ligand binding makes it difficult to develop competitive inhibitors targeting PXR ligand binding pocket (LBP), it is essential to identify allosteric sites for effective PXR antagonism. Here, molecular dynamics (MD) simulation studies unravelled the existence of two different conformational states, namely “expanded” and “contracted”, in apo PXR ligand binding domain (LBD). Ligand binding events shifted this conformational equilibrium and locked the LBD in a single “ligand‐adaptable” conformational state. Ensemble‐based computational solvent mapping identified a transiently open potential small molecule binding pocket between α5 and α8 helices, named “α8 pocket”, whose opening‐closing mechanism directly correlated with the conformational shift in LBD. A virtual hit identified through structure‐based virtual screening against α8 pocket locks the pocket in its open conformation. MD simulations further revealed that the presence of small molecule at allosteric site disrupts the LBD dynamics and locks the LBD in a “tightly‐contracted” conformation. The molecular details provided here could guide new structural studies to understand PXR activation and antagonism. PMID:27515410

Syn- and anti-conformations of 5'-deoxy- and 5'-O-methyl-uridine 2',3'-cyclic monophosphate.

PubMed

Grabarkiewicz, Tomasz; Hoffmann, Marcin

2006-01-01

Two uridine 2',3'-cyclic monophosphate (cUMP) derivatives, 5'-deoxy (DcUMP) and 5'-O-methyl (McUMP), were studied by means of quantum chemical methods. Aqueous solvent effects were estimated based on the isodensity-surface polarized-continuum model (IPCM). Gas phase calculations revealed only slight energy differences between the syn- and anti-conformers of both compounds: the relative energies of the syn-structure are -0.9 and 0.2 kcal mol(-1) for DcUMP and McUMP, respectively. According to the results from the IPCM calculations, however, both syn-conformers become about 14 kcal mol(-1) more stable in aqueous solution than their corresponding anti-structures. Additionally, the effects of a countercation and protonation on DcUMP were studied, revealing that the syn-structure is also favored over the anti-one for these systems.

Comparison of Palivizumab-Like Antibody Binding to Different Conformations of the RSV F Protein in RSV-Infected Adult Hematopoietic Cell Transplant Recipients.

PubMed

Ye, Xunyan; Iwuchukwu, Obinna P; Avadhanula, Vasanthi; Aideyan, Letisha O; McBride, Trevor J; Ferlic-Stark, Laura L; Patel, Kirtida D; Piedra, Felipe-Andres; Shah, Dimpy P; Chemaly, Roy F; Piedra, Pedro A

2018-03-28

Most respiratory syncytial virus (RSV) vaccine candidates include fusion (F) protein in different conformations. Antigenic site II found in the different F conformations is the target of palivizumab, the only US Food and Drug Administration approved monoclonal antibody (mAb). Serum palivizumab-like antibody (PLA) is a potential serologic correlate of immunity. Our objective was to determine if different conformations of F protein in a palivizumab competitive antibody (PCA) assay affect the PLA concentrations. Four PCA assays were standardized using mAbs. Each contained prefusion, postfusion, or intermediate F forms. PLA concentrations were measured in acute and convalescent sera from 22 RSV/A and 18 RSV/B-infected adult hematopoietic cell transplant (HCT) recipients. PLA concentrations were calculated using a 4-parameter logistic regression model and analyzed for statistical significance. PCA assays revealed significantly greater PLA concentrations in convalescent sera; comparable increases in PLA concentration in RSV/A and RSV/B-infected HCT recipients; and significantly reduced PLA concentrations in HCT recipients who shed RSV ≥14 days. A significant positive correlation was observed between PCA assays and RSV neutralizing antibody titers. F protein conformation does not appear to have a measurable impact on PCA assays for measuring PLA induced by RSV/A or RSV/B infection.

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

PubMed Central

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

2013-01-01

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

Estimating conformation content of a protein using citrate-stabilized Au nanoparticles

NASA Astrophysics Data System (ADS)

Deka, Jashmini; Paul, Anumita; Chattopadhyay, Arun

2010-08-01

Herein we report the use of the optical properties of citrate-stabilized gold nanoparticles (Au NPs) for estimation of native or denatured conformation content in a mixture of a protein in solution. The UV-vis extinction spectrum of citrate-stabilized Au NPs is known to broaden differently in the presence of native and denatured states of α-amylase, bovine serum albumin (BSA) or amyloglucosidase (AMG). On the other hand, herein we show that when a mixture of native and denatured protein was present in the medium, the broadening of the spectrum differed for different fractional content of the conformations. Also, the total area under the extinction spectrum varied linearly with the change in the mole fraction content of a state and for a constant total protein concentration. Transmission electron microscopy (TEM) measurements revealed different levels of agglomeration for different fractional contents of the native or denatured state of a protein. In addition, time-dependent denaturation of a protein could be followed using the present method. The rate constants calculated for denaturation indicated a possible fast change in conformation of a protein before complete thermal denaturation. The observations have been explained based on the changes in extinction coefficient (thereby oscillator strength) upon interaction of citrate-stabilized NPs with proteins being in different states and levels of agglomeration.Herein we report the use of the optical properties of citrate-stabilized gold nanoparticles (Au NPs) for estimation of native or denatured conformation content in a mixture of a protein in solution. The UV-vis extinction spectrum of citrate-stabilized Au NPs is known to broaden differently in the presence of native and denatured states of α-amylase, bovine serum albumin (BSA) or amyloglucosidase (AMG). On the other hand, herein we show that when a mixture of native and denatured protein was present in the medium, the broadening of the spectrum differed for different fractional content of the conformations. Also, the total area under the extinction spectrum varied linearly with the change in the mole fraction content of a state and for a constant total protein concentration. Transmission electron microscopy (TEM) measurements revealed different levels of agglomeration for different fractional contents of the native or denatured state of a protein. In addition, time-dependent denaturation of a protein could be followed using the present method. The rate constants calculated for denaturation indicated a possible fast change in conformation of a protein before complete thermal denaturation. The observations have been explained based on the changes in extinction coefficient (thereby oscillator strength) upon interaction of citrate-stabilized NPs with proteins being in different states and levels of agglomeration. Electronic supplementary information (ESI) available: Additional UV-vis and fluorescence spectra and graphs based on UV-vis studies. See DOI: 10.1039/c0nr00154f

Elucidation of Different Binding Modes of Purine Nucleosides to Human Deoxycytidine Kinase

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

Sabini, Elisabetta; Hazra, Saugata; Konrad, Manfred

2008-07-30

Purine nucleoside analogues of medicinal importance, such as cladribine, require phosphorylation by deoxycytidine kinase (dCK) for pharmacological activity. Structural studies of ternary complexes of human dCK show that the enzyme conformation adjusts to the different hydrogen-bonding properties between dA and dG and to the presence of substituent at the 2-position present in dG and cladribine. Specifically, the carbonyl group in dG elicits a previously unseen conformational adjustment of the active site residues Arg104 and Asp133. In addition, dG and cladribine adopt the anti conformation, in contrast to the syn conformation observed with dA. Kinetic analysis reveals that cladribine is phosphorylatedmore » at the highest efficiency with UTP as donor. We attribute this to the ability of cladribine to combine advantageous properties from dA (favorable hydrogen-bonding pattern) and dG (propensity to bind to the enzyme in its anti conformation), suggesting that dA analogues with a substituent at the 2-position are likely to be better activated by human dCK.« less

Backbone conformations and side chain flexibility of two somatostatin mimics investigated by molecular dynamics simulations.

PubMed

Interlandi, Gianluca

2009-05-15

Molecular dynamics simulations with two designed somatostatin mimics, SOM230 and SMS 201-995, were performed in explicit water for a total aggregated time of 208 ns. Analysis of the runs with SOM230 revealed the presence of two clusters of conformations. Strikingly, the two sampled conformers correspond to the two main X-ray structures in the asymmetric unit of SMS 201-995. Structural comparison between the residues of SOM230 and SMS 201-995 provides an explanation for the high binding affinity of SOM230 to four of five somatostatin receptors. Similarly, cluster analysis of the simulations with SMS 201-995 shows that the backbone of the peptide interconverts between its two main crystallographic conformers. The conformations of SMS 201-995 sampled in the two clusters violated two different sets of NOE distance constraints in agreement with a previous NMR study. Differences in side chain fluctuations between SOM230 and SMS 201-995 observed in the simulations may contribute to the relatively higher binding affinity of SOM230 to most somatostatin receptors.

Molecular dynamics of conformation-specific dopamine transporter-inhibitor complexes.

PubMed

Jean, Bernandie; Surratt, Christopher K; Madura, Jeffry D

2017-09-01

The recreational psychostimulant cocaine inhibits dopamine reuptake from the synapse, resulting in excessive stimulation of postsynaptic dopamine receptors in brain areas associated with reward and addiction. Cocaine binds to and stabilizes the outward- (extracellular-) facing conformation of the dopamine transporter (DAT) protein, while the low abuse potential DAT inhibitor benztropine prefers the inward- (cytoplasmic-) facing conformation. A correlation has been previously postulated between psychostimulant abuse potential and preference for the outward-facing DAT conformation. The 3β-aryltropane cocaine analogs LX10 and LX11, however, differ only in stereochemistry and share a preference for the outward-facing DAT, yet are reported to vary widely in abuse potential in an animal model. In search of the molecular basis for DAT conformation preference, complexes of cocaine, benztropine, LX10 or LX11 bound to each DAT conformation were subjected to 100ns of all-atom molecular dynamics simulation. Results were consistent with previous findings from cysteine accessibility assays used to assess an inhibitor's DAT conformation preference. The respective 2β- and 2α-substituted phenyltropanes of LX10 and LX11 interacted with hydrophobic regions of the DAT S1 binding site that were inaccessible to cocaine. Solvent accessibility measurements also revealed subtle differences in inhibitor positioning within a given DAT conformation. This work serves to advance our understanding of the conformational selectivity of DAT inhibitors and suggests that MD may be useful in antipsychostimulant therapeutic design. Copyright © 2017 Elsevier Inc. All rights reserved.

Modeling of a C-end rule peptide adsorbed onto gold nanoparticles.

PubMed

Triguero, Jordi; Flores-Ortega, Alejandra; Zanuy, David; Alemán, Carlos

2018-01-01

The RPAR peptide, a prototype C-end Rule (CendR) sequence that binds to neuropilin-1 (NRP-1), has potential therapeutic uses as internalization trigger in anticancer nanodevices. Recently, the functionalization of gold nanoparticles with CendR peptides has been proved to be a successful strategy to target the NRP-1 receptor in prostate cancer cells. In this work, we investigate the influence of two gold surface facets, (100) and (111), on the conformational preferences of RPAR using molecular dynamics simulations. Both clustering and conformational analyses revealed that the peptide backbone becomes very rigid upon adsorption onto gold, which is a very fast and favored process, the only flexibility being attributed to the side chains of the two Arg residues. Thus, the different components of RPAR tend to adopt an elongated shape, which is characterized by the pseudo-extended conformation of both the backbone and the Arg side chains. This conformation is very different from the already known bioactive conformation, indicating that RPAR is drastically affected by the substrate. Interestingly, the preferred conformations of the peptide adsorbed onto gold facets are not stabilized by salt bridges and/or specific intramolecular hydrogen bonds, which represent an important difference with respect to the conformations found in other environments (e.g. the peptide in solution and interacting with NRP-1 receptor). However, the conformational changes induced by the substrate are not detrimental for the use of gold nanoparticles as appropriate vehicles for the transport and targeted delivery of the RPAR. Thus, once their high affinity for the NRP-1 receptor induces the targeted delivery of the elongated peptide molecules from the gold nanoparticles, the lack of intramolecular interactions facilitates their evolution towards the bioactive conformation, increasing the therapeutic efficacy of the peptide. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Unique aspects of the structure and dynamics of elementary Iβ cellulose microfibrils revealed by computational simulations.

PubMed

Oehme, Daniel P; Downton, Matthew T; Doblin, Monika S; Wagner, John; Gidley, Michael J; Bacic, Antony

2015-05-01

The question of how many chains an elementary cellulose microfibril contains is critical to understanding the molecular mechanism(s) of cellulose biosynthesis and regulation. Given the hexagonal nature of the cellulose synthase rosette, it is assumed that the number of chains must be a multiple of six. We present molecular dynamics simulations on three different models of Iβ cellulose microfibrils, 18, 24, and 36 chains, to investigate their structure and dynamics in a hydrated environment. The 36-chain model stays in a conformational space that is very similar to the initial crystalline phase, while the 18- and 24-chain models sample a conformational space different from the crystalline structure yet similar to conformations observed in recent high-temperature molecular dynamics simulations. Major differences in the conformations sampled between the different models result from changes to the tilt of chains in different layers, specifically a second stage of tilt, increased rotation about the O2-C2 dihedral, and a greater sampling of non-TG exocyclic conformations, particularly the GG conformation in center layers and GT conformation in solvent-exposed exocyclic groups. With a reinterpretation of nuclear magnetic resonance data, specifically for contributions made to the C6 peak, data from the simulations suggest that the 18- and 24-chain structures are more viable models for an elementary cellulose microfibril, which also correlates with recent scattering and diffraction experimental data. These data inform biochemical and molecular studies that must explain how a six-particle cellulose synthase complex rosette synthesizes microfibrils likely comprised of either 18 or 24 chains. © 2015 American Society of Plant Biologists. All Rights Reserved.

NMR-based conformational analysis of perezone and analogues.

PubMed

Zepeda, L Gerardo; Burgueño-Tapia, Eleuterio; Pérez-Hernández, Nury; Cuevas, Gabriel; Joseph-Nathan, Pedro

2013-04-01

Complete assignment of the (1)H NMR chemical shift and coupling constant values of perezone (1), O-methylperezone (2) and 6-hydroxyperezone (3) was carried out by total-line-shape-fitting calculations using the PERCH iterative spectra analysis software (PERCH Solutions Ltd., Kuopio, Finland). The resulting simulated spectra for the three compounds showed strong similarity to their corresponding experimental spectra. Particularly, all vicinal, allylic and homoallylic coupling constant values for the side chain of the three compounds were very similar, thus revealing that the conformation of these three molecules in solution is indeed almost identical. This fact is in agreement with extended side chain conformations over folded chain conformations because 1, 2 and 3 undergo completely different intramolecular cycloaddition reactions. In addition, results of double pulsed field gradient spin echo NOESY 1D experiments performed on perezone (1) were unable to provide evidence for folded conformers. Copyright © 2013 John Wiley & Sons, Ltd.

Molecular structure and conformational preferences of 1-bromo-1-silacyclohexane, CH2(CH2CH2)2SiH-Br, as studies by gas-phase electron diffraction and quantum chemistry

NASA Astrophysics Data System (ADS)

Belyakov, A. V.; Baskakov, A. A.; Naraev, V. N.; Rykov, A. N.; Oberhammer, H.; Arnason, I.; Wallevik, S. O.

2012-10-01

The molecular structure of axial and equatorial conformer of the 1-bromo-1-silacyclohexane molecule, CH2(CH2CH2)2SiH-Br, as well as thermodynamic equilibrium between these species are investigated by means of gas-phase electron diffraction and quantum chemistry on the MP2(full)/SDB-AUG-cc-PVTZ level of theory. It is revealed that according to electron diffraction data, the compound exists in the gasphase as a mixture of conformers possessing the chair conformation of the six-membered ring and C s symmetry and differing in the axial and equatorial position of the Si-Br bond (ax. = 80(5) mol %, eq. = 20(7) mol %) at 352 K, that corresponds to the value of A = ( G {ax/○} - G {eq/○}) = -0.82(32) kcal/mol. It is found that observed data agree well with theoretical ones. Using Natural Bond Orbital (NBO) analysis it is revealed that axial conformer of 1-bromo-1-silacyclohexane molecule is an example of the stabilization of the form that is unfavorable from the point of view of steric effects and effects of conjugations. It is concluded that stabilization is achieved due to electrostatic interactions.

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

Changela, Anita; DiGate, Russell J.; Mondragon, Alfonso

Escherichia coli DNA topoisomerase III belongs to the type IA family of DNA topoisomerases, which transiently cleave single-stranded DNA (ssDNA) via a 5{prime} phosphotyrosine intermediate. We have solved crystal structures of wild-type E. coli topoisomerase III bound to an eight-base ssDNA molecule in three different pH environments. The structures reveal the enzyme in three distinct conformational states while bound to DNA. One conformation resembles the one observed previously with a DNA-bound, catalytically inactive mutant of topoisomerase III where DNA binding realigns catalytic residues to form a functional active site. Another conformation represents a novel intermediate in which DNA is boundmore » along the ssDNA-binding groove but does not enter the active site, which remains in a catalytically inactive, closed state. A third conformation shows an intermediate state where the enzyme is still in a closed state, but the ssDNA is starting to invade the active site. For the first time, the active site region in the presence of both the catalytic tyrosine and ssDNA substrate is revealed for a type IA DNA topoisomerase, although there is no evidence of ssDNA cleavage. Comparative analysis of the various conformational states suggests a sequence of domain movements undertaken by the enzyme upon substrate binding.« less

Conformal anomaly of generalized form factors and finite loop integrals

NASA Astrophysics Data System (ADS)

Chicherin, Dmitry; Sokatchev, Emery

2018-04-01

We reveal a new mechanism of conformal symmetry breaking at Born level. It occurs in generalized form factors with several local operators and an on-shell state of massless particles. The effect is due to hidden singularities on collinear configurations of the momenta. This conformal anomaly is different from the holomorphic anomaly of amplitudes. We present a number of examples in four and six dimensions. We find an application of the new conformal anomaly to finite loop momentum integrals with one or more massless legs. The collinear region around a massless leg creates a contact anomaly, made visible by the loop integration. The anomalous conformal Ward identity for an ℓ-loop integral is a 2nd-order differential equation whose right-hand side is an (ℓ - 1)-loop integral. It could serve as a new useful tool to find/test analytic expressions for conformal integrals. We illustrate this point with several examples of known integrals. We propose a new differential equation for the four-dimensional scalar double box.

Different Conformations of Surface Cellulose Molecules in Native Cellulose Microfibrils Revealed by Layer-by-Layer Peeling.

PubMed

Funahashi, Ryunosuke; Okita, Yusuke; Hondo, Hiromasa; Zhao, Mengchen; Saito, Tsuguyuki; Isogai, Akira

2017-11-13

Layer-by-layer peeling of surface molecules of native cellulose microfibrils was performed using a repeated sequential process of 2,2,6,6-tetramethylpiperidine-1-oxyl radical-mediated oxidation followed by hot alkali extraction. Both highly crystalline algal and tunicate celluloses and low-crystalline cotton and wood celluloses were investigated. Initially, the C6-hydroxy groups of the outermost surface molecules of each algal cellulose microfibril facing the exterior had the gauche-gauche (gg) conformation, whereas those facing the interior had the gauche-trans (gt) conformation. All the other C6-hydroxy groups of the cellulose molecules inside the microfibrils contributing to crystalline cellulose I had the trans-gauche (tg) conformation. After surface peeling, the originally second-layer molecules from the microfibril surface became the outermost surface molecules, and the original tg conformation changed to gg and gt conformations. The plant cellulose microfibrils likely had disordered structures for both the outermost surface and second-layer molecules, as demonstrated using the same layer-by-layer peeling technique.

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.

X-ray Crystal Structure of Aristolochene Synthase from Aspergillus terreus and Evolution of Templates for the Cyclization of Farnesyl Diphosphate

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

Shishova,E.; Di Costanzo, L.; Cane, D.

2007-01-01

Aristolochene synthase from Aspergillus terreus catalyzes the cyclization of the universal sesquiterpene precursor, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. The 2.2 {angstrom} resolution X-ray crystal structure of aristolochene synthase reveals a tetrameric quaternary structure in which each subunit adopts the {alpha}-helical class I terpene synthase fold with the active site in the 'open', solvent-exposed conformation. Intriguingly, the 2.15 {angstrom} resolution crystal structure of the complex with Mg{sup 2+}{sub 3}-pyrophosphate reveals ligand binding only to tetramer subunit D, which is stabilized in the 'closed' conformation required for catalysis. Tetramer assembly may hinder conformational changes required for the transition frommore » the inactive open conformation to the active closed conformation, thereby accounting for the attenuation of catalytic activity with an increase in enzyme concentration. In both conformations, but especially in the closed conformation, the active site contour is highly complementary in shape to that of aristolochene, and a catalytic function is proposed for the pyrophosphate anion based on its orientation with regard to the presumed binding mode of aristolochene. A similar active site contour is conserved in aristolochene synthase from Penicillium roqueforti despite the substantial divergent evolution of these two enzymes, while strikingly different active site contours are found in the sesquiterpene cyclases 5-epi-aristolochene synthase and trichodiene synthase. Thus, the terpenoid cyclase active site plays a critical role as a template in binding the flexible polyisoprenoid substrate in the proper conformation for catalysis. Across the greater family of terpenoid cyclases, this template is highly evolvable within a conserved {alpha}-helical fold for the synthesis of terpene natural products of diverse structure and stereochemistry.« less

Nuclear export receptor CRM1 recognizes diverse conformations in nuclear export signals.

PubMed

Fung, Ho Yee Joyce; Fu, Szu-Chin; Chook, Yuh Min

2017-03-10

Nuclear export receptor CRM1 binds highly variable nuclear export signals (NESs) in hundreds of different cargoes. Previously we have shown that CRM1 binds NESs in both polypeptide orientations (Fung et al., 2015). Here, we show crystal structures of CRM1 bound to eight additional NESs which reveal diverse conformations that range from loop-like to all-helix, which occupy different extents of the invariant NES-binding groove. Analysis of all NES structures show 5-6 distinct backbone conformations where the only conserved secondary structural element is one turn of helix that binds the central portion of the CRM1 groove. All NESs also participate in main chain hydrogen bonding with human CRM1 Lys568 side chain, which acts as a specificity filter that prevents binding of non-NES peptides. The large conformational range of NES backbones explains the lack of a fixed pattern for its 3-5 hydrophobic anchor residues, which in turn explains the large array of peptide sequences that can function as NESs.

Spectroscopic evidence of β-turn in N-glycated peptidomimetics related to leucine-enkephalin

NASA Astrophysics Data System (ADS)

Vass, E.; Hollósi, M.; Kveder, M.; Kojić-Prodić, B.; Čudić, M.; Horvat, Š.

2000-11-01

The conformational differences caused by N-glycation of the amide bond in endogenous opioid pentapeptide leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu) have been explored in solution using FTIR spectroscopy, NMR and molecular modelling. The compounds studied include protected and unprotected enkephalin analogues N-alkylated at the second (Gly 2) amino acid residue with a 6-deoxy- D-galactose moiety ( 1- 3). Comparison of the amide I component bands in the FTIR spectra, measured in trifluoroethanol (TFE), CHCl 3 and DMSO, revealed significant differences in the intensity as well as shifts in component band frequencies for glycopeptides 1- 3. We found that only the FTIR spectrum of the fully protected compound 1 indicated the presence of a higher population of β-turns, while the spectra of the partially protected and unprotected glycopeptides 2 and 3 reflected the dominance of unordered or open structures, with some low population of turns. The observed NOE connectivities in CDCl 3 for both isomers of the fully protected compound 1, the all-trans one and another with Tyr 1-Gly 2 peptide bond in cis conformation, indicate the presence of a β-like turn conformation. Molecular dynamics simulations of the glycopeptide 1 obtained by unconstrained energy minimization of trans- and cis- 1 shows that one of trans form conformations is consistent with β-turn whereas cis isomer has revealed less-compact turn.

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

Raghuwanshi, Vikram Singh; Garusinghe, Uthpala Manavi; Ilavsky, Jan

Controlling nanoparticles (NPs) aggregation in cellulose/NPs composites allows to optimise NPs driven properties and their applications. Polyelectrolytes are used to control NPs aggregation and their retention within the fibrous matrix. Here in this study, we aim at evaluating how a polyelectrolyte (Cationic Polyacrylamide; CPAM, molecular weight: 13 MDa, charge: 50%, Radius of gyration: 30–36 nm) adsorbs and re-conforms onto the surface of silica(SiO 2) NPs differing in diameter (8, 22 and 74 nm) and to investigate the respective NPs aggregation in cellulose matrices. SEM shows the local area distribution of NPs in composites. Ultra-SAXS (USAXS) allows to evaluate the averagemore » NPs size distribution and the inter-particle interactions at length scale ranging from 1 to 1000 nm. USAXS data analysis reveals that CPAM covers multiple NPs of the smaller diameter (8 nm), presumably with a single chain to form large size NPs aggregates. As the NPs diameter is increased to 22 nm, CPAM re-conforms over NP surface forming a large shell of thickness 5.5 nm. For the composites with NPs of diameter 74 nm, the CPAM chain re-conforms further onto NP surface and the surrounding shell thickness decreases to 2.2 nm. Lastly, structure factor analysis reveals higher structural ordering for NPs as increases their diameter, which is caused by different conformations adopted by CPAM onto NPs surface.« less

Effect of nanoparticles size and polyelectrolyte on nanoparticles aggregation in a cellulose fibrous matrix

DOE PAGES

Raghuwanshi, Vikram Singh; Garusinghe, Uthpala Manavi; Ilavsky, Jan; ...

2017-09-18

Controlling nanoparticles (NPs) aggregation in cellulose/NPs composites allows to optimise NPs driven properties and their applications. Polyelectrolytes are used to control NPs aggregation and their retention within the fibrous matrix. Here in this study, we aim at evaluating how a polyelectrolyte (Cationic Polyacrylamide; CPAM, molecular weight: 13 MDa, charge: 50%, Radius of gyration: 30–36 nm) adsorbs and re-conforms onto the surface of silica(SiO 2) NPs differing in diameter (8, 22 and 74 nm) and to investigate the respective NPs aggregation in cellulose matrices. SEM shows the local area distribution of NPs in composites. Ultra-SAXS (USAXS) allows to evaluate the averagemore » NPs size distribution and the inter-particle interactions at length scale ranging from 1 to 1000 nm. USAXS data analysis reveals that CPAM covers multiple NPs of the smaller diameter (8 nm), presumably with a single chain to form large size NPs aggregates. As the NPs diameter is increased to 22 nm, CPAM re-conforms over NP surface forming a large shell of thickness 5.5 nm. For the composites with NPs of diameter 74 nm, the CPAM chain re-conforms further onto NP surface and the surrounding shell thickness decreases to 2.2 nm. Lastly, structure factor analysis reveals higher structural ordering for NPs as increases their diameter, which is caused by different conformations adopted by CPAM onto NPs surface.« less

Ion mobility-mass spectrometry reveals conformational flexibility in the deubiquitinating enzyme USP5.

PubMed

Scott, Daniel; Layfield, Robert; Oldham, Neil J

2015-08-01

Many proteins exhibit conformation flexibility as part of their biological function, whether through the presence of a series of well-defined states or by the existence of intrinsic disorder. Ion mobility spectrometry, in combination with MS (IM-MS), offers a rapid and sensitive means of probing ensembles of protein structures through measurement of gas-phase collisional cross sections. We have applied IM-MS analysis to the multidomain deubiquitinating enzyme ubiquitin specific protease 5 (USP5), which is believed to exhibit significant conformational flexibility. Native ESI-MS measurement of the 94-kDa USP5 revealed two distinct charge-state distributions: [M + 17H](+) to [M + 21H](+) and [M + 24H](+) to [M + 29H](+). The collisional cross sections of these ions revealed clear groupings of 52 ± 4 nm(2) for the lower charges and 66 ± 6 nm(2) for the higher charges. Molecular dynamics simulation of a compact form of USP5, based on a crystal structure, produced structures of 53-54 nm(2) following 2 ns in the gas phase, while simulation of an extended form (based on small-angle X-ray scattering data) led to structures of 64 nm(2). These data demonstrate that IM-MS is a valuable tool in studying proteins with different discrete conformational states. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Desulfurization of 2-thiouracil nucleosides: conformational studies of 4-pyrimidinone nucleosides.

PubMed

Kraszewska, Karina; Kaczyńska, Iwona; Jankowski, Stefan; Karolak-Wojciechowska, Janina; Sochacka, Elzbieta

2011-04-01

4-Pyrimidinone ribofuranoside (H(2)o(4)U) and 4-pyrimidinone 2'-deoxyribofuranoside (dH(2)o(4)U) were synthesized by the oxidative desulfurization of parent 2-thiouracil nucleosides with m-chloroperbenzoic acid. The crystal structures of H(2)o(4)U and dH(2)o(4)U and their conformations in solution were determined and compared with corresponding 2-thiouracil and uracil nucleosides. The absence of a large 2-thiocarbonyl/2-carbonyl group in the nucleobase moiety results in C2'-endo puckering of the ribofuranose ring (S conformer) in the crystal structure of H(2)o(4)U, which is not typical of RNA nucleosides. Interestingly, the hydrogen bonding network in the crystals of dH(2)o(4)U stabilizes the sugar moiety conformation in the C3'-endo form (N conformer), rarely found in DNA nucleosides. In aqueous solution, dH(2)o(4)U reveals a similar population of the C2'-endo conformation (65%) to that of 2'-deoxy-2-thiouridine (62%), while the 62% population of the S conformer for H(2)o(4)U is significantly different from that of the parent 2-thiouridine, for which the N conformer is dominant (71%). Such a difference may be of biological importance, as the desulfurization process of natural tRNA 2-thiouridines may occur under conditions of oxidative stress in the cell and may influence the decoding process. Copyright © 2011 Elsevier Ltd. All rights reserved.

Biologically important conformational features of DNA as interpreted by quantum mechanics and molecular mechanics computations of its simple fragments.

PubMed

Poltev, V; Anisimov, V M; Dominguez, V; Gonzalez, E; Deriabina, A; Garcia, D; Rivas, F; Polteva, N A

2018-02-01

Deciphering the mechanism of functioning of DNA as the carrier of genetic information requires identifying inherent factors determining its structure and function. Following this path, our previous DFT studies attributed the origin of unique conformational characteristics of right-handed Watson-Crick duplexes (WCDs) to the conformational profile of deoxydinucleoside monophosphates (dDMPs) serving as the minimal repeating units of DNA strand. According to those findings, the directionality of the sugar-phosphate chain and the characteristic ranges of dihedral angles of energy minima combined with the geometric differences between purines and pyrimidines determine the dependence on base sequence of the three-dimensional (3D) structure of WCDs. This work extends our computational study to complementary deoxydinucleotide-monophosphates (cdDMPs) of non-standard conformation, including those of Z-family, Hoogsteen duplexes, parallel-stranded structures, and duplexes with mispaired bases. For most of these systems, except Z-conformation, computations closely reproduce experimental data within the tolerance of characteristic limits of dihedral parameters for each conformation family. Computation of cdDMPs with Z-conformation reveals that their experimental structures do not correspond to the internal energy minimum. This finding establishes the leading role of external factors in formation of the Z-conformation. Energy minima of cdDMPs of non-Watson-Crick duplexes demonstrate different sequence-dependence features than those known for WCDs. The obtained results provide evidence that the biologically important regularities of 3D structure distinguish WCDs from duplexes having non-Watson-Crick nucleotide pairing.

Ubiquitin dynamics in complexes reveal molecular recognition mechanisms beyond induced fit and conformational selection.

PubMed

Peters, Jan H; de Groot, Bert L

2012-01-01

Protein-protein interactions play an important role in all biological processes. However, the principles underlying these interactions are only beginning to be understood. Ubiquitin is a small signalling protein that is covalently attached to different proteins to mark them for degradation, regulate transport and other functions. As such, it interacts with and is recognised by a multitude of other proteins. We have conducted molecular dynamics simulations of ubiquitin in complex with 11 different binding partners on a microsecond timescale and compared them with ensembles of unbound ubiquitin to investigate the principles of their interaction and determine the influence of complex formation on the dynamic properties of this protein. Along the main mode of fluctuation of ubiquitin, binding in most cases reduces the conformational space available to ubiquitin to a subspace of that covered by unbound ubiquitin. This behaviour can be well explained using the model of conformational selection. For lower amplitude collective modes, a spectrum of zero to almost complete coverage of bound by unbound ensembles was observed. The significant differences between bound and unbound structures are exclusively situated at the binding interface. Overall, the findings correspond neither to a complete conformational selection nor induced fit scenario. Instead, we introduce a model of conformational restriction, extension and shift, which describes the full range of observed effects.

Solution‐crystallization and related phenomena in 9,9‐dialkyl‐fluorene polymers. II. Influence of side‐chain structure

PubMed Central

Perevedentsev, Aleksandr; Stavrinou, Paul N.; Smith, Paul

2015-01-01

ABSTRACT Solution‐crystallization is studied for two polyfluorene polymers possessing different side‐chain structures. Thermal analysis and temperature‐dependent optical spectroscopy are used to clarify the nature of the crystallization process, while X‐ray diffraction and scanning electron microscopy reveal important differences in the resulting microstructures. It is shown that the planar‐zigzag chain conformation termed the β‐phase, which is observed for certain linear‐side‐chain polyfluorenes, is necessary for the formation of so‐called polymer‐solvent compounds for these polymers. Introduction of alternating fluorene repeat units with branched side‐chains prevents formation of the β‐phase conformation and results in non‐solvated, i.e. melt‐crystallization‐type, polymer crystals. Unlike non‐solvated polymer crystals, for which the chain conformation is stabilized by its incorporation into a crystalline lattice, the β‐phase conformation is stabilized by complexation with solvent molecules and, therefore, its formation does not require specific inter‐chain interactions. The presented results clarify the fundamental differences between the β‐phase and other conformational/crystalline forms of polyfluorenes. © 2015 The Authors. Journal of Polymer Science Part B: Polymer Physics published by Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2015, 53, 1492–1506 PMID:27546983

Conformational Specific Infrared and Ultraviolet Spectroscopy of Cold YA(D-Pro)AA\\cdotH+ Ions: a Sterochemical "twist" on the Proline Effect

NASA Astrophysics Data System (ADS)

Harrilal, Christopher P.; DeBlase, Andrew F.; Burke, Nicole L.; McLuckey, Scott A.; Zwier, Timothy S.

2016-06-01

The "proline effect" is a well-known fragmentation phenomenon in mass spectrometry, in which y-fragments are produced preferentially over b-fragments during the collision induced dissociation of protonated L-proline containing peptide ions. This specific fragmentation channel is favored because of the high basicity of the secondary amine intermediate and the ring instability in alternative bn+ products [ASMS 2014, 25, 1705]. In contrast, peptides containing the D-Pro stereoisomer have been shown to largely favor the production of b4+ ions over y3+ ions. This strongly suggests that differences in the conformational preferences between the D-Pro and L-Pro diastereomers are likely to be responsible but structural evidence has been lacking to date. Using tandem mass spectrometry and IR-UV double resonant action spectroscopy we are able to compare the 3D structures of cold [YA(D-Pro)AA+H]+ to [YA(L-Pro)AA+H]+ ions. The UV action spectra reveals two major conformers in [YA(D-Pro)AA+H]+ and one major conformer in [YA(L-Pro)AA+H]+. Clear differences in the hydrogen bonding patterns are apparent between the two conformers observed in the D-Pro specie which are both distinct from the L-Pro diastereomer. Furthermore, conformer and diastereomer specific photofragmentation patterns are observed. It is also noted that a ten-fold photofragment enhancement unique to one of the D-Pro conformers is observed upon absorption of a resonant IR photon after UV excitation. Differences in the excited state photophysics between the two D-Pro conformers suggest that vibrational excitation of S1 turns on coupling to the dissociative -Tyr channel in one conformer, while this coupling is already present in the vibronic ground state of the other. Calculated harmonic spectra (M052X/6-31+G*) of conformers obtained from Monte Carlo searches to the experimental spectra.

Thermally induced conformational changes in polyethylene studied by two-dimensional near-infrared infrared hetero-spectral correlation spectroscopy

NASA Astrophysics Data System (ADS)

Watanabe, Shin; Noda, Isao; Ozaki, Yukihiro

2008-07-01

The amount of nonplanar gauche bonds was monitored as a function of increasing temperature in three different polyethylene (PE) samples by means of mid-infrared (MIR) and near-infrared (NIR) spectroscopy. The hetero-spectral two-dimensional (2D) correlation analysis was carried out between the NIR spectral region of 4365-4235 cm -1 and the well-established MIR spectral region of 1375-1265 cm -1, where bands due to nonplanar conformer are detected. This approach allowed us to identify the NIR band at 4265 cm -1, which behaves in a way similar to MIR bands originating from conformational-defect sequences. By combining the result of our current study and that of our previous report obtained on different types of PE, it is suggested that the NIR band originates from conformational-defect sequences in PE. This finding opens up a unique and useful way to study the state of conformational disorder in PE crystal by NIR spectroscopy, monitoring the intensity of the NIR band at 4265 cm -1. The use of NIR spectroscopy allows researchers to directly probe the degree in the formation of conformational-defect sequences in thick, real-world PE samples that cannot be studied by conventional MIR spectroscopy. The 2D correlation spectroscopy analysis among the MIR CH 2 wagging conformational-defect-mode bands on linear low-density PE (LLDPE) and low-density PE (LDPE) revealed the formation of nonplanar conformer represented by the band at 1368 cm -1 proceeds prior to those by other band at 1308 cm -1. This result agrees well with our previous finding on high-density PE (HDPE). We therefore propose with strong confidence that the bands at 1368 and 1308 cm -1 arise from different conformational-defect sequences, even though both of the bands have been proposed to arise from the same conformer of gtg' ( kink) + gtg sequence.

Conformers, infrared spectrum, UV-induced photochemistry, and near-IR-induced generation of two rare conformers of matrix-isolated phenylglycine.

PubMed

Borba, Ana; Gómez-Zavaglia, Andrea; Fausto, Rui

2014-10-21

The conformational space of α-phenylglycine (PG) have been investigated theoretically at both the DFT/B3LYP/6-311++G(d,p) and MP2/6-311++G(d,p) levels of approximation. Seventeen different minima were found on the investigated potential energy surfaces, which are characterized by different dominant intramolecular interactions: type I conformers are stabilized by hydrogen bonds of the type N-H···O=C, type II by a strong O-H···N hydrogen bond, type III by weak N-H···O-H hydrogen bonds, and type IV by a C=O···H-C contact. The calculations indicate also that entropic effects are relevant in determining the equilibrium populations of the conformers of PG in the gas phase, in particular in the case of conformers of type II, where the strong intramolecular O-H···N hydrogen bond considerably diminishes entropy by reducing the conformational mobility of the molecule. In consonance with the relative energies of the conformers and barriers for conformational interconversion, only 3 conformers of PG were observed for the compound isolated in cryogenic Ar, Xe, and N2 matrices: the conformational ground state (ICa), and forms ICc and IITa. All other significantly populated conformers existing in the gas phase prior to deposition convert either to conformer ICa or to conformer ICc during matrix deposition. The experimental observation of ICc had never been achieved hitherto. Narrowband near-IR irradiation of the first overtone of νOH vibrational mode of ICa and ICc in nitrogen matrices (at 6910 and 6930 cm(-1), respectively) led to selective generation of two additional conformers of high-energy, ITc and ITa, respectively, which were also observed experimentally for the first time. In addition, these experiments also provided the key information for the detailed vibrational characterization of the 3 conformers initially present in the matrices. On the other hand, UV irradiation (λ = 255 nm) of PG isolated in a xenon matrix revealed that PG undergoes facile photofragmentation through two photochemical pathways that are favored for different initial conformations of the reactant: (a) decarboxylation, leading to CO2 plus benzylamine (the dominant photofragmentation channel in PG cis-COOH conformers ICa and ICc) and (b) decarbonylation, with generation of CO plus benzonitrile, H2O and H2 (prevalent in the case of the trans-COOH conformer, IITa).

Conformers, infrared spectrum, UV-induced photochemistry, and near-IR-induced generation of two rare conformers of matrix-isolated phenylglycine

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

Borba, Ana, E-mail: anaborba@ci.uc.pt; Fausto, Rui; Gómez-Zavaglia, Andrea

The conformational space of α-phenylglycine (PG) have been investigated theoretically at both the DFT/B3LYP/6-311++G(d,p) and MP2/6-311++G(d,p) levels of approximation. Seventeen different minima were found on the investigated potential energy surfaces, which are characterized by different dominant intramolecular interactions: type I conformers are stabilized by hydrogen bonds of the type N–H···O=C, type II by a strong O–H···N hydrogen bond, type III by weak N–H···O–H hydrogen bonds, and type IV by a C=O···H–C contact. The calculations indicate also that entropic effects are relevant in determining the equilibrium populations of the conformers of PG in the gas phase, in particular in the casemore » of conformers of type II, where the strong intramolecular O–H···N hydrogen bond considerably diminishes entropy by reducing the conformational mobility of the molecule. In consonance with the relative energies of the conformers and barriers for conformational interconversion, only 3 conformers of PG were observed for the compound isolated in cryogenic Ar, Xe, and N{sub 2} matrices: the conformational ground state (ICa), and forms ICc and IITa. All other significantly populated conformers existing in the gas phase prior to deposition convert either to conformer ICa or to conformer ICc during matrix deposition. The experimental observation of ICc had never been achieved hitherto. Narrowband near-IR irradiation of the first overtone of νOH vibrational mode of ICa and ICc in nitrogen matrices (at 6910 and 6930 cm{sup −1}, respectively) led to selective generation of two additional conformers of high-energy, ITc and ITa, respectively, which were also observed experimentally for the first time. In addition, these experiments also provided the key information for the detailed vibrational characterization of the 3 conformers initially present in the matrices. On the other hand, UV irradiation (λ = 255 nm) of PG isolated in a xenon matrix revealed that PG undergoes facile photofragmentation through two photochemical pathways that are favored for different initial conformations of the reactant: (a) decarboxylation, leading to CO{sub 2} plus benzylamine (the dominant photofragmentation channel in PG cis-COOH conformers ICa and ICc) and (b) decarbonylation, with generation of CO plus benzonitrile, H{sub 2}O and H{sub 2} (prevalent in the case of the trans-COOH conformer, IITa)« less

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

PubMed

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

2015-04-27

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

Structure of apo-CAP reveals that large conformational changes are necessary for DNA binding

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

Sharma, Hitesh; Yu, Shaoning; Kong, Jilie

2009-10-21

The binding of cAMP to the Escherichia coli catabolite gene activator protein (CAP) produces a conformational change that enables it to bind specific DNA sequences and regulate transcription, which it cannot do in the absence of the nucleotide. The crystal structures of the unliganded CAP containing a D138L mutation and the unliganded WT CAP were determined at 2.3 and 3.6 {angstrom} resolution, respectively, and reveal that the two DNA binding domains have dimerized into one rigid body and their two DNA recognition helices become buried. The WT structure shows multiple orientations of this rigid body relative to the nucleotide bindingmore » domain supporting earlier biochemical data suggesting that the inactive form exists in an equilibrium among different conformations. Comparison of the structures of the liganded and unliganded CAP suggests that cAMP stabilizes the active DNA binding conformation of CAP through the interactions that the N{sup 6} of the adenosine makes with the C-helices. These interactions are associated with the reorientation and elongation of the C-helices that precludes the formation of the inactive structure.« less

Interaction of dihydrofolate reductase with methotrexate: Ensemble and single-molecule kinetics

NASA Astrophysics Data System (ADS)

Rajagopalan, P. T. Ravi; Zhang, Zhiquan; McCourt, Lynn; Dwyer, Mary; Benkovic, Stephen J.; Hammes, Gordon G.

2002-10-01

The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule was placed on a protein loop that closes over methotrexate when binding occurs, thus causing a quenching of the fluorescence. The biotin was used to attach the enzyme in an active form to a glass surface for single-molecule studies. The equilibrium dissociation constant for the binding of methotrexate to the enzyme is 9.5 nM. The stopped-flow studies revealed that methotrexate binds to two different conformations of the enzyme, and the association and dissociation rate constants were determined. The single-molecule investigation revealed a conformational change in the enzyme-methotrexate complex that was not observed in the stopped-flow studies. The ensemble averaged rate constants for this conformation change in both directions is about 2-4 s1 and is attributed to the opening and closing of the enzyme loop over the bound methotrexate. Thus the mechanism of methotrexate binding to DHFR involves multiple steps and protein conformational changes.

Effects of proline cis-trans isomerization on TB domain secondary structure.

PubMed Central

Yuan, X.; Werner, J. M.; Knott, V.; Handford, P. A.; Campbell, I. D.; Downing, K.

1998-01-01

The transforming growth factor beta (TGF-beta) binding protein-like (TB) domain is found principally in proteins localized to extracellular matrix fibrils, including human fibrillin-1, the defective protein in the Marfan syndrome. Analysis of the nuclear magnetic resonance (NMR) data for the sixth TB module from human fibrillin-1 has revealed the existence of two stable conformers that differ in the isomerization states of two proline residues. Unusually, the two isoforms do not readily interconvert and are stable on the time scale of milliseconds. We have computed independent structures of the major and minor conformers of TB6 to assess how the domain fold adjusts to incorporate alternatively cis- or trans-prolines. Based on previous observations, it has been suggested that multiple conformers can only be accommodated in flexible regions of protein structure. In contrast, P22, which exists in trans in the major form and cis in the minor form of TB6, is in a rigid region of the domain, which is confirmed by backbone dynamics measurements. Overall, the structures of the major and minor conformers are similar. However, the secondary structure topologies of the two forms differ as a direct consequence of the changes in proline conformation. PMID:9792099

Gender Differences and Psychosocial Factors Associated with Alcohol Involvement and Dysphoria in Adolescence.

ERIC Educational Resources Information Center

Locke, Thomas F.; Newcomb, Michael D.

2003-01-01

Examines correlations between Alcohol Involvement, Dysphoria, and their combined effects, with Social Conformity, Perceived Opportunity, Relationship Satisfaction, Parental Divorce, and Family Support/Bonding in a sample of late adolescents. Results revealed gender differences between Alcohol Involvement and Dysphoria. (Contains 74 references, 2…

Liquid crystal polymers: evidence of hairpin defects in nematic main chains, comparison with side chain polymers

NASA Astrophysics Data System (ADS)

Li, M. H.; Brûlet, A.; Keller, P.; Cotton, J. P.

1996-09-01

This article describes the conformation of two species of liquid crystalline polymers as revealed by small angle neutron scattering. The results obtained with side chain polymers are recalled. The procedure used to analyze the scattering data of main chains in the nematic phase is reported in this paper. It permits a demonstration of the existence of hairpins. Comparison of both polymer species shows that in the isotropic phase, the two polymers adopt a random coil conformation. In the nematic phase, the conformations are very different; the side chains behave as a melt of penetrable random coils whereas the main chains behave as a nematic phase of non penetrable cylinders.

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

Chien, Ellen Y.T.; Liu, Wei; Zhao, Qiang

Dopamine modulates movement, cognition, and emotion through activation of dopamine G protein-coupled receptors in the brain. The crystal structure of the human dopamine D3 receptor (D3R) in complex with the small molecule D2R/D3R-specific antagonist eticlopride reveals important features of the ligand binding pocket and extracellular loops. On the intracellular side of the receptor, a locked conformation of the ionic lock and two distinctly different conformations of intracellular loop 2 are observed. Docking of R-22, a D3R-selective antagonist, reveals an extracellular extension of the eticlopride binding site that comprises a second binding pocket for the aryl amide of R-22, which differsmore » between the highly homologous D2R and D3R. This difference provides direction to the design of D3R-selective agents for treating drug abuse and other neuropsychiatric indications.« less

Application of Solid-State NMR to Reveal Structural Differences in Cefazolin Sodium Pentahydrate from Different Manufacturing Processes

NASA Astrophysics Data System (ADS)

Tian, Ye; Wang, Wei D.; Zou, Wen-Bo; Qian, Jian-Qin; Hu, Chang-Qin

2018-04-01

The solid form of an active pharmaceutical ingredient is important when developing a new chemical entity. A solid understanding of the crystal structure and morphology that affect the mechanical and physical characteristics of pharmaceutical powders determines the manufacturing process. Solid-state NMR, thermogravimetric analysis, X-ray diffraction, and Fourier-transform infrared spectroscopy were combined with theoretical calculation to investigate different crystal packings of α-cefazolin sodium from three different vendors and conformational polymorphism was identified to exist in the α-cefazolin sodium. Marginal differences observed among CEZ-Na pentahydrate 1, 2, and 3 were speculated as the proportion of conformation 2. Understanding the differences in the polymorphic structure of α-cefazolin sodium may help with making modifications to incorporate new knowledge with a product’s development.

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

PubMed

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

2017-09-09

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

The complete conformational free energy landscape of β-xylose reveals a two-fold catalytic itinerary for β-xylanases.

PubMed

Iglesias-Fernández, Javier; Raich, Lluís; Ardèvol, Albert; Rovira, Carme

2015-02-01

Unraveling the conformational catalytic itinerary of glycoside hydrolases (GHs) is a growing topic of interest in glycobiology, with major impact in the design of GH inhibitors. β-xylanases are responsible for the hydrolysis of glycosidic bonds in β-xylans, a group of hemicelluloses of high biotechnological interest that are found in plant cell walls. The precise conformations followed by the substrate during catalysis in β-xylanases have not been unambiguously resolved, with three different pathways being proposed from structural analyses. In this work, we compute the conformational free energy landscape (FEL) of β-xylose to predict the most likely catalytic itineraries followed by β-xylanases. The calculations are performed by means of ab initio metadynamics, using the Cremer-Pople puckering coordinates as collective variables. The computed FEL supports only two of the previously proposed itineraries, 2 S O → [ 2,5 B] ǂ → 5 S 1 and 1 S 3 → [ 4 H 3 ] ǂ → 4 C 1 , which clearly appear in low energy regions of the FEL. Consistently, 2 S O and 1 S 3 are conformations preactivated for catalysis in terms of free energy/anomeric charge and bond distances. The results however exclude the O E → [ O S 2 ] ǂ → B 2,5 itinerary that has been recently proposed for a family 11 xylanase. Classical and ab initio QM/MM molecular dynamics simulations reveal that, in this case, the observed O E conformation has been enforced by enzyme mutation. These results add a word of caution on using modified enzymes to inform on catalytic conformational itineraries of glycoside hydrolases.

The complete conformational free energy landscape of β-xylose reveals a two-fold catalytic itinerary for β-xylanases† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c4sc02240h Click here for additional data file.

PubMed Central

Iglesias-Fernández, Javier; Raich, Lluís; Ardèvol, Albert

2015-01-01

Unraveling the conformational catalytic itinerary of glycoside hydrolases (GHs) is a growing topic of interest in glycobiology, with major impact in the design of GH inhibitors. β-xylanases are responsible for the hydrolysis of glycosidic bonds in β-xylans, a group of hemicelluloses of high biotechnological interest that are found in plant cell walls. The precise conformations followed by the substrate during catalysis in β-xylanases have not been unambiguously resolved, with three different pathways being proposed from structural analyses. In this work, we compute the conformational free energy landscape (FEL) of β-xylose to predict the most likely catalytic itineraries followed by β-xylanases. The calculations are performed by means of ab initio metadynamics, using the Cremer–Pople puckering coordinates as collective variables. The computed FEL supports only two of the previously proposed itineraries, 2SO → [2,5B]ǂ → 5S1 and 1S3 → [4H3]ǂ → 4C1, which clearly appear in low energy regions of the FEL. Consistently, 2SO and 1S3 are conformations preactivated for catalysis in terms of free energy/anomeric charge and bond distances. The results however exclude the OE → [OS2]ǂ → B2,5 itinerary that has been recently proposed for a family 11 xylanase. Classical and ab initio QM/MM molecular dynamics simulations reveal that, in this case, the observed OE conformation has been enforced by enzyme mutation. These results add a word of caution on using modified enzymes to inform on catalytic conformational itineraries of glycoside hydrolases. PMID:29560204

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 energy landscape of the acyl pocket loop in acetylcholinesterase: a Monte Carlo-generalized Born model study.

PubMed

Carlacci, Louis; Millard, Charles B; Olson, Mark A

2004-10-01

The X-ray crystal structure of the reaction product of acetylcholinesterase (AChE) with the inhibitor diisopropylphosphorofluoridate (DFP) showed significant structural displacement in a loop segment of residues 287-290. To understand this conformational selection, a Monte Carlo (MC) simulation study was performed of the energy landscape for the loop segment. A computational strategy was applied by using a combined simulated annealing and room temperature Metropolis sampling approach with solvent polarization modeled by a generalized Born (GB) approximation. Results from thermal annealing reveal a landscape topology of broader basin opening and greater distribution of energies for the displaced loop conformation, while the ensemble average of conformations at 298 K favored a shift in populations toward the native by a free-energy difference in good agreement with the estimated experimental value. Residue motions along a reaction profile of loop conformational reorganization are proposed where Arg-289 is critical in determining electrostatic effects of solvent interaction versus Coulombic charging.

Quantification Bias Caused by Plasmid DNA Conformation in Quantitative Real-Time PCR Assay

PubMed Central

Lin, Chih-Hui; Chen, Yu-Chieh; Pan, Tzu-Ming

2011-01-01

Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification. PMID:22194997

Probing messenger RNA conformational heterogeneity using single-molecule fluorescence anisotropy

NASA Astrophysics Data System (ADS)

Sinha, Deepak; Sastry, Srikanth; Shivashankar, G. V.

2006-03-01

In this letter we describe a method to probe biomolecular conformations and their dynamics at the single molecule level. We show, using fluorescence anisotropy based methods, that the hydrodynamic volume of biomolecules captures the intrinsic heterogeneity within a population. Population distributions of conformations and their dynamics are studied by making anisotropy measurements on one molecule at a time within a confocal volume. The mean anisotropy of mRNA is lowered on addition of salt while the spread remains the same. The intrinsic heterogeneity is revealed when conformational transitions are frozen, resulting in a drastic increase in the spread of the anisotropy. These studies reveal that mRNA samples a broad range of conformations.

Anomalous charge transport in conjugated polymers reveals underlying mechanisms of trapping and percolation

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

Mollinger, Sonya A.; Salleo, Alberto; Spakowitz, Andrew J.

While transport in conjugated polymers has many similarities to that in crystalline inorganic materials, several key differences reveal the unique relationship between the morphology of polymer films and the charge mobility. We develop a model that directly incorporates the molecular properties of the polymer film and correctly predicts these unique transport features. At low degree of polymerization, the increase of the mobility with the polymer chain length reveals trapping at chain ends, and saturation of the mobility at high degree of polymerization results from conformational traps within the chains. Similarly, the inverse field dependence of the mobility reveals that transportmore » on single polymer chains is characterized by the ability of the charge to navigate around kinks and loops in the chain. Lastly, these insights emphasize the connection between the polymer conformations and the transport and thereby offer a route to designing improved device morphologies through molecular design and materials processing.« less

Anomalous charge transport in conjugated polymers reveals underlying mechanisms of trapping and percolation

DOE PAGES

Mollinger, Sonya A.; Salleo, Alberto; Spakowitz, Andrew J.

2016-11-10

While transport in conjugated polymers has many similarities to that in crystalline inorganic materials, several key differences reveal the unique relationship between the morphology of polymer films and the charge mobility. We develop a model that directly incorporates the molecular properties of the polymer film and correctly predicts these unique transport features. At low degree of polymerization, the increase of the mobility with the polymer chain length reveals trapping at chain ends, and saturation of the mobility at high degree of polymerization results from conformational traps within the chains. Similarly, the inverse field dependence of the mobility reveals that transportmore » on single polymer chains is characterized by the ability of the charge to navigate around kinks and loops in the chain. Lastly, these insights emphasize the connection between the polymer conformations and the transport and thereby offer a route to designing improved device morphologies through molecular design and materials processing.« less

Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms

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

Simonetti, Angelita; Marzi, Stefano; Fabbretti, Attilio

2013-06-01

The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region. Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2more » is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue.« less

Deciphering the shape and deformation of secondary structures through local conformation analysis

PubMed Central

2011-01-01

Background Protein deformation has been extensively analysed through global methods based on RMSD, torsion angles and Principal Components Analysis calculations. Here we use a local approach, able to distinguish among the different backbone conformations within loops, α-helices and β-strands, to address the question of secondary structures' shape variation within proteins and deformation at interface upon complexation. Results Using a structural alphabet, we translated the 3 D structures of large sets of protein-protein complexes into sequences of structural letters. The shape of the secondary structures can be assessed by the structural letters that modeled them in the structural sequences. The distribution analysis of the structural letters in the three protein compartments (surface, core and interface) reveals that secondary structures tend to adopt preferential conformations that differ among the compartments. The local description of secondary structures highlights that curved conformations are preferred on the surface while straight ones are preferred in the core. Interfaces display a mixture of local conformations either preferred in core or surface. The analysis of the structural letters transition occurring between protein-bound and unbound conformations shows that the deformation of secondary structure is tightly linked to the compartment preference of the local conformations. Conclusion The conformation of secondary structures can be further analysed and detailed thanks to a structural alphabet which allows a better description of protein surface, core and interface in terms of secondary structures' shape and deformation. Induced-fit modification tendencies described here should be valuable information to identify and characterize regions under strong structural constraints for functional reasons. PMID:21284872

Deciphering the shape and deformation of secondary structures through local conformation analysis.

PubMed

Baussand, Julie; Camproux, Anne-Claude

2011-02-01

Protein deformation has been extensively analysed through global methods based on RMSD, torsion angles and Principal Components Analysis calculations. Here we use a local approach, able to distinguish among the different backbone conformations within loops, α-helices and β-strands, to address the question of secondary structures' shape variation within proteins and deformation at interface upon complexation. Using a structural alphabet, we translated the 3 D structures of large sets of protein-protein complexes into sequences of structural letters. The shape of the secondary structures can be assessed by the structural letters that modeled them in the structural sequences. The distribution analysis of the structural letters in the three protein compartments (surface, core and interface) reveals that secondary structures tend to adopt preferential conformations that differ among the compartments. The local description of secondary structures highlights that curved conformations are preferred on the surface while straight ones are preferred in the core. Interfaces display a mixture of local conformations either preferred in core or surface. The analysis of the structural letters transition occurring between protein-bound and unbound conformations shows that the deformation of secondary structure is tightly linked to the compartment preference of the local conformations. The conformation of secondary structures can be further analysed and detailed thanks to a structural alphabet which allows a better description of protein surface, core and interface in terms of secondary structures' shape and deformation. Induced-fit modification tendencies described here should be valuable information to identify and characterize regions under strong structural constraints for functional reasons.

The free energy landscape of pseudorotation in 3'-5' and 2'-5' linked nucleic acids.

PubMed

Li, Li; Szostak, Jack W

2014-02-19

The five-membered furanose ring is a central component of the chemical structure of biological nucleic acids. The conformations of the furanose ring can be analytically described using the concept of pseudorotation, and for RNA and DNA they are dominated by the C2'-endo and C3'-endo conformers. While the free energy difference between these two conformers can be inferred from NMR measurements, a free energy landscape of the complete pseudorotation cycle of nucleic acids in solution has remained elusive. Here, we describe a new free energy calculation method for molecular dynamics (MD) simulations using the two pseudorotation parameters directly as the collective variables. To validate our approach, we calculated the free energy surface of ribose pseudorotation in guanosine and 2'-deoxyguanosine. The calculated free energy landscape reveals not only the relative stability of the different pseudorotation conformers, but also the main transition path between the stable conformations. Applying this method to a standard A-form RNA duplex uncovered the expected minimum at the C3'-endo state. However, at a 2'-5' linkage, the minimum shifts to the C2'-endo conformation. The free energy of the C3'-endo conformation is 3 kcal/mol higher due to a weaker hydrogen bond and a reduced base stacking interaction. Unrestrained MD simulations suggest that the conversion from C3'-endo to C2'-endo and vice versa is on the nanosecond and microsecond time scale, respectively. These calculations suggest that 2'-5' linkages may enable folded RNAs to sample a wider spectrum of their pseudorotation conformations.

Trapping a 96° domain rotation in two distinct conformations by engineered disulfide bridges

PubMed Central

Schultz-Heienbrok, Robert; Maier, Timm; Sträter, Norbert

2004-01-01

Engineering disulfide bridges is a common technique to lock a protein movement in a defined conformational state. We have designed two double mutants of Escherichia coli 5′-nucleotidase to trap the enzyme in both an open (S228C, P513C) and a closed (P90C, L424C) conformation by the formation of disulfide bridges. The mutant proteins have been expressed, purified, and crystallized, to structurally characterize the designed variants. The S228C, P513C is a double mutant crystallized in two different crystal forms with three independent conformers, which differ from each other by a rotation of up to 12° of the C-terminal domain with respect to the N-terminal domain. This finding, as well as an analysis of the domain motion in the crystal, indicates that the enzyme still exhibits considerable residual domain flexibility. In the double mutant that was designed to trap the enzyme in the closed conformation, the structure analysis reveals an unexpected intermediate conformation along the 96° rotation trajectory between the open and closed enzyme forms. A comparison of the five independent conformers analyzed in this study shows that the domain movement of the variant enzymes is characterized by a sliding movement of the residues of the domain interface along the interface, which is in contrast to a classical closure motion where the residues of the domain interface move perpendicular to the interface. PMID:15215524

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.

Ligand Selectivity Mechanism and Conformational Changes in Guanine Riboswitch by Molecular Dynamics Simulations and Free Energy Calculations.

PubMed

Hu, Guodong; Ma, Aijing; Wang, Jihua

2017-04-24

Riboswitches regulate gene expression through direct and specific interactions with small metabolite molecules. Binding of a ligand to its RNA target is high selectivity and affinity and induces conformational changes of the RNA's secondary and tertiary structure. The structural difference of two purine riboswitches aptamers is caused by only one single mutation, where cytosine 74 in the guanine riboswitch is corresponding to a uracil 74 in adenine riboswitch. Here we employed molecular dynamics (MD) simulation, molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) and thermodynamic integration computational methodologies to evaluate the energetic and conformational changes of ligands binding to purine riboswitches. The snapshots used in MM-PBSA calculation were extracted from ten 50 ns MD simulation trajectories for each complex. These free energy results are in consistent with the experimental data and rationalize the selectivity of the riboswitches for different ligands. In particular, it is found that the loss in binding free energy upon mutation is mainly electrostatic in guanine (GUA) and riboswitch complex. Furthermore, new hydrogen bonds are found in mutated complexes. To reveal the conformational properties of guanine riboswitch, we performed a total of 6 μs MD simulations in both the presence and the absence of the ligand GUA. The MD simulations suggest that the conformation of guanine riboswitch depends on the distance of two groups in the binding pocket of ligand. The conformation is in a close conformation when U51-A52 is close to C74-U75.

Discriminating the structure of exo-2-aminonorbornane using nuclear quadrupole coupling interactions.

PubMed

Écija, Patricia; Cocinero, Emilio J; Lesarri, Alberto; Millán, Judith; Basterretxea, Francisco; Fernández, José A; Castaño, Fernando

2011-04-28

The intrinsic conformational and structural properties of the bicycle exo-2-aminonorbornane have been probed in a supersonic jet expansion using Fourier-transform microwave (FT-MW) spectroscopy and quantum chemical calculations. The rotational spectrum revealed two different conformers arising from the internal rotation of the amino group, exhibiting small (MHz) hyperfine patterns originated by the (14)N nuclear quadrupole coupling interaction. Complementary ab initio (MP2) and DFT (B3LYP and M05-2X) calculations provided comparative predictions for the structural properties, rotational and centrifugal distortion data, hyperfine parameters, and isomerization barriers. Due to the similarity of the rotational constants, the structural assignment of the observed rotamers and the calculation of the torsion angles of the amino group were based on the conformational dependence of the (14)N nuclear quadrupole coupling hyperfine tensor. In the most stable conformation (ss), the two amino N-H bonds are staggered with respect to the adjacent C-H bond. In the second conformer (st), only one of the N-H bonds is staggered and the other is trans. A third predicted conformer (ts) was not detected, consistent with a predicted conformational relaxation to conformer ss through a low barrier of 5.2 kJ mol(-1).

Nature vs. nurture in human sociality: multi-level genomic analyses of social conformity.

PubMed

Chen, Biqing; Zhu, Zijian; Wang, Yingying; Ding, Xiaohu; Guo, Xiaobo; He, Mingguang; Fang, Wan; Zhou, Qin; Zhou, Shanbi; Lei, Han; Huang, Ailong; Chen, Tingmei; Ni, Dongsheng; Gu, Yuping; Liu, Jianing; Rao, Yi

2018-05-01

Social conformity is fundamental to human societies and has been studied for more than six decades, but our understanding of its mechanisms remains limited. Individual differences in conformity have been attributed to social and cultural environmental influences, but not to genes. Here we demonstrate a genetic contribution to conformity after analyzing 1,140 twins and single-nucleotide polymorphism (SNP)-based studies of 2,130 young adults. A two-step genome-wide association study (GWAS) revealed replicable associations in 9 genomic loci, and a meta-analysis of three GWAS with a sample size of ~2,600 further confirmed one locus, corresponding to the NAV3 (Neuron Navigator 3) gene which encodes a protein important for axon outgrowth and guidance. Further multi-level (haplotype, gene, pathway) GWAS strongly associated genes including NAV3, PTPRD (protein tyrosine phosphatase receptor type D), ARL10 (ADP ribosylation factor-like GTPase 10), and CTNND2 (catenin delta 2), with conformity. Magnetic resonance imaging of 64 subjects shows correlation of activation or structural features of brain regions with the SNPs of these genes, supporting their functional significance. Our results suggest potential moderate genetic influence on conformity, implicate several specific genetic elements in conformity and will facilitate further research on cellular and molecular mechanisms underlying human conformity.

Modulation of Cell Proliferation and Differentiation through Substrate-dependent Changes in Fibronectin Conformation

PubMed Central

García, Andrés J.; Vega, María D.; Boettiger, David

1999-01-01

Integrin-mediated cell adhesion to extracellular matrices provides signals essential for cell cycle progression and differentiation. We demonstrate that substrate-dependent changes in the conformation of adsorbed fibronectin (Fn) modulated integrin binding and controlled switching between proliferation and differentiation. Adsorption of Fn onto bacterial polystyrene (B), tissue culture polystyrene (T), and collagen (C) resulted in differences in Fn conformation as indicated by antibody binding. Using a biochemical method to quantify bound integrins in cultured cells, we found that differences in Fn conformation altered the quantity of bound α5 and β1 integrin subunits but not αv or β3. C2C12 myoblasts grown on these Fn-coated substrates proliferated to different levels (B > T > C). Immunostaining for muscle-specific myosin revealed minimal differentiation on B, significant levels on T, and extensive differentiation on C. Differentiation required binding to the RGD cell binding site in Fn and was blocked by antibodies specific for this site. Switching between proliferation and differentiation was controlled by the levels of α5β1 integrin bound to Fn, and differentiation was inhibited by anti-α5, but not anti-αv, antibodies, suggesting distinct integrin-mediated signaling pathways. Control of cell proliferation and differentiation through conformational changes in extracellular matrix proteins represents a versatile mechanism to elicit specific cellular responses for biological and biotechnological applications. PMID:10069818

The physical basis of how prion conformations determine strain phenotypes

NASA Astrophysics Data System (ADS)

Tanaka, Motomasa; Collins, Sean R.; Toyama, Brandon H.; Weissman, Jonathan S.

2006-08-01

A principle that has emerged from studies of protein aggregation is that proteins typically can misfold into a range of different aggregated forms. Moreover, the phenotypic and pathological consequences of protein aggregation depend critically on the specific misfolded form. A striking example of this is the prion strain phenomenon, in which prion particles composed of the same protein cause distinct heritable states. Accumulating evidence from yeast prions such as [PSI+] and mammalian prions argues that differences in the prion conformation underlie prion strain variants. Nonetheless, it remains poorly understood why changes in the conformation of misfolded proteins alter their physiological effects. Here we present and experimentally validate an analytical model describing how [PSI+] strain phenotypes arise from the dynamic interaction among the effects of prion dilution, competition for a limited pool of soluble protein, and conformation-dependent differences in prion growth and division rates. Analysis of three distinct prion conformations of yeast Sup35 (the [PSI+] protein determinant) and their in vivo phenotypes reveals that the Sup35 amyloid causing the strongest phenotype surprisingly shows the slowest growth. This slow growth, however, is more than compensated for by an increased brittleness that promotes prion division. The propensity of aggregates to undergo breakage, thereby generating new seeds, probably represents a key determinant of their physiological impact for both infectious (prion) and non-infectious amyloids.

Structure and dynamics of double helical DNA in torsion angle hyperspace: a molecular mechanics approach.

PubMed

Borkar, Aditi; Ghosh, Indira; Bhattacharyya, Dhananjay

2010-04-01

Analysis of the conformational space populated by the torsion angles and the correlation between the conformational energy and the sequence of DNA are important for fully understanding DNA structure and function. Presence of seven variable torsion angles about single covalent bonds in DNA main chain puts a big challenge for such analysis. We have carried out restrained energy minimization studies for four representative dinucleosides, namely d(ApA):d(TpT), d(CpG):d(CpG), d(GpC):d(GpC) and d(CpA):d(TpG) to determine the energy hyperspace of DNA in context to the values of the torsion angles and the structural properties of the DNA conformations populating the favorable regions of this energy hyperspace. The torsion angles were manipulated by constraining their values at the reference points and then performing energy minimization. The energy minima obtained on the potential energy contour plots mostly correspond to the conformations populated in crystal structures of DNA. Some novel favorable conformations that are not present in crystal structure data are also found. The plots also suggest few low energy routes for conformational transitions or the associated energy barrier heights. Analyses of base pairing and stacking possibility reveal structural changes accompanying these transitions as well as the flexibility of different base steps towards variations in different torsion angles.

On the Roles of Substrate Binding and Hinge Unfolding in Conformational Changes of Adenylate Kinase

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

Brokaw, Jason B.; Chu, Jhih-wei

2010-11-17

We characterized the conformational change of adenylate kinase (AK) between open and closed forms by conducting five all-atom molecular-dynamics simulations, each of 100 ns duration. Different initial structures and substrate binding configurations were used to probe the pathways of AK conformational change in explicit solvent, and no bias potential was applied. A complete closed-to-open and a partial open-to-closed transition were observed, demonstrating the direct impact of substrate-mediated interactions on shifting protein conformation. The sampled configurations suggest two possible pathways for connecting the open and closed structures of AK, affirming the prediction made based on available x-ray structures and earlier worksmore » of coarse-grained modeling. The trajectories of the all-atom molecular-dynamics simulations revealed the complexity of protein dynamics and the coupling between different domains during conformational change. Calculations of solvent density and density fluctuations surrounding AK did not show prominent variation during the transition between closed and open forms. Finally, we characterized the effects of local unfolding of an important hinge near Pro177 on the closed-to-open transition of AK and identified a novel mechanism by which hinge unfolding modulates protein conformational change. The local unfolding of Pro177 hinge induces alternative tertiary contacts that stabilize the closed structure and prevent the opening transition.« less

Crowding Shifts the FMN Recognition Mechanism of Riboswitch Aptamer from Conformational Selection to Induced Fit.

PubMed

Rode, Ambadas B; Endoh, Tamaki; Sugimoto, Naoki

2018-06-04

In bacteria, the binding between the riboswitch aptamer domain and ligand is regulated by environmental cues, such as low Mg 2+ in macrophages during pathogenesis to ensure spatiotemporal expression of virulence genes. Binding was investigated between the flavin mononucleotide (FMN) riboswitch aptamer and its anionic ligand in the presence of molecular crowding agent without Mg 2+ ion, which mimics pathogenic conditions. Structural, kinetic, and thermodynamic analyses under the crowding revealed more dynamic conformational rearrangements of the FMN riboswitch aptamer compared to dilute Mg 2+ -containing solution. It is hypothesized that under crowding conditions FMN binds through an induced fit mechanism in contrast to the conformational selection mechanism previously demonstrated in dilute Mg 2+ solution. Since these two mechanisms involve different conformational intermediates and rate constants, these findings have practical significance in areas such as drug design and RNA engineering. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Conformational dimorphism of isochroman-1-ones in the solid state

NASA Astrophysics Data System (ADS)

Babjaková, Eva; Hanulíková, Barbora; Dastychová, Lenka; Kuřitka, Ivo; Nečas, Marek; Vícha, Robert

2014-12-01

Isochroman-1-one derivatives, which are relatives of coumarins, display a broad spectrum of biological activity; therefore, these derivatives attract the attention of chemists. A series of new isochroman-1-ones were prepared by the reaction of benzyl-derived Grignard reagents with acyl chlorides. All of the prepared compounds were characterized using single-crystal X-ray diffraction as well as FT-IR, NMR and MS techniques. Single crystal X-ray diffraction analysis revealed that the isochromanones can adopt two distinct conformations in the solid state. For one of the compounds, two polymorphs with unique forms crystallized separately under different temperatures. The packing of all of the examined crystals is stabilized via weak intramolecular C-H⋯π and/or C-H⋯O interactions. Although the closed conformer was predominantly found in the actual crystals, the open conformer is thermochemically more stable for all of the examined compounds according to DFT calculations.

Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy

NASA Astrophysics Data System (ADS)

Maity, Sourav; Mazzolini, Monica; Arcangeletti, Manuel; Valbuena, Alejandro; Fabris, Paolo; Lazzarino, Marco; Torre, Vincent

2015-05-01

Cyclic nucleotide-gated (CNG) channels are activated by binding of cyclic nucleotides. Although structural studies have identified the channel pore and selectivity filter, conformation changes associated with gating remain poorly understood. Here we combine single-molecule force spectroscopy (SMFS) with mutagenesis, bioinformatics and electrophysiology to study conformational changes associated with gating. By expressing functional channels with SMFS fingerprints in Xenopus laevis oocytes, we were able to investigate gating of CNGA1 in a physiological-like membrane. Force spectra determined that the S4 transmembrane domain is mechanically coupled to S5 in the closed state, but S3 in the open state. We also show there are multiple pathways for the unfolding of the transmembrane domains, probably caused by a different degree of α-helix folding. This approach demonstrates that CNG transmembrane domains have dynamic structure and establishes SMFS as a tool for probing conformational change in ion channels.

A Dualistic Conformational Response to Substrate Binding in the Human Serotonin Transporter Reveals a High Affinity State for Serotonin*

PubMed Central

Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida; Wiborg, Ove; Sinning, Steffen

2015-01-01

Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT. PMID:25614630

Molecular Clustering Interrelationships and Carbohydrate Conformation in Hull and Seeds Among Barley Cultivars

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

N Liu; P Yu

2011-12-31

The objective of this study was to use molecular spectral analyses with the diffuse reflectance Fourier transform infrared spectroscopy (DRIFT) bioanlytical technique to study carbohydrate conformation features, molecular clustering and interrelationships in hull and seed among six barley cultivars (AC Metcalfe, CDC Dolly, McLeod, CDC Helgason, CDC Trey, CDC Cowboy), which had different degradation kinetics in rumen. The molecular structure spectral analyses in both hull and seed involved the fingerprint regions of ca. 1536-1484 cm{sup -1} (attributed mainly to aromatic lignin semicircle ring stretch), ca. 1293-1212 cm{sup -1} (attributed mainly to cellulosic compounds in the hull), ca. 1269-1217 cm{sup -1}more » (attributed mainly to cellulosic compound in the seeds), and ca. 1180-800 cm{sup -1} (attributed mainly to total CHO C-O stretching vibrations) together with an agglomerative hierarchical cluster (AHCA) and principal component spectral analyses (PCA). The results showed that the DRIFT technique plus AHCA and PCA molecular analyses were able to reveal carbohydrate conformation features and identify carbohydrate molecular structure differences in both hull and seeds among the barley varieties. The carbohydrate molecular spectral analyses at the region of ca. 1185-800 cm{sup -1} together with the AHCA and PCA were able to show that the barley seed inherent structures exhibited distinguishable differences among the barley varieties. CDC Helgason had differences from AC Metcalfe, MeLeod, CDC Cowboy and CDC Dolly in carbohydrate conformation in the seed. Clear molecular cluster classes could be distinguished and identified in AHCA analysis and the separate ellipses could be grouped in PCA analysis. But CDC Helgason had no distinguished differences from CDC Trey in carbohydrate conformation. These carbohydrate conformation/structure difference could partially explain why the varieties were different in digestive behaviors in animals. The molecular spectroscopy technique used in this study could also be used for other plant-based feed and food structure studies.« less

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.

Synthesis, Spatial Structure and Analgesic Activity of Sodium 3-Benzylaminocarbonyl-1-methyl-2,2-dioxo-1H-2λ6,1-benzothiazin-4-olate Solvates

PubMed Central

Ukrainets, Igor V.; Petrushova, Lidiya A.; Shishkina, Svitlana V.; Grinevich, Lina A.; Sim, Galina

2016-01-01

In order to obtain and then test pharmocologically any possible conformers of the new feasible analgesic N-benzyl-4-hydroxy-1-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamide, its 4-O-sodium salt was synthesized using two methods. X-ray diffraction study made possible to determine that, depending on the chosen synthesis conditions, the above-mentioned compound forms either monosolvate with methanol or monohydrate, where organic anion exists in the form of three different conformers. Pharmacological testing of the two known pseudo-enantiomeric forms of the original N-benzylamide and of the two solvates of its sodium salt was performed simultaneously under the same conditions and in equimolar doses. Comparison of the results obtained while studying the peculiarities of the synthesized compounds spatial structure and biological properties revealed an important structure-action relationship. In particular, it was shown that the intensity of analgesic effect of different conformational isomers of N-benzyl-4-hydroxy-1-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamide may change considerably: while low active conformers are comparable with piroxicam, highly active conformers are more than twice as effective as meloxicam. PMID:27775559

Effect of sterically demanding substituents on the conformational stability of the collagen triple helix.

PubMed

Erdmann, Roman S; Wennemers, Helma

2012-10-17

The effect of sterically demanding groups at proline residues on the conformational stability of the collagen triple helix was examined. The thermal stabilities (T(m) and ΔG) of eight different triple helices derived from collagen model peptides with (4R)- or (4S)-configured amidoprolines bearing either methyl or bulkier tert-butyl groups in the Xaa or Yaa position were determined and served as a relative measure for the conformational stability of the corresponding collagen triple helices. The results show that sterically demanding substituents are tolerated in the collagen triple helix when they are attached to (4R)-configured amidoprolines in the Xaa position or to (4S)-configured amidoprolines in the Yaa position. Structural studies in which the preferred conformation of (4R)- or (4S)-configured amidoproline were overlaid with the Pro and Hyp residues within a crystal structure of collagen revealed that the sterically demanding groups point to the outside of these two triple helices and thereby do not interfere with the formation of the triple helix. In all of the other examined collagen derivatives with lower stability of the triple helices, the acetyl or pivaloyl residues point toward the inside of the triple helix and clash with a residue of the neighboring strand. The results also revealed that unfavorable steric dispositions affect the conformational stability of the collagen triple helix more than unfavorable ring puckers of the proline residues. The results are useful for the design of functionalized collagen based materials.

Conformation-selective inhibitors reveal differences in the activation and phosphate-binding loops of the tyrosine kinases Abl and Src

PubMed Central

Hari, Sanjay B.; Perera, B. Gayani K.; Ranjitkar, Pratistha; Seeliger, Markus A.; Maly, Dustin J.

2013-01-01

Over the last decade, an increasingly diverse array of potent and selective inhibitors that target the ATP-binding sites of protein kinases have been developed. Many of these inhibitors, like the clinically approved drug imatinib (Gleevec), stabilize a specific catalytically inactive ATP-binding site conformation of their kinases targets. Imatinib is notable in that it is highly selective for its kinase target, Abl, over other closely-related tyrosine kinases, like Src. In addition, imatinib is highly sensitive to the phosphorylation state of Abl's activation loop, which is believed to be a general characteristic of all inhibitors that stabilize a similar inactive ATP-binding site conformation. In this report, we perform a systematic analysis of a diverse series of ATP-competitive inhibitors that stabilize a similar inactive ATP-binding site conformation as imatinib with the tyrosine kinases Src and Abl. In contrast to imatinib, many of these inhibitors have very similar potencies against Src and Abl. Furthermore, only a subset of this class of inhibitors is sensitive to the phosphorylation state of the activation loop of these kinases. In attempting to explain this observation, we have uncovered an unexpected correlation between Abl's activation loop and another flexible active site feature, called the phosphate-binding loop (p-loop). These studies shed light on how imatinib is able to obtain its high target selectivity and reveal how the conformational preference of flexible active site regions can vary between closely related kinases. PMID:24106839

LeuT conformational sampling utilizing accelerated molecular dynamics and principal component analysis.

PubMed

Thomas, James R; Gedeon, Patrick C; Grant, Barry J; Madura, Jeffry D

2012-07-03

Monoamine transporters (MATs) function by coupling ion gradients to the transport of dopamine, norepinephrine, or serotonin. Despite their importance in regulating neurotransmission, the exact conformational mechanism by which MATs function remains elusive. To this end, we have performed seven 250 ns accelerated molecular dynamics simulations of the leucine transporter, a model for neurotransmitter MATs. By varying the presence of binding-pocket leucine substrate and sodium ions, we have sampled plausible conformational states representative of the substrate transport cycle. The resulting trajectories were analyzed using principal component analysis of transmembrane helices 1b and 6a. This analysis revealed seven unique structures: two of the obtained conformations are similar to the currently published crystallographic structures, one conformation is similar to a proposed open inward structure, and four conformations represent novel structures of potential importance to the transport cycle. Further analysis reveals that the presence of binding-pocket sodium ions is necessary to stabilize the locked-occluded and open-inward conformations. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A mutation uncouples the tubulin conformational and GTPase cycles, revealing allosteric control of microtubule dynamics

PubMed Central

Geyer, Elisabeth A; Burns, Alexander; Lalonde, Beth A; Ye, Xuecheng; Piedra, Felipe-Andres; Huffaker, Tim C; Rice, Luke M

2015-01-01

Microtubule dynamic instability depends on the GTPase activity of the polymerizing αβ-tubulin subunits, which cycle through at least three distinct conformations as they move into and out of microtubules. How this conformational cycle contributes to microtubule growing, shrinking, and switching remains unknown. Here, we report that a buried mutation in αβ-tubulin yields microtubules with dramatically reduced shrinking rate and catastrophe frequency. The mutation causes these effects by suppressing a conformational change that normally occurs in response to GTP hydrolysis in the lattice, without detectably changing the conformation of unpolymerized αβ-tubulin. Thus, the mutation weakens the coupling between the conformational and GTPase cycles of αβ-tubulin. By showing that the mutation predominantly affects post-GTPase conformational and dynamic properties of microtubules, our data reveal that the strength of the allosteric response to GDP in the lattice dictates the frequency of catastrophe and the severity of rapid shrinking. DOI: http://dx.doi.org/10.7554/eLife.10113.001 PMID:26439009

Ligand and receptor dynamics contribute to the mechanism of graded PPARγ agonism

PubMed Central

Hughes, Travis S.; Chalmers, Michael J.; Novick, Scott; Kuruvilla, Dana S.; Chang, Mi Ra; Kamenecka, Theodore M.; Rance, Mark; Johnson, Bruce A.; Burris, Thomas P.; Griffin, Patrick R.; Kojetin, Douglas J.

2011-01-01

SUMMARY Ligand binding to proteins is not a static process, but rather involves a number of complex dynamic transitions. A flexible ligand can change conformation upon binding its target. The conformation and dynamics of a protein can change to facilitate ligand binding. The conformation of the ligand, however, is generally presumed to have one primary binding mode, shifting the protein conformational ensemble from one state to another. We report solution NMR studies that reveal peroxisome proliferator-activated receptor γ (PPARγ) modulators can sample multiple binding modes manifesting in multiple receptor conformations in slow conformational exchange. Our NMR, hydrogen/deuterium exchange and docking studies reveal that ligand-induced receptor stabilization and binding mode occupancy correlate with the graded agonist response of the ligand. Our results suggest that ligand and receptor dynamics affect the graded transcriptional output of PPARγ modulators. PMID:22244763

Computational investigation of the effect of pH on the color of firefly bioluminescence by DFT.

PubMed

Pinto da Silva, Luís; Esteves da Silva, Joaquim C G

2011-04-04

In spite of recent advances towards understanding the mechanism of firefly bioluminescence, there is no consensus about which oxyluciferin (OxyLH(2)) species are the red and yellow-green emitters. The crystal structure of Luciola cruciata luciferase (LcLuc) revealed different conformations for the various steps of the bioluminescence reaction, with different degrees of polarity and rigidity of the active-site microenvironment. In this study, these different conformations of luciferase (Luc) are simulated and their effects on the different chemical equilibria of OxyLH(2) are investigated as a function of pH by means of density functional theory with the PBE0 functional. In particular, the thermodynamic properties and the absorption spectra of each species, as well as their relative stabilities in the ground and excited states, were computed in the different conformations of Luc. From the calculations it is possible to derive the acid dissociation and tautomeric constants, and the corresponding distribution diagrams. It is observed that the anionic keto form of OxyLH(2) is both the red and the yellow-green emitter. Consequently, the effect of Luc conformations on the structural and electronic properties of the Keto-(-1) form are studied. Finally, insights into the Luc-catalyzed light-emitting reaction are derived from the calculations. The multicolor bioluminescence can be explained by interactions of the emitter with active-site molecules, the effects of which on light emission are modulated by the internal dielectric constant of the different conformations. These interactions can suffer also from rearrangement due to entry of external solvent and changes in the protonation state of some amino acid residues and adenosine monophosphate (AMP). Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Investigation of an "alternate water supply system" in enzymatic hydrolysis in the processive endocellulase Cel7A from Rasamsonia emersonii by molecular dynamics simulation.

PubMed

Sun, Xun; Qian, Meng-Dan; Guan, Shan-Shan; Shan, Ya-Ming; Dong, Ying; Zhang, Hao; Wang, Song; Han, Wei-Wei

2017-02-01

Cel7A from Rasamsonia emersonii is one of the processive endocellulases classified under family 7 glycoside hydrolase. Molecular dynamics simulations were carried out to obtain the optimized sliding and hydrolyzing conformations, in which the reducing ends of sugar chains are located on different sites. Hydrogen bonds are investigated to clarify the interactions between protein and substrate in either conformation. Nine hydrogen bonding interactions are identified in the sliding conformation, and six similar interactions are also found correspondingly in the hydrolyzing conformation. In addition, four strong hydrophobic interactions are also determined. The domain cross-correlation map analysis shows movement correlation of protein including autocorrelation between residues. The root mean square fluctuations analysis represents the various flexibilities of different fragment in the two conformations. Comparing the two conformations reveals the water-supply mechanism of selective hydrolysis of cellulose in Cel7A. The mechanism can be described as follow. When the reducing end of substrate slides from the unhydrolyzing site (sliding conformation) to the hydrolyzing site (hydrolyzing conformation), His225 is pushed down and rotated, the rotation leads to the movement of Glu209 with the interstrand hydrogen bonding in β-sheet. It further makes Asp211 close to the hydrolysis center and provides a water molecule bounding on its carboxyl in the previous unhydrolyzing site. After the hydrolysis takes place and the product is excluded from the enzyme, the Asp211 comes back to its initial position. In summary, Asp211 acts as an elevator to transport outer water molecules into the hydrolysis site for every other glycosidic bond. © 2016 Wiley Periodicals, Inc.

The Free Energy Landscape of Pseudorotation in 3′–5′ and 2′–5′ Linked Nucleic Acids

PubMed Central

2014-01-01

The five-membered furanose ring is a central component of the chemical structure of biological nucleic acids. The conformations of the furanose ring can be analytically described using the concept of pseudorotation, and for RNA and DNA they are dominated by the C2′-endo and C3′-endo conformers. While the free energy difference between these two conformers can be inferred from NMR measurements, a free energy landscape of the complete pseudorotation cycle of nucleic acids in solution has remained elusive. Here, we describe a new free energy calculation method for molecular dynamics (MD) simulations using the two pseudorotation parameters directly as the collective variables. To validate our approach, we calculated the free energy surface of ribose pseudorotation in guanosine and 2′-deoxyguanosine. The calculated free energy landscape reveals not only the relative stability of the different pseudorotation conformers, but also the main transition path between the stable conformations. Applying this method to a standard A-form RNA duplex uncovered the expected minimum at the C3′-endo state. However, at a 2′–5′ linkage, the minimum shifts to the C2′-endo conformation. The free energy of the C3′-endo conformation is 3 kcal/mol higher due to a weaker hydrogen bond and a reduced base stacking interaction. Unrestrained MD simulations suggest that the conversion from C3′-endo to C2′-endo and vice versa is on the nanosecond and microsecond time scale, respectively. These calculations suggest that 2′–5′ linkages may enable folded RNAs to sample a wider spectrum of their pseudorotation conformations. PMID:24499340

Tilting the balance between canonical and noncanonical conformations for the H1 hypervariable loop of a llama VHH through point mutations.

PubMed

Mahajan, Sai Pooja; Velez-Vega, Camilo; Escobedo, Fernando A

2013-01-10

Nanobodies are single-domain antibodies found in camelids. These are the smallest naturally occurring binding domains and derive functionality via three hypervariable loops (H1-H3) that form the binding surface. They are excellent candidates for antibody engineering because of their favorable characteristics like small size, high solubility, and stability. To rationally engineer antibodies with affinity for a specific target, the hypervariable loops can be tailored to obtain the desired binding surface. As a first step toward such a goal, we consider the design of loops with a desired conformation. In this study, we focus on the H1 loop of the anti-hCG llama nanobody that exhibits a noncanonical conformation. We aim to "tilt" the stability of the H1 loop structure from a noncanonical conformation to a (humanized) type 1 canonical conformation by studying the effect of selected mutations to the amino acid sequence of the H1, H2, and proximal residues. We use all-atomistic, explicit-solvent, biased molecular dynamic simulations to simulate the wild-type and mutant loops in a prefolded framework. We thus find mutants with increasing propensity to form a stable type 1 canonical conformation of the H1 loop. Free energy landscapes reveal the existence of conformational isomers of the canonical conformation that may play a role in binding different antigenic surfaces. We also elucidate the approximate mechanism and kinetics of transitions between such conformational isomers by using a Markovian model. We find that a particular three-point mutant has the strongest thermodynamic propensity to form the H1 type 1 canonical structure but also to exhibit transitions between conformational isomers, while a different, more rigid three-point mutant has the strongest propensity to be kinetically trapped in such a canonical structure.

Non-invasive vibrational SFG spectroscopy reveals that bacterial adhesion can alter the conformation of grafted "brush" chains on SAM.

PubMed

Bulard, Emilie; Guo, Ziang; Zheng, Wanquan; Dubost, Henri; Fontaine-Aupart, Marie-Pierre; Bellon-Fontaine, Marie-Noëlle; Herry, Jean-Marie; Briandet, Romain; Bourguignon, Bernard

2011-04-19

Understanding bacterial adhesion on a surface is a crucial step to design new materials with improved properties or to control biofilm formation and eradication. Sum Frequency Generation (SFG) vibrational spectroscopy has been employed to study in situ the conformational response of a self-assembled monolayer (SAM) of octadecanethiol (ODT) on a gold film to the adhesion of hydrophilic and hydrophobic ovococcoid model bacteria. The present work highlights vibrational SFG spectroscopy as a powerful and unique non-invasive biophysical technique to probe and control bacteria interaction with ordered surfaces. Indeed, the SFG vibrational spectral changes reveal different ODT SAM conformations in air and upon exposure to aqueous solution or bacterial adhesion. Furthermore, this effect depends on the bacterial cell surface properties. The SFG spectral modeling demonstrates that hydrophobic bacteria flatten the ODT SAM alkyl chain terminal part, whereas the hydrophilic ones raise this ODT SAM terminal part. Microorganism-induced alteration of grafted chains can thus affect the desired interfacial functionality, a result that should be considered for the design of new reactive materials. © 2011 American Chemical Society

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

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.

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

PubMed

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

2012-03-15

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

A latent class regression analysis of men's conformity to masculine norms and psychological distress.

PubMed

Wong, Y Joel; Owen, Jesse; Shea, Munyi

2012-01-01

How are specific dimensions of masculinity related to psychological distress in specific groups of men? To address this question, the authors used latent class regression to assess the optimal number of latent classes that explained differential relationships between conformity to masculine norms and psychological distress in a racially diverse sample of 223 men. The authors identified a 2-class solution. Both latent classes demonstrated very different associations between conformity to masculine norms and psychological distress. In Class 1 (labeled risk avoiders; n = 133), conformity to the masculine norm of risk-taking was negatively related to psychological distress. In Class 2 (labeled detached risk-takers; n = 90), conformity to the masculine norms of playboy, self-reliance, and risk-taking was positively related to psychological distress, whereas conformity to the masculine norm of violence was negatively related to psychological distress. A post hoc analysis revealed that younger men and Asian American men (compared with Latino and White American men) had significantly greater odds of being in Class 2 versus Class 1. The implications of these findings for future research and clinical practice are examined. (c) 2012 APA, all rights reserved.

Protein flexibility and conformational entropy in ligand design targeting the carbohydrate recognition domain of galectin-3.

PubMed

Diehl, Carl; Engström, Olof; Delaine, Tamara; Håkansson, Maria; Genheden, Samuel; Modig, Kristofer; Leffler, Hakon; Ryde, Ulf; Nilsson, Ulf J; Akke, Mikael

2010-10-20

Rational drug design is predicated on knowledge of the three-dimensional structure of the protein-ligand complex and the thermodynamics of ligand binding. Despite the fundamental importance of both enthalpy and entropy in driving ligand binding, the role of conformational entropy is rarely addressed in drug design. In this work, we have probed the conformational entropy and its relative contribution to the free energy of ligand binding to the carbohydrate recognition domain of galectin-3. Using a combination of NMR spectroscopy, isothermal titration calorimetry, and X-ray crystallography, we characterized the binding of three ligands with dissociation constants ranging over 2 orders of magnitude. (15)N and (2)H spin relaxation measurements showed that the protein backbone and side chains respond to ligand binding by increased conformational fluctuations, on average, that differ among the three ligand-bound states. Variability in the response to ligand binding is prominent in the hydrophobic core, where a distal cluster of methyl groups becomes more rigid, whereas methyl groups closer to the binding site become more flexible. The results reveal an intricate interplay between structure and conformational fluctuations in the different complexes that fine-tunes the affinity. The estimated change in conformational entropy is comparable in magnitude to the binding enthalpy, demonstrating that it contributes favorably and significantly to ligand binding. We speculate that the relatively weak inherent protein-carbohydrate interactions and limited hydrophobic effect associated with oligosaccharide binding might have exerted evolutionary pressure on carbohydrate-binding proteins to increase the affinity by means of conformational entropy.

Free energy surface of two- and three-dimensional transitions of Au 12 nanoclusters obtained by ab initio metadynamics

NASA Astrophysics Data System (ADS)

Santarossa, Gianluca; Vargas, Angelo; Iannuzzi, Marcella; Baiker, Alfons

2010-05-01

The description of the conformational space generated by metal nanoparticles is a fundamental issue for the study of their physicochemical properties. In this investigation, an exhaustive exploration and a unified view of the conformational space of a gold nanocluster is provided using a Au 12 cluster as an example. Such system is characterized by coexisting planar/quasiplanar and tridimensional conformations separated by high-energy barriers. The conformational space of Au 12 has been explored by means of Born-Oppenheimer ab initio metadynamics, i.e., a molecular dynamics simulation coupled with a history dependent potential to accelerate events that might occur on a long time scale compared to the time step used in the simulations (rare events). The sampled conformations have complex, in general not intuitive topologies that we have classified as planar/quasiplanar or tridimensional, belonging to different regions of the free energy surface. Three conformational free energy basins were identified, one for the planar/quasiplanar and two for the tridimensional structures. At thermodynamic equilibrium, the planar/quasi-planar and tridimensional conformations were found to coexist, to be fluxional and to be separated by high-free-energy barriers. The comparison between the free energy and the potential energy revealed the relevance of the entropic contribution in the equilibrium distribution of the conformations of the cluster.

Critical Role of Interdomain Interactions in the Conformational Change and Catalytic Mechanism of Endoplasmic Reticulum Aminopeptidase 1.

PubMed

Stamogiannos, Athanasios; Maben, Zachary; Papakyriakou, Athanasios; Mpakali, Anastasia; Kokkala, Paraskevi; Georgiadis, Dimitris; Stern, Lawrence J; Stratikos, Efstratios

2017-03-14

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that is important for the generation of antigenic epitopes and major histocompatibility class I-restricted adaptive immune responses. ERAP1 processes a vast variety of different peptides but still shows length and sequence selectivity, although the mechanism behind these properties is poorly understood. X-ray crystallographic analysis has revealed that ERAP1 can assume at least two distinct conformations in which C-terminal domain IV is either proximal or distal to active site domain II. To improve our understanding of the role of this conformational change in the catalytic mechanism of ERAP1, we used site-directed mutagenesis to perturb key salt bridges between domains II and IV. Enzymatic analysis revealed that these mutations, although located away from the catalytic site, greatly reduce the catalytic efficiency and change the allosteric kinetic behavior. The variants were more efficiently activated by small peptides and bound a competitive inhibitor with weaker affinity and faster dissociation kinetics. Molecular dynamics analysis suggested that the mutations affect the conformational distribution of ERAP1, reducing the population of closed states. Small-angle X-ray scattering indicated that both the wild type and the ERAP1 variants are predominantly in an open conformational state in solution. Overall, our findings suggest that electrostatic interactions between domains II and IV in ERAP1 are crucial for driving a conformational change that regulates the structural integrity of the catalytic site. The extent of domain opening in ERAP1 probably underlies its specialization for antigenic peptide precursors and should be taken into account in inhibitor development efforts.

Conformational states and folding pathways of peptides revealed by principal-independent component analyses.

PubMed

Nguyen, Phuong H

2007-05-15

Principal component analysis is a powerful method for projecting multidimensional conformational space of peptides or proteins onto lower dimensional subspaces in which the main conformations are present, making it easier to reveal the structures of molecules from e.g. molecular dynamics simulation trajectories. However, the identification of all conformational states is still difficult if the subspaces consist of more than two dimensions. This is mainly due to the fact that the principal components are not independent with each other, and states in the subspaces cannot be visualized. In this work, we propose a simple and fast scheme that allows one to obtain all conformational states in the subspaces. The basic idea is that instead of directly identifying the states in the subspace spanned by principal components, we first transform this subspace into another subspace formed by components that are independent of one other. These independent components are obtained from the principal components by employing the independent component analysis method. Because of independence between components, all states in this new subspace are defined as all possible combinations of the states obtained from each single independent component. This makes the conformational analysis much simpler. We test the performance of the method by analyzing the conformations of the glycine tripeptide and the alanine hexapeptide. The analyses show that our method is simple and quickly reveal all conformational states in the subspaces. The folding pathways between the identified states of the alanine hexapeptide are analyzed and discussed in some detail. 2007 Wiley-Liss, Inc.

Interpreting the adsorption of serum albumin and lactoglobulin onto ZnS nanopaticles: effect of conformational rigidity of the proteins.

PubMed

Saikia, Jiban; Saha, Bedabrata; Das, Gopal

2014-02-15

The work we have undertaken is to investigate the adsorption of two different proteins (BSA and BLG) having near same IEP and differing in their conformational flexibility, onto the surface of ZnS nanoparticles (ZnS NPs). BSA and BLG both have an IEP value around pH~5. BSA is more prone to conformational deformation and considered "soft" while BLG holds the conformational rigidity and considered as "hard" protein. To ascertain the differences in surface coverage and conformation of the protein onto ZnS surface (PZC ~ 3.7), we have evaluated the adsorption profile at pH 7, where the entire surface behaves negatively. An integrated approach was taken by incorporating zeta (ζ) potential, fluorescence and CD for analyzing the adsorption process. In both systems, an increase in protein surface coverage was observed with the increase in free protein concentration in the solution and ζ values approaching that of native protein at high surface coverage. An alteration in the tertiary structure was observed for both BSA and BLG. The CD spectra analysis reveals that the secondary structure of the BSA was more deviated from the native protein structure, accommodating the increased adsorption value. For BLG no such prominent structural alteration was observed. These findings help us to understand better, how adjustment of the protein adsorption amount can be achieved onto the surface of nanoparticles having like charges. Copyright © 2013 Elsevier Inc. All rights reserved.

Temperature-dependent conformational variation of chromophoric dissolved organic matter and its consequent interaction with phenanthrene.

PubMed

Chen, Wei; Liu, Xiao-Yang; Yu, Han-Qing

2017-03-01

Temperature variation caused by climate change, seasonal variation and geographic locations affects the physicochemical compositions of chromophoric dissolved organic matter (CDOM), resulting in difference in the fates of CDOM-related environmental pollutants. Exploration into the thermal induced structural transition of CDOM can help to better understand their environmental impacts, but information on this aspect is still lacking. Through integrating fluorescence excitation-emission matrix coupled parallel factor analysis with synchronous fluorescence two-dimensional correlation spectroscopy, this study provides an in-depth insight into the temperature-dependent conformational transitions of CDOM and their impact on its hydrophobic interaction with persistent organic pollutants (with phenanthrene as an example) in water. The fluorescence components in CDOM change linearly to water temperature with different extents and different temperature regions. The thermal induced transition priority in CDOM is protein-like component → fulvic-like component → humic-like component. Furthermore, the impact of thermal-induced conformational transition of CDOM on its hydrophobic interaction with phenanthrene is observed and explored. The fluorescence-based analytic results reveal that the conjugation degree of the aromatic groups in the fulvic- and humic-like substances, and the unfolding of the secondary structure in the protein-like substances with aromatic structure, contribute to the conformation variation. This integrated approach jointly enhances the characterization of temperature-dependent conformational variation of CDOM, and provides a promising way to elucidate the environmental behaviours of CDOM. Copyright © 2017 Elsevier Ltd. All rights reserved.

Subtle Change in the Charge Distribution of Surface Residues May Affect the Secondary Functions of Cytochrome c*

PubMed Central

Paul, Simanta Sarani; Sil, Pallabi; Haldar, Shubhasis; Mitra, Samaresh; Chattopadhyay, Krishnananda

2015-01-01

Although the primary function of cytochrome c (cyt c) is electron transfer, the protein caries out an additional secondary function involving its interaction with membrane cardiolipin (CDL), its peroxidase activity, and the initiation of apoptosis. Whereas the primary function of cyt c is essentially conserved, its secondary function varies depending on the source of the protein. We report here a detailed experimental and computational study, which aims to understand, at the molecular level, the difference in the secondary functions of cyt c obtained from horse heart (mammalian) and Saccharomyces cerevisiae (yeast). The conformational landscape of cyt c has been found to be heterogeneous, consisting of an equilibrium between the compact and extended conformers as well as the oligomeric species. Because the determination of relative populations of these conformers is difficult to obtain by ensemble measurements, we used fluorescence correlation spectroscopy (FCS), a method that offers single-molecule resolution. The population of different species is found to depend on multiple factors, including the protein source, the presence of CDL and urea, and their concentrations. The complex interplay between the conformational distribution and oligomerization plays a crucial role in the variation of the pre-apoptotic regulation of cyt c observed from different sources. Finally, computational studies reveal that the variation in the charge distribution at the surface and the charge reversal sites may be the key determinant of the conformational stability of cyt c. PMID:25873393

Diversity of Secondary Structure in Catalytic Peptides with β-Turn-Biased Sequences

PubMed Central

2016-01-01

X-ray crystallography has been applied to the structural analysis of a series of tetrapeptides that were previously assessed for catalytic activity in an atroposelective bromination reaction. Common to the series is a central Pro-Xaa sequence, where Pro is either l- or d-proline, which was chosen to favor nucleation of canonical β-turn secondary structures. Crystallographic analysis of 35 different peptide sequences revealed a range of conformational states. The observed differences appear not only in cases where the Pro-Xaa loop-region is altered, but also when seemingly subtle alterations to the flanking residues are introduced. In many instances, distinct conformers of the same sequence were observed, either as symmetry-independent molecules within the same unit cell or as polymorphs. Computational studies using DFT provided additional insight into the analysis of solid-state structural features. Select X-ray crystal structures were compared to the corresponding solution structures derived from measured proton chemical shifts, 3J-values, and 1H–1H-NOESY contacts. These findings imply that the conformational space available to simple peptide-based catalysts is more diverse than precedent might suggest. The direct observation of multiple ground state conformations for peptides of this family, as well as the dynamic processes associated with conformational equilibria, underscore not only the challenge of designing peptide-based catalysts, but also the difficulty in predicting their accessible transition states. These findings implicate the advantages of low-barrier interconversions between conformations of peptide-based catalysts for multistep, enantioselective reactions. PMID:28029251

Conformational analysis of cellobiose by electronic structure theories.

PubMed

French, Alfred D; Johnson, Glenn P; Cramer, Christopher J; Csonka, Gábor I

2012-03-01

Adiabatic Φ/ψ maps for cellobiose were prepared with B3LYP density functional theory. A mixed basis set was used for minimization, followed with 6-31+G(d) single-point calculations, with and without SMD continuum solvation. Different arrangements of the exocyclic groups (38 starting geometries) were considered for each Φ/ψ point. The vacuum calculations agreed with earlier computational and experimental results on the preferred gas phase conformation (anti-Φ(H), syn-ψ(H)), and the results from the solvated calculations were consistent with the (syn Φ(H)/ψ(H) conformations from condensed phases (crystals or solutions). Results from related studies were compared, and there is substantial dependence on the solvation model as well as arrangements of exocyclic groups. New stabilizing interactions were revealed by Atoms-In-Molecules theory. Published by Elsevier Ltd.

Chronic Beryllium Disease: Revealing the Role of Beryllium Ion and Small Peptides Binding to HLA-DP2

PubMed Central

Petukh, Marharyta; Wu, Bohua; Stefl, Shannon; Smith, Nick; Hyde-Volpe, David; Wang, Li; Alexov, Emil

2014-01-01

Chronic Beryllium (Be) Disease (CBD) is a granulomatous disorder that predominantly affects the lung. The CBD is caused by Be exposure of individuals carrying the HLA-DP2 protein of the major histocompatibility complex class II (MHCII). While the involvement of Be in the development of CBD is obvious and the binding site and the sequence of Be and peptide binding were recently experimentally revealed [1], the interplay between induced conformational changes and the changes of the peptide binding affinity in presence of Be were not investigated. Here we carry out in silico modeling and predict the Be binding to be within the acidic pocket (Glu26, Glu68 and Glu69) present on the HLA-DP2 protein in accordance with the experimental work [1]. In addition, the modeling indicates that the Be ion binds to the HLA-DP2 before the corresponding peptide is able to bind to it. Further analysis of the MD generated trajectories reveals that in the presence of the Be ion in the binding pocket of HLA-DP2, all the different types of peptides induce very similar conformational changes, but their binding affinities are quite different. Since these conformational changes are distinctly different from the changes caused by peptides normally found in the cell in the absence of Be, it can be speculated that CBD can be caused by any peptide in presence of Be ion. However, the affinities of peptides for Be loaded HLA-DP2 were found to depend of their amino acid composition and the peptides carrying acidic group at positions 4 and 7 are among the strongest binders. Thus, it is proposed that CBD is caused by the exposure of Be of an individual carrying the HLA-DP2*0201 allele and that the binding of Be to HLA-DP2 protein alters the conformational and ionization properties of HLA-DP2 such that the binding of a peptide triggers a wrong signaling cascade. PMID:25369028

Conformational plasticity of DM43, a metalloproteinase inhibitor from Didelphis marsupialis: chemical and pressure-induced equilibrium (un)folding studies.

PubMed

Chapeaurouge, Alex; Martins, Samantha M; Holub, Oliver; Rocha, Surza L G; Valente, Richard H; Neves-Ferreira, Ana G C; Ferreira, Sérgio T; Domont, Gilberto B; Perales, Jonas

2009-10-01

We have investigated the folding of DM43, a homodimeric metalloproteinase inhibitor isolated from the serum of the South American opossum Didelphis marsupialis. Denaturation of the protein induced by GdnHCl (guanidine hydrochloride) was monitored by extrinsic and intrinsic fluorescence spectroscopy. While the equilibrium (un)folding of DM43 followed by tryptophan fluorescence was well described by a cooperative two-state transition, bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid) fluorescence measurements revealed an intensity maximum at the midpoint of the unfolding transition (2 M GdnHCl), indicating a partially folded intermediate state. We further investigated the DM43 intermediate stabilized at 2 M GdnHCl using size exclusion chromatography. This analysis revealed that the folding intermediate can be best described as partially folded DM43 monomers. Thermodynamic analysis of the GdnHCl-induced denaturation of DM43 revealed Gibbs free-energy changes of 13.57 kcal/mol for dimer dissociation and 1.86 kcal/mol for monomer unfolding, pointing to a critical role of dimerization as a determinant of the structure and stability of this protein. In addition, by using hydrostatic pressure (up to 3.5 kbar) we were able to stabilize partially folded states different from those stabilized in the presence of GdnHCl. Taken together, these results indicate that the conformational plasticity of DM43 could provide this protein with the ability to adapt its conformation to a variety of different environments and biological partners during its biological lifetime.

Structural diversity of supercoiled DNA

PubMed Central

Irobalieva, Rossitza N.; Fogg, Jonathan M.; Catanese, Daniel J.; Sutthibutpong, Thana; Chen, Muyuan; Barker, Anna K.; Ludtke, Steven J.; Harris, Sarah A.; Schmid, Michael F.; Chiu, Wah; Zechiedrich, Lynn

2015-01-01

By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function. PMID:26455586

Structural diversity of supercoiled DNA

NASA Astrophysics Data System (ADS)

Irobalieva, Rossitza N.; Fogg, Jonathan M.; Catanese, Daniel J.; Sutthibutpong, Thana; Chen, Muyuan; Barker, Anna K.; Ludtke, Steven J.; Harris, Sarah A.; Schmid, Michael F.; Chiu, Wah; Zechiedrich, Lynn

2015-10-01

By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function.

Ligand Size and Conformation Affect the Behavior of Nanoparticles Coated with in Vitro and in Vivo Protein Corona.

PubMed

Zhang, Huajin; Wu, Tianmu; Yu, Wenqi; Ruan, Shaobo; He, Qin; Gao, Huile

2018-03-14

Protein corona is immediately established on the surface of nanoparticles upon their introduction into biological milieu. Several studies have shown that the targeting efficiency of ligand-modified nanoparticles is attenuated or abolished owing to the protein adsorption. Here, transferrin receptor-targeting ligands, including LT7 (CHAIYPRH), DT7 (hrpyiahc, all d-form amino acids), and transferrin, were used to identify the influence of the ligand size and conformation on protein corona formation. The results showed that the targeting capacity of ligand-modified nanoparticles was lost after incubation with plasma in vitro, whereas it was partially retained after in vivo corona formation. Results from sodium dodecyl sulfate polyacrylamide gel electrophoresis and liquid chromatography-mass spectrometry revealed the difference in the composition of in vitro and in vivo corona, wherein the ligand size and conformation played a critical role. Differences were observed in cellular internalization and exocytosis profiles on the basis of the ligand and corona source.

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

PubMed

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

2014-01-09

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

Conformational preferences of DNA following damage by aristolochic acids: Structural and energetic insights into the different mutagenic potential of the ALI and ALII-N(6)-dA adducts.

PubMed

Kathuria, Preetleen; Sharma, Purshotam; Abendong, Minette N; Wetmore, Stacey D

2015-04-21

Aristolochic acids (AAI and AAII), produced by the Aristolochiaceae family of plants, are classified as group I (human) carcinogens by the International Agency for Research on Cancer. These acids are metabolized in cells to yield aristolactams (ALI and ALII, respectively), which further form bulky adducts with the purine nucleobases. Specifically, the adenine lesions are more persistent in cells and have been associated with chronic renal diseases and related carcinogenesis. To understand the structural basis of the nephrotoxicity induced by AAs, the ALI-N(6)-dA and ALII-N(6)-dA lesions are systematically studied using computational methods. Density functional theory calculations indicate that the aristolactam moiety intrinsically prefers a planar conformation with respect to adenine. Nucleoside and nucleotide models suggest that the anti and syn orientations about the glycosidic bond are isoenergetic for both adducts. Molecular dynamics simulations and free energy calculations reveal that the anti base-displaced intercalated conformation is the most stable conformer for both types of AL-N(6)-dA adducted DNA, which agrees with previous experimental work on the ALII-N(6)-dA adduct and thereby validates our approach. Interestingly, this conformer differs from the dominant conformations adopted by other N6-linked adenine lesions, including those derived from polycyclic aromatic hydrocarbons. Furthermore, the second most stable syn base-displaced intercalated conformation lies closer in energy to the anti base-displaced intercalated conformation for ALI-N(6)-dA compared to ALII-N(6)-dA. This indicates that a mixture of conformations may be detectable for ALI-N(6)-dA in DNA. If this enhanced conformational flexibility of double-stranded DNA persists when bound to a lesion-bypass polymerase, this provides a possible structural explanation for the previously observed greater nephrotoxic potential for the ALI versus ALII-N(6)-dA adduct. In addition, the structural characteristics of the preferred conformations of adducted DNA explain the resistance of these adducts to repair and thereby add to our current understanding of the toxicity of AAs within living cells.

Conformational changes and metastable states induced in proteins by green light

NASA Astrophysics Data System (ADS)

Comorosan, Sorin; Popescu, Irinel; Polosan, Silviu; Pirvu, Cristian; Ionescu, Elena; Paslaru, Liliana; Apostol, Marian

2015-01-01

In this paper we report conformational changes recorded on a protein molecule (α-amylase) under green light irradiation. In order to explain the experimental results we advanced the hypothesis that green light induces electric dipoles in the protein, which interact with each other, generating conformational modifications toward a more compact design, with different physical properties. The experiments were carried out with un-polarized light (λ = 520 nm) from a light-emitting-diode (1000 lm, 20 W, 105 mW on the target). In view of the character of our hypothesis, and corroborated with all our experimental results, we suggest that this phenomenon may be more extended and general, specific for a larger class of proteins, occurring on the protein macromolecules under the green light. The effects of α-amylase protein irradiation were revealed by circular dichroism, fluorescence, Raman and FTIR-spectroscopies, zeta potential, cyclic voltammetry, electric impedance spectroscopy and atomic force microscopy. Tentatively, we term the novel conformations as P∗ (polarized) proteins.

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

PubMed Central

Formoso, Elena; Limongelli, Vittorio; Parrinello, Michele

2015-01-01

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

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

NASA Astrophysics Data System (ADS)

Formoso, Elena; Limongelli, Vittorio; Parrinello, Michele

2015-02-01

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

Molecular dynamics simulations of void defects in the energetic material HMX.

PubMed

Duan, Xiao Hui; Li, Wen Peng; Pei, Chong Hua; Zhou, Xiao Qing

2013-09-01

A molecular dynamics (MD) simulation was carried out to characterize the dynamic evolution of void defects in crystalline octahydro-1, 3, 5, 7-tetranitro-1, 3, 5, 7-tetrazocine (HMX). Different models were constructed with the same concentration of vacancies (10 %) to discuss the size effects of void. Energetic ground state properties were determined by annealing simulations. The void formation energy per molecule removed was found to be 55-63 kcal/mol(-1), and the average binding energy per molecule was between 32 and 34 kcal/mol(-1) according to the change in void size. Voids with larger size had lower formation energy. Local binding energies for molecules directly on the void surface decreased greatly compared to those in defect-free lattice, and then gradually increased until the distance away from the void surface was around 10 Å. Analysis of 1 ns MD simulations revealed that the larger the void size, the easier is void collapse. Mean square displacements (MSDs) showed that HMX molecules that had collapsed into void present liquid structure characteristics. Four unique low-energy conformers were found for HMX molecules in void: two whose conformational geometries corresponded closely to those found in HMX polymorphs and two, additional, lower energy conformers that were not seen in the crystalline phases. The ratio of different conformers changed with the simulated temperature, in that the ratio of α conformer increased with the increase in temperature.

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

Maaloum, M.; Muller, P.; Beker, A-F.

Almost two decades ago, measurements of force versus extension on isolated double-stranded DNA molecules revealed a force plateau. This unusual stretching phenomenon in DNA suggests that the long molecules may be extended from the usual B form into a new conformation. Different models have been proposed to describe the nature of DNA in its stretched form, S-DNA. Using atomic force microscopy combined with a molecular combing method, we identified the structure of {lambda}-phage DNA for different stretching values. We provide strong evidence for the existence of a first-order transition between B form and S form. Beyond a certain extension ofmore » the natural length, DNA molecules adopt a new double-helix conformation characterized by a diameter of 1.2 nm and a helical pitch of18 nm.« less

Base-Displaced Intercalated Conformation of the 2-Amino-3-methylimidazo[4,5-f]quinoline N2-dG DNA Adduct Positioned at the Nonreiterated G1 in the NarI Restriction Site

PubMed Central

2016-01-01

The conformation of an N2-dG adduct arising from the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a potent food mutagen, was determined in 5′-d(C1T2C3X4G5C6G7C8C9A10T11C12)-3′:5′-d(G13A14T15G16G17C18G19C20C21G22A23G24)-3′; X = N2-dG-IQ, in which the modified nucleotide X4 corresponds to G1 in the 5′-d(G1G2CG3CC)-3′ NarI restriction endonuclease site. Circular dichroism (CD) revealed blue shifts relative to the unmodified duplex, consistent with adduct-induced twisting, and a hypochromic effect for the IQ absorbance in the near UV region. NMR revealed that the N2-dG-IQ adduct adopted a base-displaced intercalated conformation in which the modified guanine remained in the anti conformation about the glycosidic bond, the IQ moiety intercalated into the duplex, and the complementary base C21 was displaced into the major groove. The processing of the N2-dG-IQ lesion by hpol η is sequence-dependent; when placed at the reiterated G3 position, but not at the G1 position, this lesion exhibits a propensity for frameshift replication [Choi, J. Y., et al. (2006) J. Biol. Chem., 281, 25297–25306]. The structure of the N2-dG-IQ adduct at the nonreiterated G1 position was compared to that of the same adduct placed at the G3 position [Stavros, K. M., et al. (2014) Nucleic Acids Res., 42, 3450–3463]. CD indicted minimal spectral differences between the G1 vs G3N2-dG-IQ adducts. NMR indicated that the N2-dG-IQ adduct exhibited similar base-displaced intercalated conformations at both the G1 and G3 positions. This result differed as compared to the corresponding C8-dG-IQ adducts placed at the same positions. The C8-dG-IQ adduct adopted a minor groove conformation when placed at position G1 but a base-displaced intercalated conformation when placed at position G3 in the NarI sequence. The present studies suggest that differences in lesion bypass by hpol η may be mediated by differences in the 3′-flanking sequences, perhaps modulating the ability to accommodate transient strand slippage intermediates. PMID:26083477

Cyndi: a multi-objective evolution algorithm based method for bioactive molecular conformational generation.

PubMed

Liu, Xiaofeng; Bai, Fang; Ouyang, Sisheng; Wang, Xicheng; Li, Honglin; Jiang, Hualiang

2009-03-31

Conformation generation is a ubiquitous problem in molecule modelling. Many applications require sampling the broad molecular conformational space or perceiving the bioactive conformers to ensure success. Numerous in silico methods have been proposed in an attempt to resolve the problem, ranging from deterministic to non-deterministic and systemic to stochastic ones. In this work, we described an efficient conformation sampling method named Cyndi, which is based on multi-objective evolution algorithm. The conformational perturbation is subjected to evolutionary operation on the genome encoded with dihedral torsions. Various objectives are designated to render the generated Pareto optimal conformers to be energy-favoured as well as evenly scattered across the conformational space. An optional objective concerning the degree of molecular extension is added to achieve geometrically extended or compact conformations which have been observed to impact the molecular bioactivity (J Comput -Aided Mol Des 2002, 16: 105-112). Testing the performance of Cyndi against a test set consisting of 329 small molecules reveals an average minimum RMSD of 0.864 A to corresponding bioactive conformations, indicating Cyndi is highly competitive against other conformation generation methods. Meanwhile, the high-speed performance (0.49 +/- 0.18 seconds per molecule) renders Cyndi to be a practical toolkit for conformational database preparation and facilitates subsequent pharmacophore mapping or rigid docking. The copy of precompiled executable of Cyndi and the test set molecules in mol2 format are accessible in Additional file 1. On the basis of MOEA algorithm, we present a new, highly efficient conformation generation method, Cyndi, and report the results of validation and performance studies comparing with other four methods. The results reveal that Cyndi is capable of generating geometrically diverse conformers and outperforms other four multiple conformer generators in the case of reproducing the bioactive conformations against 329 structures. The speed advantage indicates Cyndi is a powerful alternative method for extensive conformational sampling and large-scale conformer database preparation.

Optical and tunneling microscopy and spectroscopy at the ultimate spatial limit

NASA Astrophysics Data System (ADS)

Chen, Chi

2009-12-01

The combination of optical detection system with a scanning tunneling microscope (STM) leads to the possibility of resolving radiative transition probability with the ultrahigh spatial resolution of STM in real space. This opens an innovative approach toward revealing the correlation between molecular structure, electronic characteristics, and optical properties. This thesis describes a series of experiments that manifests this correlation, including atomic silver chains and single porphine molecules. In atomic silver chains, the number and positions of the emission maxima in the photon images match the nodes in the dI/d V images of "particle-in-a-box" states. This surprising correlation between the emission maxima and nodes in the density of states is a manifestation of Fermi's golden rule in real space for radiative transitions, which provides an understanding of the mechanism of STM induced light emission. From single porphine molecules, orthogonal spatial contrast of two types of vibronic coupling is resolved by both photon spectroscopy and vibronic-mode-selected photon images. Intramolecular transitions from the two orthogonal LUMOs individually couple to different molecular normal modes. This is the first demonstration of the photon emission probability of a single molecule and its direct correlations with the molecular orbitals. This also provides the first real space experimental evidence to separate the tangled effects of molecular conformations and nano-environments on the inhomogeneity of molecular emission. DSB molecules are found to have two conformational isomers and one of them shows surface chirality. All these conformers and enantiomers can be switched to each other by electron injection. Different DSB conformers present distinct manipulation dynamics, which demonstrate how different conformations and their preferred adsorption geometries can have pronounced influence on the molecular mechanics on the surface. Overall, this thesis studies the very fundamental nature of single molecules and artificial nanostructures by integrating all kinds of important functions of STM: topography, spectroscopy, manipulation, and photon emission. Detailed correlations between the emission patterns and orbital structures are revealed by the ultimate spatial resolution of our "STM photon microscopy".

Protein Conformational Populations and Functionally Relevant Sub-states

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

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

2013-01-01

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

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.

Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding.

PubMed

Gui, Miao; Song, Wenfei; Zhou, Haixia; Xu, Jingwei; Chen, Silian; Xiang, Ye; Wang, Xinquan

2017-01-01

The global outbreak of SARS in 2002-2003 was caused by the infection of a new human coronavirus SARS-CoV. The infection of SARS-CoV is mediated mainly through the viral surface glycoproteins, which consist of S1 and S2 subunits and form trimer spikes on the envelope of the virions. Here we report the ectodomain structures of the SARS-CoV surface spike trimer in different conformational states determined by single-particle cryo-electron microscopy. The conformation 1 determined at 4.3 Å resolution is three-fold symmetric and has all the three receptor-binding C-terminal domain 1 (CTD1s) of the S1 subunits in "down" positions. The binding of the "down" CTD1s to the SARS-CoV receptor ACE2 is not possible due to steric clashes, suggesting that the conformation 1 represents a receptor-binding inactive state. Conformations 2-4 determined at 7.3, 5.7 and 6.8 Å resolutions are all asymmetric, in which one RBD rotates away from the "down" position by different angles to an "up" position. The "up" CTD1 exposes the receptor-binding site for ACE2 engagement, suggesting that the conformations 2-4 represent a receptor-binding active state. This conformational change is also required for the binding of SARS-CoV neutralizing antibodies targeting the CTD1. This phenomenon could be extended to other betacoronaviruses utilizing CTD1 of the S1 subunit for receptor binding, which provides new insights into the intermediate states of coronavirus pre-fusion spike trimer during infection.

Thermodynamic analysis of the disorder-to-α-helical transition of 18.5-kDa myelin basic protein reveals an equilibrium intermediate representing the most compact conformation.

PubMed

Vassall, Kenrick A; Jenkins, Andrew D; Bamm, Vladimir V; Harauz, George

2015-05-22

The intrinsically disordered, 18.5-kDa isoform of myelin basic protein (MBP) is a peripheral membrane protein that is essential to proper myelin formation in the central nervous system. MBP acts in oligodendrocytes both to adjoin membrane leaflets to each other in forming myelin and as a hub in numerous protein-protein and protein-membrane interaction networks. Like many intrinsically disordered proteins (IDPs), MBP multifunctionality arises from its high conformational plasticity and its ability to undergo reversible disorder-to-order transitions. One such transition is the disorder-to-α-helical conformational change that is induced upon MBP-membrane binding. Here, we have investigated the disorder-to-α-helical transition of MBP-derived α-peptides and the full-length 18.5-kDa protein. This transition was induced through titration of the membrane-mimetic solvent trifluoroethanol into both protein and peptide solutions, and conformational change was monitored using circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid binding, tryptophan fluorescence quenching, and Förster (fluorescence) resonance energy transfer measurements. The data suggest that the disorder-to-α-helical transition of MBP follows a 3-state model: disordered↔intermediate↔α-helical, with each of the identified equilibrium states likely representing a conformational ensemble. The disordered state is characterized by slight compaction with little regular secondary structure, whereas the intermediate is also disordered but globally more compact. Surprisingly, the α-helical conformation is less compact than the intermediate. This study suggests that multifunctionality in MBP could arise from differences in the population of energetically distinct ensembles under different conditions and also provides an example of an IDP that undergoes cooperative global conformation change. Copyright © 2015 Elsevier Ltd. All rights reserved.

Discovery of black dye crystal structure polymorphs: Implications for dye conformational variation in dye-sensitized solar cells

DOE PAGES

Cole, Jacqueline M.; Low, Kian Sing; Gong, Yun

2015-11-24

Here, we present the discovery of a new crystal structure polymorph (1) and pseudopolymorph (2) of the Black Dye, one of the world’s leading dyes for dye-sensitized solar cells, DSSCs (10.4% device performance efficiency). This reveals that Black Dye molecules can adopt multiple low-energy conformers. This is significant since it challenges existing models of the Black Dye···TiO 2 adsorption process that renders a DSSC working electrode; these have assumed a single molecular conformation that refers to the previously reported Black Dye crystal structure (3). The marked structural differences observed between 1, 2, and 3 make the need for modeling multiplemore » conformations more acute. Additionally, the ordered form of the Black Dye (1) provides a more appropriate depiction of its anionic structure, especially regarding its anchoring group and NCS bonding descriptions. The tendency toward NCS ligand isomerism, evidenced via the disordered form 2, has consequences for electron injection and electron recombination in Black Dye embedded DSSC devices. Dyes 2 and 3 differ primarily by the absence or presence of a solvent of crystallization, respectively; solvent environment effects on the dye are thereby elucidated. This discovery of multiple Black Dye conformers from diffraction, with atomic-level definition, complements recently reported nanoscopic evidence for multiple dye conformations existing at a dye···TiO 2 interface, for a chemically similar DSSC dye; those results emanated from imaging and spectroscopy, but were unresolved at the submolecular level. Taken together, these findings lead to the general notion that multiple dye conformations should be explicitly considered when modeling dye···TiO 2 interfaces in DSSCs, at least for ruthenium-based dye complexes.« less

Free energy changes and components implicit in the MWC allosteric model for the cooperative oxygen binding of hemoglobin.#

PubMed Central

Bucci, Enrico

2013-01-01

Hill’s plots of oxygen binding isotherms reveal the presence of a transition between two different oxygen affinities at the beginning and end of the isotherm. They correspond to the two conformations anticipated by the MWC model, namely the T and R conformations at the beginning and end of oxygen binding, when the lower affinity of the T form develops into the higher affinity of the R form. The difference between the binding Gibbs free energies changes of the two affinities (ΔGL) is the free energy of binding cooperativity. Notably ΔGL is positive in favor of the T form, that moves to a higher energy level upon oxygen release. Osmotic stress reveals a higher volume/surface ratio of deoxyHb, with a positive ΔGW also in favor of the T form . Increasing protein concentration shifts the isotherms to the right indicating the formation of intermediate polymeric forms. Enthalpy of the intermediates show a strong absorption of heat at the third oxygenation step due to polymers formation with quinary, and above, structures. The disassembly of intermediate polymers releases energy with a negative ΔG that compensates and allow the positivity of ΔGL. High energy polymers are the barrier preventing the relaxation of the T and R conformations into one another. The MWC allosteric model is the best justification of oxygen binding cooperativity . PMID:23710673

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

Conformation-selective resonant photoelectron imaging from dipole-bound states of cold 3-hydroxyphenoxide

NASA Astrophysics Data System (ADS)

Zhu, Guo-Zhu; Huang, Dao-Ling; Wang, Lai-Sheng

2017-07-01

We report a photoelectron imaging and photodetachment study of cryogenically cooled 3-hydroxyphenoxide (3HOP) anions, m-HO(C6H4)O-. In a previous preliminary study, two conformations of the cold 3HOP anions with different dipole bound states were observed [D. L. Huang et al., J. Phys. Chem. Lett. 6, 2153 (2015)]. Five near-threshold vibrational resonances were revealed in the photodetachment spectrum from the dipole-bound excited states of the two conformations. Here, we report a more extensive investigation of the two conformers with observation of thirty above-threshold vibrational resonances in a wide spectral range between 18 850 and 19 920 cm-1 (˜1000 cm-1 above the detachment thresholds). By tuning the detachment laser to the vibrational resonances in the photodetachment spectrum, high-resolution conformation-selective resonant photoelectron images are obtained. Using information of the autodetachment channels and theoretical vibrational frequencies, we are able to assign the resonant peaks in the photodetachment spectrum: seventeen are assigned to vibrational levels of anti-3HOP, eight to syn-3HOP, and five to overlapping vibrational levels of both conformers. From the photodetachment spectrum and the conformation-selective resonant photoelectron spectra, we have obtained fourteen fundamental vibrational frequencies for the neutral syn- and anti-m-HO(C6H4)Oṡ radicals. The possibility to produce conformation-selected neutral beams using resonant photodetachment via dipole-bound excited states of anions is discussed.

Conformational Dynamics of a Y-Family DNA Polymerase during Substrate Binding and Catalysis As Revealed by Interdomain Förster Resonance Energy Transfer

PubMed Central

2015-01-01

Numerous kinetic, structural, and theoretical studies have established that DNA polymerases adjust their domain structures to enclose nucleotides in their active sites and then rearrange critical active site residues and substrates for catalysis, with the latter conformational change acting to kinetically limit the correct nucleotide incorporation rate. Additionally, structural studies have revealed a large conformational change between the apoprotein and the DNA–protein binary state for Y-family DNA polymerases. In previous studies [Xu, C., Maxwell, B. A., Brown, J. A., Zhang, L., and Suo, Z. (2009) PLoS Biol.7, e1000225], a real-time Förster resonance energy transfer (FRET) method was developed to monitor the global conformational transitions of DNA polymerase IV from Sulfolobus solfataricus (Dpo4), a prototype Y-family enzyme, during nucleotide binding and incorporation by measuring changes in distance between locations on the enzyme and the DNA substrate. To elucidate further details of the conformational transitions of Dpo4 during substrate binding and catalysis, in this study, the real-time FRET technique was used to monitor changes in distance between various pairs of locations in the protein itself. In addition to providing new insight into the conformational changes as revealed in previous studies, the results here show that the previously described conformational change between the apo and DNA-bound states of Dpo4 occurs in a mechanistic step distinct from initial formation or dissociation of the binary complex of Dpo4 and DNA. PMID:24568554

Inter-ring rotations of AAA ATPase p97 revealed by electron cryomicroscopy

PubMed Central

Yeung, Heidi O.; Förster, Andreas; Bebeacua, Cecilia; Niwa, Hajime; Ewens, Caroline; McKeown, Ciarán; Zhang, Xiaodong; Freemont, Paul S.

2014-01-01

The type II AAA+ protein p97 is involved in numerous cellular activities, including endoplasmic reticulum-associated degradation, transcription activation, membrane fusion and cell-cycle control. These activities are at least in part regulated by the ubiquitin system, in which p97 is thought to target ubiquitylated protein substrates within macromolecular complexes and assist in their extraction or disassembly. Although ATPase activity is essential for p97 function, little is known about how ATP binding or hydrolysis is coupled with p97 conformational changes and substrate remodelling. Here, we have used single-particle electron cryomicroscopy (cryo-EM) to study the effect of nucleotides on p97 conformation. We have identified conformational heterogeneity within the cryo-EM datasets from which we have resolved two major p97 conformations. A comparison of conformations reveals inter-ring rotations upon nucleotide binding and hydrolysis that may be linked to the remodelling of target protein complexes. PMID:24598262

Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands

NASA Astrophysics Data System (ADS)

Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A.; Bernardi, Anna; Colombo, Giorgio

2016-04-01

Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators.

Color deviation controlling of phosphor conformal coating by advanced spray painting technology for white LEDs.

PubMed

Yang, Liang; Wang, Simin; Lv, Zhicheng; Liu, Sheng

2013-04-01

An advanced phosphor conformal coating technology is proposed, good correlated color temperature (CCT) and chromaticity uniformity samples are fabricated through phosphor spray painting technology. Spray painting technology is also suitable for phosphor conformal coating of whole LED wafers. The samples of different CCTs are obtained through controlling the phosphor film thickness in the range of 6-80 μm; CCT variation of samples can be controlled in the range of ±200 K. The experimental Δuv reveals that the spray painting method can obtain a much smaller CCT variation (Δuv of 1.36e(-3)) than the conventional dispensing method (Δuv of 11.86e(-3)) when the light is emitted at angles from -90° to +90°, and chromaticity area uniformity is also improved significantly.

Investigating Differences in Gas-Phase Conformations of 25-Hydroxyvitamin D3 Sodiated Epimers using Ion Mobility-Mass Spectrometry and Theoretical Modeling

NASA Astrophysics Data System (ADS)

Chouinard, Christopher D.; Cruzeiro, Vinícius Wilian D.; Beekman, Christopher R.; Roitberg, Adrian E.; Yost, Richard A.

2017-08-01

Drift tube ion mobility coupled with mass spectrometry was used to investigate the gas-phase structure of 25-hydroxyvitamin D3 (25OHD3) and D2 (25OHD2) epimers, and to evaluate its potential in rapid separation of these compounds. Experimental results revealed two distinct drift species for the 25OHD3 sodiated monomer, whereas only one of these conformations was observed for its epimer (epi25OHD3). The unique species allowed 25OHD3 to be readily distinguished, and the same pattern was observed for 25OHD2 epimers. Theoretical modeling of 25OHD3 epimers identified energetically stable gas-phase structures, indicating that both compounds may adopt a compact "closed" conformation, but that 25OHD3 may also adopt a slightly less energetically favorable "open" conformation that is not accessible to its epimer. Calculated theoretical collision cross-sections for these structures agreed with experimental results to <2%. Experimentation indicated that additional energy in the ESI source (i.e., increased temperature, spray voltage) affected the ratio of 25OHD3 conformations, with the less energetically favorable "open" conformation increasing in relative intensity. Finally, LC-IM-MS results yielded linear quantitation of 25OHD3, in the presence of the epimer interference, at biologically relevant concentrations. This study demonstrates that ion mobility can be used in tandem with theoretical modeling to determine structural differences that contribute to drift separation. These separation capabilities provide potential for rapid (<60 ms) identification of 25OHD3 and 25OHD2 in mixtures with their epimers.

Molecular Dynamic Simulation of Space and Earth-Grown Crystal Structures of Thermostable T1 Lipase Geobacillus zalihae Revealed a Better Structure.

PubMed

Ishak, Siti Nor Hasmah; Aris, Sayangku Nor Ariati Mohamad; Halim, Khairul Bariyyah Abd; Ali, Mohd Shukuri Mohamad; Leow, Thean Chor; Kamarudin, Nor Hafizah Ahmad; Masomian, Malihe; Rahman, Raja Noor Zaliha Raja Abd

2017-09-25

Less sedimentation and convection in a microgravity environment has become a well-suited condition for growing high quality protein crystals. Thermostable T1 lipase derived from bacterium Geobacillus zalihae has been crystallized using the counter diffusion method under space and earth conditions. Preliminary study using YASARA molecular modeling structure program for both structures showed differences in number of hydrogen bond, ionic interaction, and conformation. The space-grown crystal structure contains more hydrogen bonds as compared with the earth-grown crystal structure. A molecular dynamics simulation study was used to provide insight on the fluctuations and conformational changes of both T1 lipase structures. The analysis of root mean square deviation (RMSD), radius of gyration, and root mean square fluctuation (RMSF) showed that space-grown structure is more stable than the earth-grown structure. Space-structure also showed more hydrogen bonds and ion interactions compared to the earth-grown structure. Further analysis also revealed that the space-grown structure has long-lived interactions, hence it is considered as the more stable structure. This study provides the conformational dynamics of T1 lipase crystal structure grown in space and earth condition.

Calcium-controlled conformational choreography in the N-terminal half of adseverin

NASA Astrophysics Data System (ADS)

Chumnarnsilpa, Sakesit; Robinson, Robert C.; Grimes, Jonathan M.; Leyrat, Cedric

2015-09-01

Adseverin is a member of the calcium-regulated gelsolin superfamily of actin-binding proteins. Here we report the crystal structure of the calcium-free N-terminal half of adseverin (iA1-A3) and the Ca2+-bound structure of A3, which reveal structural similarities and differences with gelsolin. Solution small-angle X-ray scattering combined with ensemble optimization revealed a dynamic Ca2+-dependent equilibrium between inactive, intermediate and active conformations. Increasing calcium concentrations progressively shift this equilibrium from a main population of inactive conformation to the active form. Molecular dynamics simulations of iA1-A3 provided insights into Ca2+-induced destabilization, implicating a critical role for the A2 type II calcium-binding site and the A2A3 linker in the activation process. Finally, mutations that disrupt the A1/A3 interface increase Ca2+-independent F-actin severing by A1-A3, albeit at a lower efficiency than observed for gelsolin domains G1-G3. Together, these data address the calcium dependency of A1-A3 activity in relation to the calcium-independent activity of G1-G3.

Evaluation of the HARDMAN comparability methodology for manpower, personnel and training

NASA Technical Reports Server (NTRS)

Zimmerman, W.; Butler, R.; Gray, V.; Rosenberg, L.

1984-01-01

The methodology evaluation and recommendation are part of an effort to improve Hardware versus Manpower (HARDMAN) methodology for projecting manpower, personnel, and training (MPT) to support new acquisition. Several different validity tests are employed to evaluate the methodology. The methodology conforms fairly well with both the MPT user needs and other accepted manpower modeling techniques. Audits of three completed HARDMAN applications reveal only a small number of potential problem areas compared to the total number of issues investigated. The reliability study results conform well with the problem areas uncovered through the audits. The results of the accuracy studies suggest that the manpower life-cycle cost component is only marginally sensitive to changes in other related cost variables. Even with some minor problems, the methodology seem sound and has good near term utility to the Army. Recommendations are provided to firm up the problem areas revealed through the evaluation.

Nobiletin: a citrus flavonoid displaying potent physiological activity.

PubMed

Noguchi, Shuji; Atsumi, Haruka; Iwao, Yasunori; Kan, Toshiyuki; Itai, Shigeru

2016-02-01

Nobiletin [systematic name: 2-(3,4-dimethoxyphenyl)-5,6,7,8-tetramethoxy-4H-chromen-4-one; C21H22O8] is a flavonoid found in citrus peels, and has been reported to show a wide range of physiological properties, including anti-inflammatory, anticancer and antidementia activities. We have solved the crystal structure of nobiletin, which revealed that the chromene and arene rings of its flavone moiety, as well as the two methoxy groups bound to its arene ring, were coplanar. In contrast, the C atoms of the four methoxy groups bound to the chromene ring are out of the plane, making the molecule conformationally chiral. A comparison of the crystal structures of nobiletin revealed that it could adopt a variety of different conformations through rotation of the covalent bond between the chromene and arene rings, and the orientations of methoxy groups bound to the chromene ring.

Entropy–enthalpy transduction caused by conformational shifts can obscure the forces driving protein–ligand binding

PubMed Central

Fenley, Andrew T.; Muddana, Hari S.; Gilson, Michael K.

2012-01-01

Molecular dynamics simulations of unprecedented duration now can provide new insights into biomolecular mechanisms. Analysis of a 1-ms molecular dynamics simulation of the small protein bovine pancreatic trypsin inhibitor reveals that its main conformations have different thermodynamic profiles and that perturbation of a single geometric variable, such as a torsion angle or interresidue distance, can select for occupancy of one or another conformational state. These results establish the basis for a mechanism that we term entropy–enthalpy transduction (EET), in which the thermodynamic character of a local perturbation, such as enthalpic binding of a small molecule, is camouflaged by the thermodynamics of a global conformational change induced by the perturbation, such as a switch into a high-entropy conformational state. It is noted that EET could occur in many systems, making measured entropies and enthalpies of folding and binding unreliable indicators of actual thermodynamic driving forces. The same mechanism might also account for the high experimental variance of measured enthalpies and entropies relative to free energies in some calorimetric studies. Finally, EET may be the physical mechanism underlying many cases of entropy–enthalpy compensation. PMID:23150595

“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

New open conformation of SMYD3 implicates conformational selection and allostery

PubMed Central

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

2016-01-01

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

Variable sequences outside the SAM-binding core critically influence the conformational dynamics of the SAM-III/SMK box riboswitch

PubMed Central

Lu, Changrui; Smith, Angela M; Ding, Fang; Chowdhury, Anirban; Henkin, Tina M; Ke, Ailong

2012-01-01

The SMK box (SAM-III) translational riboswitches were identified in S-adenosyl-L-methionine (SAM) synthetase metK genes in members of the Lactobacillales. This riboswitch switches between two alternative conformations in response to the intracellular SAM concentration and controls metK expression at the level of translation initiation. We previously reported the crystal structure of the SAM-bound SMK box riboswitch. In this study we combined SHAPE chemical probing with mutagenesis to probe the ligand-induced conformational switching mechanism. We revealed that while the majority of the apo SMK box RNA molecules exist in an alternatively base paired (ON) conformation, a subset of them pre-organize into a SAM-bound-like (READY) conformation, which upon SAM exposure is selectively stabilized into the SAM-bound (OFF) conformation through an induced-fit mechanism. Mutagenesis showed that the ON state is only slightly more stable than the READY state, as several single-nucleotide substitutions in a hypervariable region outside the SAM-binding core can alter the folding landscape to favor the READY state. Such SMK variants display a “constitutively-OFF” behavior both in vitro and in vivo. Time-resolved and temperature-dependent SHAPE analyses revealed adaptation of the SMK box RNA to its mesothermal working environment. The latter analysis revealed that the SAM-bound SMK box RNA follows a two-step folding/unfolding process. PMID:21549712

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

PubMed

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

2017-06-16

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

Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography*

PubMed Central

Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; Lampe, Jed N.; Nishida, Clinton R.; de Montellano, Paul R. Ortiz

2015-01-01

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states. PMID:25670859

Protein conformation determines the sensibility to high pressure treatment of infectious scrapie prions.

PubMed

Heindl, Philipp; García, Avelina Fernández; Butz, Peter; Pfaff, Eberhard; Tauscher, Bernhard

2006-03-01

Application of high pressure can be used for gentle pasteurizing of food, minimizing undesirable alterations such as vitamin losses and changes in taste and color. In addition, pressure has become a useful tool for investigating structural changes in proteins. Treatments of proteins with high pressure can reveal conformations that are not obtainable by other physical variables like temperature, since pressure favors structural transitions accompanied with smaller volumes. Here, we discuss both the potential use of high pressure to inactivate infectious TSE material and the application of this thermodynamic parameter for the investigation of prion folding. This review summarizes our findings on the effects of pressure on the structure of native infectious scrapie prions in hamster brain homogenates and on the structure of infectious prion rods isolated from diseased hamsters brains. Native prions were found to be pressure sensitive, whereas isolated prions revealed an extreme pressure-resistant structure. The discussion will be focused on the different pressure behavior of these prion isoforms, which points out differences in the protein structure that have not been taken into consideration before.

Characterizing Peptide β-HAIRPIN Loops via Cold Ion Spectroscopy of Model Compounds

NASA Astrophysics Data System (ADS)

Lawler, John T.; DeBlase, Andrew F.; Harrilal, Christopher P.; Fischer, Joshua L.; McLuckey, Scott A.; Zwier, Timothy S.

2017-06-01

The introduction of non-native D-amino acids into peptides is known to reduce conformational entropy in peptides. D-proline has been shown to promote the formation of β-hairpin loops when paired with Gly, providing a framework for building these loops with different lengths of anti-parallel beta-sheet. This study seeks to characterize and compare the conformational preferences of a model protonated pentapeptide containing DPG, [YAP^{D}GA+H]^{+}, with its L-Pro counterpart via conformation specific cold ion spectroscopy as a foundation for future consideration of larger beta-hairpin models. The UV spectrum of YAP^{D}GA of the Tyr chromophore is beautifully sharp, but contains a complicated set of transitions that could arise from the presence of more than one conformer. To assess this possibility, we recorded non-conformation specific IR "gain" spectra in the hydride stretch region. The IR spectrum so obtained displays a set of five strong IR transitions that bear a close resemblance to those found in one of the conformers of its close analog, [YAP^{D}AA+H]^{+}, signaling that a single conformer dominates the population. Two transitions at 3392 and 3464 cm-1 are slightly shifted versions of the C10 and C14 hydrogen bonds found in one of the conformers of [YAP^{D}AA+H]^{+}, and are characteristic of formation of a β-hairpin loop. Notably, in [YAP^{D}GA+H]^{+}, there is at most a minor second conformer with a free carboxylic acid OH, appearing weakly in the IR "gain" spectrum. As expected, the UV spectrum of YAP^{L}GA is more congested, which suggests the presence of multiple conformers. Further investigation into this peptide will reveal the conformational preferences of the L-pro containing molecule. Preliminary data affirms that D-proline containing peptides show reduced conformational states when compared to their natural counterparts.

A Network of Conformational Transitions in the Apo Form of NDM-1 Enzyme Revealed by MD Simulation and a Markov State Model.

PubMed

Gao, Kaifu; Zhao, Yunjie

2017-04-13

New Delhi metallo-β-lactamase-1 (NDM-1) is a novel β-lactamase enzyme that confers enteric bacteria with nearly complete resistance to all β-lactam antibiotics, so it raises a formidable and global threat to human health. However, the binding mechanism between apo-NDM-1 and antibiotics as well as related conformational changes remains poorly understood, which largely hinders the overcoming of its antibiotic resistance. In our study, long-time conventional molecular dynamics simulation and Markov state models were applied to reveal both the dynamical and conformational landscape of apo-NDM-1: the MD simulation demonstrates that loop L3, which is responsible for antibiotic binding, is the most flexible and undergoes dramatic conformational changes; moreover, the Markov state model built from the simulation maps four metastable states including open, semiopen, and closed conformations of loop L3 as well as frequent transitions between the states. Our findings propose a possible conformational selection model for the binding mechanism between apo-NDM-1 and antibiotics, which facilitates the design of novel inhibitors and antibiotics.

Atomic view of the histidine environment stabilizing higher-pH conformations of pH-dependent proteins

PubMed Central

Valéry, Céline; Deville-Foillard, Stéphanie; Lefebvre, Christelle; Taberner, Nuria; Legrand, Pierre; Meneau, Florian; Meriadec, Cristelle; Delvaux, Camille; Bizien, Thomas; Kasotakis, Emmanouil; Lopez-Iglesias, Carmen; Gall, Andrew; Bressanelli, Stéphane; Le Du, Marie-Hélène; Paternostre, Maïté; Artzner, Franck

2015-01-01

External stimuli are powerful tools that naturally control protein assemblies and functions. For example, during viral entry and exit changes in pH are known to trigger large protein conformational changes. However, the molecular features stabilizing the higher pH structures remain unclear. Here we elucidate the conformational change of a self-assembling peptide that forms either small or large nanotubes dependent on the pH. The sub-angstrom high-pH peptide structure reveals a globular conformation stabilized through a strong histidine-serine H-bond and a tight histidine-aromatic packing. Lowering the pH induces histidine protonation, disrupts these interactions and triggers a large change to an extended β-sheet-based conformation. Re-visiting available structures of proteins with pH-dependent conformations reveals both histidine-containing aromatic pockets and histidine-serine proximity as key motifs in higher pH structures. The mechanism discovered in this study may thus be generally used by pH-dependent proteins and opens new prospects in the field of nanomaterials. PMID:26190377

Revealing Hidden Conformational Space of LOV Protein VIVID Through Rigid Residue Scan Simulations

NASA Astrophysics Data System (ADS)

Zhou, Hongyu; Zoltowski, Brian D.; Tao, Peng

2017-04-01

VIVID(VVD) protein is a Light-Oxygen-Voltage(LOV) domain in circadian clock system. Upon blue light activation, a covalent bond is formed between VVD residue Cys108 and its cofactor flavin adenine dinucleotide(FAD), and prompts VVD switching from Dark state to Light state with significant conformational deviation. However, the mechanism of this local environment initiated global protein conformational change remains elusive. We employed a recently developed computational approach, rigid residue scan(RRS), to systematically probe the impact of the internal degrees of freedom in each amino acid residue of VVD on its overall dynamics by applying rigid body constraint on each residue in molecular dynamics simulations. Key residues were identified with distinctive impacts on Dark and Light states, respectively. All the simulations display wide range of distribution on a two-dimensional(2D) plot upon structural root-mean-square deviations(RMSD) from either Dark or Light state. Clustering analysis of the 2D RMSD distribution leads to 15 representative structures with drastically different conformation of N-terminus, which is also a key difference between Dark and Light states of VVD. Further principle component analyses(PCA) of RRS simulations agree with the observation of distinctive impact from individual residues on Dark and Light states.

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.

Inherent flexibility determines the transition mechanisms of the EF-hands of calmodulin.

PubMed

Tripathi, Swarnendu; Portman, John J

2009-02-17

We explore how inherent flexibility of a protein molecule influences the mechanism controlling allosteric transitions by using a variational model inspired from work in protein folding. The striking differences in the predicted transition mechanism for the opening of the two domains of calmodulin (CaM) emphasize that inherent flexibility is key to understanding the complex conformational changes that occur in proteins. In particular, the C-terminal domain of CaM (cCaM), which is inherently less flexible than its N-terminal domain (nCaM), reveals "cracking" or local partial unfolding during the open/closed transition. This result is in harmony with the picture that cracking relieves local stresses caused by conformational deformations of a sufficiently rigid protein. We also compare the conformational transition in a recently studied even-odd paired fragment of CaM. Our results rationalize the different relative binding affinities of the EF-hands in the engineered fragment compared with the intact odd-even paired EF-hands (nCaM and cCaM) in terms of changes in flexibility along the transition route. Aside from elucidating general theoretical ideas about the cracking mechanism, these studies also emphasize how the remarkable intrinsic plasticity of CaM underlies conformational dynamics essential for its diverse functions.

Revealing Hidden Conformational Space of LOV Protein VIVID Through Rigid Residue Scan Simulations

PubMed Central

Zhou, Hongyu; Zoltowski, Brian D.; Tao, Peng

2017-01-01

VIVID(VVD) protein is a Light-Oxygen-Voltage(LOV) domain in circadian clock system. Upon blue light activation, a covalent bond is formed between VVD residue Cys108 and its cofactor flavin adenine dinucleotide(FAD), and prompts VVD switching from Dark state to Light state with significant conformational deviation. However, the mechanism of this local environment initiated global protein conformational change remains elusive. We employed a recently developed computational approach, rigid residue scan(RRS), to systematically probe the impact of the internal degrees of freedom in each amino acid residue of VVD on its overall dynamics by applying rigid body constraint on each residue in molecular dynamics simulations. Key residues were identified with distinctive impacts on Dark and Light states, respectively. All the simulations display wide range of distribution on a two-dimensional(2D) plot upon structural root-mean-square deviations(RMSD) from either Dark or Light state. Clustering analysis of the 2D RMSD distribution leads to 15 representative structures with drastically different conformation of N-terminus, which is also a key difference between Dark and Light states of VVD. Further principle component analyses(PCA) of RRS simulations agree with the observation of distinctive impact from individual residues on Dark and Light states. PMID:28425502

Crystal structure of the thioesterification conformation of Bacillus subtilis o-succinylbenzoyl-CoA synthetase reveals a distinct substrate-binding mode.

PubMed

Chen, Yaozong; Li, Tin Lok; Lin, Xingbang; Li, Xin; Li, Xiang David; Guo, Zhihong

2017-07-21

o -Succinylbenzoyl-CoA (OSB-CoA) synthetase (MenE) is an essential enzyme in bacterial vitamin K biosynthesis and an important target in the development of new antibiotics. It is a member of the adenylating enzymes (ANL) family, which reconfigure their active site in two different active conformations, one for the adenylation half-reaction and the other for a thioesterification half-reaction, in a domain-alternation catalytic mechanism. Although several aspects of the adenylating mechanism in MenE have recently been uncovered, its thioesterification conformation remains elusive. Here, using a catalytically competent Bacillus subtilis mutant protein complexed with an OSB-CoA analogue, we determined MenE high-resolution structures to 1.76 and 1.90 Å resolution in a thioester-forming conformation. By comparison with the adenylation conformation, we found that MenE's C-domain rotates around the Ser-384 hinge by 139.5° during domain-alternation catalysis. The structures also revealed a thioesterification active site specifically conserved among MenE orthologues and a substrate-binding mode distinct from those of many other acyl/aryl-CoA synthetases. Of note, using site-directed mutagenesis, we identified several residues that specifically contribute to the thioesterification half-reaction without affecting the adenylation half-reaction. Moreover, we observed a substantial movement of the activated succinyl group in the thioesterification half-reaction. These findings provide new insights into the domain-alternation catalysis of a bacterial enzyme essential for vitamin K biosynthesis and of its adenylating homologues in the ANL enzyme family. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Myotonic dystrophy type 1 RNA crystal structures reveal heterogeneous 1 × 1 nucleotide UU internal loop conformations.

PubMed

Kumar, Amit; Park, HaJeung; Fang, Pengfei; Parkesh, Raman; Guo, Min; Nettles, Kendall W; Disney, Matthew D

2011-11-15

RNA internal loops often display a variety of conformations in solution. Herein, we visualize conformational heterogeneity in the context of the 5'CUG/3'GUC repeat motif present in the RNA that causes myotonic dystrophy type 1 (DM1). Specifically, two crystal structures of a model DM1 triplet repeating construct, 5'r[UUGGGC(CUG)(3)GUCC](2), refined to 2.20 and 1.52 Å resolution are disclosed. Here, differences in the orientation of the 5' dangling UU end between the two structures induce changes in the backbone groove width, which reveals that noncanonical 1 × 1 nucleotide UU internal loops can display an ensemble of pairing conformations. In the 2.20 Å structure, CUGa, the 5' UU forms a one hydrogen-bonded pair with a 5' UU of a neighboring helix in the unit cell to form a pseudoinfinite helix. The central 1 × 1 nucleotide UU internal loop has no hydrogen bonds, while the terminal 1 × 1 nucleotide UU internal loops each form a one-hydrogen bond pair. In the 1.52 Å structure, CUGb, the 5' UU dangling end is tucked into the major groove of the duplex. While the canonically paired bases show no change in base pairing, in CUGb the terminal 1 × 1 nucleotide UU internal loops now form two hydrogen-bonded pairs. Thus, the shift in the major groove induced by the 5' UU dangling end alters noncanonical base patterns. Collectively, these structures indicate that 1 × 1 nucleotide UU internal loops in DM1 may sample multiple conformations in vivo. This observation has implications for the recognition of this RNA, and other repeating transcripts, by protein and small molecule ligands.

Myotonic Dystrophy Type 1 RNA Crystal Structures Reveal Heterogeneous 1 × 1 Nucleotide UU Internal Loop Conformations

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

Kumar, Amit; Park, HaJeung; Fang, Pengfei

2012-03-27

RNA internal loops often display a variety of conformations in solution. Herein, we visualize conformational heterogeneity in the context of the 5'CUG/3'GUC repeat motif present in the RNA that causes myotonic dystrophy type 1 (DM1). Specifically, two crystal structures of a model DM1 triplet repeating construct, 5'r[{und UU}GGGC(C{und U}G){sub 3}GUCC]{sub 2}, refined to 2.20 and 1.52 {angstrom} resolution are disclosed. Here, differences in the orientation of the 5' dangling UU end between the two structures induce changes in the backbone groove width, which reveals that noncanonical 1 x 1 nucleotide UU internal loops can display an ensemble of pairing conformations.more » In the 2.20 {angstrom} structure, CUGa, the 5' UU forms a one hydrogen-bonded pair with a 5' UU of a neighboring helix in the unit cell to form a pseudoinfinite helix. The central 1 x 1 nucleotide UU internal loop has no hydrogen bonds, while the terminal 1 x 1 nucleotide UU internal loops each form a one-hydrogen bond pair. In the 1.52 {angstrom} structure, CUGb, the 5' UU dangling end is tucked into the major groove of the duplex. While the canonically paired bases show no change in base pairing, in CUGb the terminal 1 x 1 nucleotide UU internal loops now form two hydrogen-bonded pairs. Thus, the shift in the major groove induced by the 5' UU dangling end alters noncanonical base patterns. Collectively, these structures indicate that 1 x 1 nucleotide UU internal loops in DM1 may sample multiple conformations in vivo. This observation has implications for the recognition of this RNA, and other repeating transcripts, by protein and small molecule ligands.« less

Conductance of P2X4 purinergic receptor is determined by conformational equilibrium in the transmembrane region.

PubMed

Minato, Yuichi; Suzuki, Shiho; Hara, Tomoaki; Kofuku, Yutaka; Kasuya, Go; Fujiwara, Yuichiro; Igarashi, Shunsuke; Suzuki, Ei-Ichiro; Nureki, Osamu; Hattori, Motoyuki; Ueda, Takumi; Shimada, Ichio

2016-04-26

Ligand-gated ion channels are partially activated by their ligands, resulting in currents lower than the currents evoked by the physiological full agonists. In the case of P2X purinergic receptors, a cation-selective pore in the transmembrane region expands upon ATP binding to the extracellular ATP-binding site, and the currents evoked by α,β-methylene ATP are lower than the currents evoked by ATP. However, the mechanism underlying the partial activation of the P2X receptors is unknown although the crystal structures of zebrafish P2X4 receptor in the apo and ATP-bound states are available. Here, we observed the NMR signals from M339 and M351, which were introduced in the transmembrane region, and the endogenous alanine and methionine residues of the zebrafish P2X4 purinergic receptor in the apo, ATP-bound, and α,β-methylene ATP-bound states. Our NMR analyses revealed that, in the α,β-methylene ATP-bound state, M339, M351, and the residues that connect the ATP-binding site and the transmembrane region, M325 and A330, exist in conformational equilibrium between closed and open conformations, with slower exchange rates than the chemical shift difference (<100 s(-1)), suggesting that the small population of the open conformation causes the partial activation in this state. Our NMR analyses also revealed that the transmembrane region adopts the open conformation in the state bound to the inhibitor trinitrophenyl-ATP, and thus the antagonism is due to the closure of ion pathways, except for the pore in the transmembrane region: i.e., the lateral cation access in the extracellular region.

Conductance of P2X4 purinergic receptor is determined by conformational equilibrium in the transmembrane region

PubMed Central

Minato, Yuichi; Suzuki, Shiho; Hara, Tomoaki; Kofuku, Yutaka; Kasuya, Go; Fujiwara, Yuichiro; Igarashi, Shunsuke; Suzuki, Ei-ichiro; Nureki, Osamu; Hattori, Motoyuki; Ueda, Takumi; Shimada, Ichio

2016-01-01

Ligand-gated ion channels are partially activated by their ligands, resulting in currents lower than the currents evoked by the physiological full agonists. In the case of P2X purinergic receptors, a cation-selective pore in the transmembrane region expands upon ATP binding to the extracellular ATP-binding site, and the currents evoked by α,β-methylene ATP are lower than the currents evoked by ATP. However, the mechanism underlying the partial activation of the P2X receptors is unknown although the crystal structures of zebrafish P2X4 receptor in the apo and ATP-bound states are available. Here, we observed the NMR signals from M339 and M351, which were introduced in the transmembrane region, and the endogenous alanine and methionine residues of the zebrafish P2X4 purinergic receptor in the apo, ATP-bound, and α,β-methylene ATP-bound states. Our NMR analyses revealed that, in the α,β-methylene ATP-bound state, M339, M351, and the residues that connect the ATP-binding site and the transmembrane region, M325 and A330, exist in conformational equilibrium between closed and open conformations, with slower exchange rates than the chemical shift difference (<100 s−1), suggesting that the small population of the open conformation causes the partial activation in this state. Our NMR analyses also revealed that the transmembrane region adopts the open conformation in the state bound to the inhibitor trinitrophenyl-ATP, and thus the antagonism is due to the closure of ion pathways, except for the pore in the transmembrane region: i.e., the lateral cation access in the extracellular region. PMID:27071117

Conformational dynamics of L-lysine, L-arginine, L-ornithine binding protein reveals ligand-dependent plasticity.

PubMed

Silva, Daniel-Adriano; Domínguez-Ramírez, Lenin; Rojo-Domínguez, Arturo; Sosa-Peinado, Alejandro

2011-07-01

The molecular basis of multiple ligand binding affinity for amino acids in periplasmic binding proteins (PBPs) and in the homologous domain for class C G-protein coupled receptors is an unsolved question. Here, using unrestrained molecular dynamic simulations, we studied the ligand binding mechanism present in the L-lysine, L-arginine, L-ornithine binding protein. We developed an analysis based on dihedral angles for the description of the conformational changes upon ligand binding. This analysis has an excellent correlation with each of the two main movements described by principal component analysis (PCA) and it's more convenient than RMSD measurements to describe the differences in the conformational ensembles observed. Furthermore, an analysis of hydrogen bonds showed specific interactions for each ligand studied as well as the ligand interaction with the aromatic residues Tyr-14 and Phe-52. Using uncharged histidine tautomers, these interactions are not observed. On the basis of these results, we propose a model in which hydrogen bond interactions place the ligand in the correct orientation to induce a cation-π interaction with Tyr-14 and Phe-52 thereby stabilizing the closed state. Our results also show that this protein adopts slightly different closed conformations to make available specific hydrogen bond interactions for each ligand thus, allowing a single mechanism to attain multiple ligand specificity. These results shed light on the experimental evidence for ligand-dependent conformational plasticity not explained by the previous crystallographic data. Copyright © 2011 Wiley-Liss, Inc.

Human insulin analogues modified at the B26 site reveal a hormone conformation that is undetected in the receptor complex

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

Žáková, Lenka; Kletvíková, Emília; Lepšík, Martin

[AsnB26]- and [GlyB26]-insulin mutants attain a B26-turn like fold without assistance of chemical modifications. Their structures match the insulin receptor interface and expand the spectrum of insulin conformations. The structural characterization of the insulin–insulin receptor (IR) interaction still lacks the conformation of the crucial B21–B30 insulin region, which must be different from that in its storage forms to ensure effective receptor binding. Here, it is shown that insulin analogues modified by natural amino acids at the TyrB26 site can represent an active form of this hormone. In particular, [AsnB26]-insulin and [GlyB26]-insulin attain a B26-turn-like conformation that differs from that inmore » all known structures of the native hormone. It also matches the receptor interface, avoiding substantial steric clashes. This indicates that insulin may attain a B26-turn-like conformation upon IR binding. Moreover, there is an unexpected, but significant, binding specificity of the AsnB26 mutant for predominantly the metabolic B isoform of the receptor. As it is correlated with the B26 bend of the B-chain of the hormone, the structures of AsnB26 analogues may provide the first structural insight into the structural origins of differential insulin signalling through insulin receptor A and B isoforms.« less

Conformational study on cyclic melanocortin ligands and new insight into their binding mode at the MC4 receptor.

PubMed

Grieco, Paolo; Brancaccio, Diego; Novellino, Ettore; Hruby, Victor J; Carotenuto, Alfonso

2011-09-01

The melanocortin receptors are involved in many physiological functions, including pigmentation, sexual function, feeding behavior, and energy homeostasis, making them potential targets to treat obesity, sexual dysfunction, etc. Understanding the basis of the ligand-receptor interactions is crucial for the design of potent and selective ligands for these receptors. The conformational preferences of the cyclic melanocortin ligands MTII (Ac-Nle(4)-c[Asp(5)-His(6)-DPhe(7)-Arg(8)-Trp(9)-Lys(10)]-NH(2)) and SHU9119 (Ac-Nle(4)-c[Asp(5)-His(6)-DNal(2')(7)-Arg(8)-Trp(9)-Lys(10)]-NH(2)), which show agonist and antagonist activity at the h-MC4R, respectively, were comprehensively investigated by solution NMR spectroscopy in different environments. In particular, water and water/DMSO (8:2) solutions were used as isotropic solutions and an aqueous solution of DPC (dodecylphosphocholine) micelles was used as a membrane mimetic environment. NMR-derived conformations of these two ligands were docked within h-MC4R models. NMR and docking studies revealed intriguing differences which can help explain the different activities of these two ligands. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

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

Utschig, L. M.; Dalosto, S. D.; Thurnauer, M. C.

Metal ion binding to a surface site on photosynthetic reaction centers (RCs) modulates light-induced electron and proton transfer events in the RC. Whereas many studies have elucidated aspects of metal ion modulation events in Rhodobacter sphaeroides RCs, much less is understood about the surface site in Blastochloris viridis (Blc. viridis) RCs. Interestingly, electron paramagnetic resonance studies revealed two spectroscopically distinct Cu{sup 2+} surface site environments in Blc. viridis RCs. Herein, Cu{sup 2+} has been used to spectroscopically probe the structure of these Cu{sup 2+} site(s) in response to freezing conditions, temperature, and charge separation. One Cu{sup 2+} environment in Blc.more » viridis RCs, termed CuA, exhibits temperature-dependent conformational flexibility. Different conformation states of the CuA{sup 2+} site are trapped when the RC is frozen in the dark either by fast-freeze or slow-freeze procedure. The second Cu{sup 2+} environment, termed CuB, is structurally invariant to different freezing conditions and shows resolved hyperfine coupling to three nitrogen atoms. Cu{sup 2+} is most likely binding at the same location on the RC, but in different coordination environments which may reflect two distinct conformational states of the isolated Blc. viridis RC protein.« less

Conformational Plasticity of the Cell-Penetrating Peptide SAP As Revealed by Solid-State 19F-NMR and Circular Dichroism Spectroscopies.

PubMed

Afonin, Sergii; Kubyshkin, Vladimir; Mykhailiuk, Pavel K; Komarov, Igor V; Ulrich, Anne S

2017-07-13

The cell-penetrating peptide SAP, which was designed as an amphipathic poly-l-proline helix II (PPII), was suggested to self-assemble into regular fibrils that are relevant for its internalization. Herein we have analyzed the structure of SAP in the membrane-bound state by solid-state 19 F-NMR, which revealed other structural states, in addition to the expected surface-aligned PPII. Trifluoromethyl-bicyclopentyl-glycine (CF 3 -Bpg) and two rigid isomers of trifluoromethyl-4,5-methanoprolines (CF 3 -MePro) were used as labels for 19 F-NMR analysis. The equilibria between different conformations of SAP were studied and were found to be shifted by the substituents at Pro-11. Synchrotron-CD results suggested that substituting Pro-11 by CF 3 -MePro governed the coil-to-PPII equilibrium in solution and in the presence of a lipid bilayer. Using CD and 19 F-NMR, we examined the slow kinetics of the association of SAP with membranes and the dependence of the SAP conformational dynamics on the lipid composition. The peptide did not bind to lipids in the solid ordered phase and aggregated only in the liquid ordered "raft"-like bilayers. Self-association could not be detected in solution or in the presence of liquid disordered membranes. Surface-bound amphipathic SAP in a nonaggregated state was structured as a mixture of nonideal extended conformations reflecting the equilibrium already present in solution, i.e., before binding to the membrane.

Circular dichroism study of the carbohydrate-modified opioid peptides

NASA Astrophysics Data System (ADS)

Horvat, Štefica; Otvos, Laszlo; Urge, Laszlo; Horvat, Jaroslav; Čudić, Mare; Varga-Defterdarović, Lidija

1999-09-01

The conformational preferences of enkephalins and the related glycoconjugates in which free or protected carbohydrate moieties were linked to the opioid peptides through an ether, ester or amide bond were investigated by circular dichroism spectroscopy in water, trifluoroethanol and water-trifluoroethanol mixtures. The analysis of the spectra revealed that the conformation of the enkephalin molecule is very sensitive to slight changes in the peptide structure around the C-terminal region. It was found that the type II β-turn structures are populated in N-terminal tetrapeptide enkephalin fragment, while leucine-enkephalin amide feature a type I (III) β-turn structure in solution. Incorporation of the sugar moiety into opioid peptide compound did not significantly influence the overall conformation of the peptide backbone, although minor intensity changes may reflect shifts in the population of the different turn systems. These small structural alterations can be responsible for the receptor-subtype selectivity of the various carbohydrate-modified enkephalin analogs.

Ferrocenyl-cymantrenyl hetero-bimetallic chalcones: Synthesis, structure and biological properties

NASA Astrophysics Data System (ADS)

Mishra, Sasmita; Tirkey, Vijaylakshmi; Ghosh, Avishek; Dash, Hirak R.; Das, Surajit; Shukla, Madhulata; Saha, Satyen; Mobin, Sheikh M.; Chatterjee, Saurav

2015-04-01

Two new ferrocenyl-cymantrenyl bimetallic chalcones, [(CO)3Mn(η5-C5H4)C(O)CHdbnd CH(η5-C5H4)Fe(η5-C5H5)] (1) and [{(CO)3Mn(η5-C5H4)C(O)CHdbnd CH(η5-C5H4)}2Fe] (2) have been synthesized. Their reactivity study with triphenylphosphine and bis-(diphenylphosphino)ferrocene led to the isolation of phosphine substituted bimetallic chalcones (3-6). Single crystal X-ray structural characterization for 1 and its phosphine analogue (3) reveals their different conformational identity with anti-conformation for 1, while syn-conformation for 3. Investigation of antimalarial and antibacterial activities was carried out for compounds 1 and 2 against two strains of Plasmodium falciparum (3D7, K1) and four bacterial strains. TD-DFT calculation was performed for compound 1 and electrochemical properties were studied for bimetallic chalcone compounds by cyclic voltammetric technique.

Inherent conformational flexibility of F1-ATPase α-subunit.

PubMed

Hahn-Herrera, Otto; Salcedo, Guillermo; Barril, Xavier; García-Hernández, Enrique

2016-09-01

The core of F1-ATPase consists of three catalytic (β) and three noncatalytic (α) subunits, forming a hexameric ring in alternating positions. A wealth of experimental and theoretical data has provided a detailed picture of the complex role played by catalytic subunits. Although major conformational changes have only been seen in β-subunits, it is clear that α-subunits have to respond to these changes in order to be able to transmit information during the rotary mechanism. However, the conformational behavior of α-subunits has not been explored in detail. Here, we have combined unbiased molecular dynamics (MD) simulations and calorimetrically measured thermodynamic signatures to investigate the conformational flexibility of isolated α-subunits, as a step toward deepening our understanding of its function inside the α3β3 ring. The simulations indicate that the open-to-closed conformational transition of the α-subunit is essentially barrierless, which is ideal to accompany and transmit the movement of the catalytic subunits. Calorimetric measurements of the recombinant α-subunit from Geobacillus kaustophilus indicate that the isolated subunit undergoes no significant conformational changes upon nucleotide binding. Simulations confirm that the nucleotide-free and nucleotide-bound subunits show average conformations similar to that observed in the F1 crystal structure, but they reveal an increased conformational flexibility of the isolated α-subunit upon MgATP binding, which might explain the evolutionary conserved capacity of α-subunits to recognize nucleotides with considerable strength. Furthermore, we elucidate the different dependencies that α- and β-subunits show on Mg(II) for recognizing ATP. Copyright © 2016 Elsevier B.V. All rights reserved.

Crystal Structures of Lys-63-linked tri- and di-ubiquitin Reveal a Highly Extended Chain Architecture

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

Weeks, S.; Grasty, K; Hernandez-Cuebas, L

2009-01-01

The covalent attachment of different types of poly-ubiquitin chains signal different outcomes for the proteins so targeted. For example, a protein modified with Lys-48-linked poly-ubiquitin chains is targeted for proteasomal degradation, whereas Lys-63-linked chains encode nondegradative signals. The structural features that enable these different types of chains to encode different signals have not yet been fully elucidated. We report here the X-ray crystal structures of Lys-63-linked tri- and di-ubiquitin at resolutions of 2.3 and 1.9 {angstrom}, respectively. The tri- and di-ubiquitin species adopt essentially identical structures. In both instances, the ubiquitin chain assumes a highly extended conformation with a left-handedmore » helical twist; the helical chain contains four ubiquitin monomers per turn and has a repeat length of {approx}110 {angstrom}. Interestingly, Lys-48 ubiquitin chains also adopt a left-handed helical structure with a similar repeat length. However, the Lys-63 architecture is much more open than that of Lys-48 chains and exposes much more of the ubiquitin surface for potential recognition events. These new crystal structures are consistent with the results of solution studies of Lys-63 chain conformation, and reveal the structural basis for differential recognition of Lys-63 versus Lys-48 chains.« less

The allosteric switching mechanism in bacteriophage MS2

NASA Astrophysics Data System (ADS)

Perkett, Matthew R.; Mirijanian, Dina T.; Hagan, Michael F.

2016-07-01

We use all-atom simulations to elucidate the mechanisms underlying conformational switching and allostery within the coat protein of the bacteriophage MS2. Assembly of most icosahedral virus capsids requires that the capsid protein adopts different conformations at precise locations within the capsid. It has been shown that a 19 nucleotide stem loop (TR) from the MS2 genome acts as an allosteric effector, guiding conformational switching of the coat protein during capsid assembly. Since the principal conformational changes occur far from the TR binding site, it is important to understand the molecular mechanism underlying this allosteric communication. To this end, we use all-atom simulations with explicit water combined with a path sampling technique to sample the MS2 coat protein conformational transition, in the presence and absence of TR-binding. The calculations find that TR binding strongly alters the transition free energy profile, leading to a switch in the favored conformation. We discuss changes in molecular interactions responsible for this shift. We then identify networks of amino acids with correlated motions to reveal the mechanism by which effects of TR binding span the protein. We find that TR binding strongly affects residues located at the 5-fold and quasi-sixfold interfaces in the assembled capsid, suggesting a mechanism by which the TR binding could direct formation of the native capsid geometry. The analysis predicts amino acids whose substitution by mutagenesis could alter populations of the conformational substates or their transition rates.

The allosteric switching mechanism in bacteriophage MS2

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

Perkett, Matthew R.; Mirijanian, Dina T.; Hagan, Michael F., E-mail: hagan@brandeis.edu

2016-07-21

We use all-atom simulations to elucidate the mechanisms underlying conformational switching and allostery within the coat protein of the bacteriophage MS2. Assembly of most icosahedral virus capsids requires that the capsid protein adopts different conformations at precise locations within the capsid. It has been shown that a 19 nucleotide stem loop (TR) from the MS2 genome acts as an allosteric effector, guiding conformational switching of the coat protein during capsid assembly. Since the principal conformational changes occur far from the TR binding site, it is important to understand the molecular mechanism underlying this allosteric communication. To this end, we usemore » all-atom simulations with explicit water combined with a path sampling technique to sample the MS2 coat protein conformational transition, in the presence and absence of TR-binding. The calculations find that TR binding strongly alters the transition free energy profile, leading to a switch in the favored conformation. We discuss changes in molecular interactions responsible for this shift. We then identify networks of amino acids with correlated motions to reveal the mechanism by which effects of TR binding span the protein. We find that TR binding strongly affects residues located at the 5-fold and quasi-sixfold interfaces in the assembled capsid, suggesting a mechanism by which the TR binding could direct formation of the native capsid geometry. The analysis predicts amino acids whose substitution by mutagenesis could alter populations of the conformational substates or their transition rates.« less

Complement Factor H and Simian Virus 40 bind the GM1 ganglioside in distinct conformations.

PubMed

Blaum, Bärbel S; Frank, Martin; Walker, Ross C; Neu, Ursula; Stehle, Thilo

2016-05-01

Mammalian cell surfaces are decorated with a variety of glycan chains that orchestrate development and defense and are exploited by pathogens for cellular attachment and entry. While glycosidic linkages are, in principle, flexible, the conformational space that a given glycan can sample is subject to spatial and electrostatic restrictions imposed by its overall chemical structure. Here, we show how the glycan moiety of the GM1 ganglioside, a branched, monosialylated pentasaccharide that serves as a ligand for various proteins, undergoes differential conformational selection in its interactions with different lectins. Using STD NMR and X-ray crystallography, we found that the innate immune regulator complement Factor H (FH) binds a previously not reported GM1 conformation that is not compatible with the GM1-binding sites of other structurally characterized GM1-binding lectins such as the Simian Virus 40 (SV40) capsid. Molecular dynamics simulations of the free glycan in explicit solvent on the 10 μs timescale reveal that the FH-bound conformation nevertheless corresponds to a minimum in the Gibbs free energy plot. In contrast to the GM1 conformation recognized by SV40, the FH-bound GM1 conformation is associated with poor NOE restraints, explaining how it escaped(1)H-(1)H NOE-restrained modeling in the past and highlighting the necessity for ensemble representations of glycan structures. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The allosteric switching mechanism in bacteriophage MS2

PubMed Central

Perkett, Matthew R.; Mirijanian, Dina T.

2016-01-01

We use all-atom simulations to elucidate the mechanisms underlying conformational switching and allostery within the coat protein of the bacteriophage MS2. Assembly of most icosahedral virus capsids requires that the capsid protein adopts different conformations at precise locations within the capsid. It has been shown that a 19 nucleotide stem loop (TR) from the MS2 genome acts as an allosteric effector, guiding conformational switching of the coat protein during capsid assembly. Since the principal conformational changes occur far from the TR binding site, it is important to understand the molecular mechanism underlying this allosteric communication. To this end, we use all-atom simulations with explicit water combined with a path sampling technique to sample the MS2 coat protein conformational transition, in the presence and absence of TR-binding. The calculations find that TR binding strongly alters the transition free energy profile, leading to a switch in the favored conformation. We discuss changes in molecular interactions responsible for this shift. We then identify networks of amino acids with correlated motions to reveal the mechanism by which effects of TR binding span the protein. We find that TR binding strongly affects residues located at the 5-fold and quasi-sixfold interfaces in the assembled capsid, suggesting a mechanism by which the TR binding could direct formation of the native capsid geometry. The analysis predicts amino acids whose substitution by mutagenesis could alter populations of the conformational substates or their transition rates. PMID:27448905

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).

Molecular and cellular aspects of rhabdovirus entry.

PubMed

Albertini, Aurélie A V; Baquero, Eduard; Ferlin, Anna; Gaudin, Yves

2012-01-01

Rhabdoviruses enter the cell via the endocytic pathway and subsequently fuse with a cellular membrane within the acidic environment of the endosome. Both receptor recognition and membrane fusion are mediated by a single transmembrane viral glycoprotein (G). Fusion is triggered via a low-pH induced structural rearrangement. G is an atypical fusion protein as there is a pH-dependent equilibrium between its pre- and post-fusion conformations. The elucidation of the atomic structures of these two conformations for the vesicular stomatitis virus (VSV) G has revealed that it is different from the previously characterized class I and class II fusion proteins. In this review, the pre- and post-fusion VSV G structures are presented in detail demonstrating that G combines the features of the class I and class II fusion proteins. In addition to these similarities, these G structures also reveal some particularities that expand our understanding of the working of fusion machineries. Combined with data from recent studies that revealed the cellular aspects of the initial stages of rhabdovirus infection, all these data give an integrated view of the entry pathway of rhabdoviruses into their host cell.

Molecular and Cellular Aspects of Rhabdovirus Entry

PubMed Central

Albertini, Aurélie A. V.; Baquero, Eduard; Ferlin, Anna; Gaudin, Yves

2012-01-01

Rhabdoviruses enter the cell via the endocytic pathway and subsequently fuse with a cellular membrane within the acidic environment of the endosome. Both receptor recognition and membrane fusion are mediated by a single transmembrane viral glycoprotein (G). Fusion is triggered via a low-pH induced structural rearrangement. G is an atypical fusion protein as there is a pH-dependent equilibrium between its pre- and post-fusion conformations. The elucidation of the atomic structures of these two conformations for the vesicular stomatitis virus (VSV) G has revealed that it is different from the previously characterized class I and class II fusion proteins. In this review, the pre- and post-fusion VSV G structures are presented in detail demonstrating that G combines the features of the class I and class II fusion proteins. In addition to these similarities, these G structures also reveal some particularities that expand our understanding of the working of fusion machineries. Combined with data from recent studies that revealed the cellular aspects of the initial stages of rhabdovirus infection, all these data give an integrated view of the entry pathway of rhabdoviruses into their host cell. PMID:22355455

Flexibility of the sugar moiety of nucleosides at high pressures

NASA Astrophysics Data System (ADS)

Lee, Scott

2007-03-01

In this poster we review our recent high pressure experiments on deoxyadenosine, adenosine, deoxycytidine and cytidine via mid-infrared absorption. These experiments reveal the presence of phase transitions near 2 GPa in these four different nucleosides. The spectroscopic evidence indicates that the sugar pucker changes at the phase transition in all four nucleosides. Differences between the deoxyribose nucleosides and the ribose nucleosides are compared to the known differences in the conformational flexibility of DNA and RNA.

In silico Analysis of Conformational Changes Induced by Mutation of Aromatic Binding Residues: Consequences for Drug Binding in the hERG K+ Channel

PubMed Central

Knape, Kirsten; Linder, Tobias; Wolschann, Peter; Beyer, Anton; Stary-Weinzinger, Anna

2011-01-01

Pharmacological inhibition of cardiac hERG K+ channels is associated with increased risk of lethal arrhythmias. Many drugs reduce hERG current by directly binding to the channel, thereby blocking ion conduction. Mutation of two aromatic residues (F656 and Y652) substantially decreases the potency of numerous structurally diverse compounds. Nevertheless, some drugs are only weakly affected by mutation Y652A. In this study we utilize molecular dynamics simulations and docking studies to analyze the different effects of mutation Y652A on a selected number of hERG blockers. MD simulations reveal conformational changes in the binding site induced by mutation Y652A. Loss of π-π-stacking between the two aromatic residues induces a conformational change of the F656 side chain from a cavity facing to cavity lining orientation. Docking studies and MD simulations qualitatively reproduce the diverse experimentally observed modulatory effects of mutation Y652A and provide a new structural interpretation for the sensitivity differences. PMID:22194911

Conformational Differences between Open and Closed States of the Eukaryotic Translation Initiation Complex

PubMed Central

Llácer, Jose L.; Hussain, Tanweer; Marler, Laura; Aitken, Colin Echeverría; Thakur, Anil; Lorsch, Jon R.; Hinnebusch, Alan G.; Ramakrishnan, V.

2015-01-01

Summary Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and eIF5. The PIC, in an open conformation, attaches to the 5′ end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2β as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition. PMID:26212456

Methods to Approach Velocity Data Reduction and Their Effects on Conformation Statistics in Viscoelastic Turbulent Channel Flows

NASA Astrophysics Data System (ADS)

Samanta, Gaurab; Beris, Antony; Handler, Robert; Housiadas, Kostas

2009-03-01

Karhunen-Loeve (KL) analysis of DNS data of viscoelastic turbulent channel flows helps us to reveal more information on the time-dependent dynamics of viscoelastic modification of turbulence [Samanta et. al., J. Turbulence (in press), 2008]. A selected set of KL modes can be used for a data reduction modeling of these flows. However, it is pertinent that verification be done against established DNS results. For this purpose, we did comparisons of velocity and conformations statistics and probability density functions (PDFs) of relevant quantities obtained from DNS and reconstructed fields using selected KL modes and time-dependent coefficients. While the velocity statistics show good agreement between results from DNS and KL reconstructions even with just hundreds of KL modes, tens of thousands of KL modes are required to adequately capture the trace of polymer conformation resulting from DNS. New modifications to KL method have therefore been attempted to account for the differences in conformation statistics. The applicability and impact of these new modified KL methods will be discussed in the perspective of data reduction modeling.

Protein NMR Studies of Substrate Binding to Human Blood Group A and B Glycosyltransferases.

PubMed

Grimm, Lena Lisbeth; Weissbach, Sophie; Flügge, Friedemann; Begemann, Nora; Palcic, Monica M; Peters, Thomas

2017-07-04

Donor and acceptor substrate binding to human blood group A and B glycosyltransferases (GTA, GTB) has been studied by a variety of protein NMR experiments. Prior crystallographic studies had shown these enzymes to adopt an open conformation in the absence of substrates. Binding either of the donor substrate UDP-Gal or of UDP induces a semiclosed conformation. In the presence of both donor and acceptor substrates, the enzymes shift towards a closed conformation with ordering of an internal loop and the C-terminal residues, which then completely cover the donor-binding pocket. Chemical-shift titrations of uniformly 2 H, 15 N-labeled GTA or GTB with UDP affected about 20 % of all crosspeaks in 1 H, 15 N TROSY-HSQC spectra, reflecting substantial plasticity of the enzymes. On the other hand, it is this conformational flexibility that impedes NH backbone assignments. Chemical-shift-perturbation experiments with δ1-[ 13 C]methyl-Ile-labeled samples revealed two Ile residues-Ile123 at the bottom of the UDP binding pocket, and Ile192 as part of the internal loop-that were significantly disturbed upon stepwise addition of UDP and H-disaccharide, also revealing long-range perturbations. Finally, methyl TROSY-based relaxation dispersion experiments do not reveal micro- to millisecond timescale motions. Although this study reveals substantial conformational plasticity of GTA and GTB, the matter of how binding of substrates shifts the enzymes into catalytically competent states remains enigmatic. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Direct observations of conformational distributions of intrinsically disordered p53 peptides using UV Raman and explicit solvent simulations

PubMed Central

Xiong, Kan; Zwier, Matthew C.; Myshakina, Nataliya S.; Burger, Virginia M.; Asher, Sanford A.; Chong, Lillian T.

2011-01-01

We report the first experimental measurements of Ramachandran Ψ-angle distributions for intrinsically disordered peptides: the N-terminal peptide fragment of tumor suppressor p53 and its P27 mutant form. To provide atomically detailed views of the conformational distributions, we performed classical, explicit-solvent molecular dynamics simulations on the microsecond timescale. Upon binding its partner protein, MDM2, wild-type p53 peptide adopts an α-helical conformation. Mutation of Pro27 to serine results in the highest affinity yet observed for MDM2-binding of the p53 peptide. Both UV resonance Raman spectroscopy (UVRR) and simulations reveal that the P27S mutation decreases the extent of PPII helical content and increases the probability for conformations that are similar to the α-helical MDM2-bound conformation. In addition, UVRR measurements were performed on peptides that were isotopically labeled at the Leu26 residue preceding the Pro27 in order to determine the conformational distributions of Leu26 in the wild-type and mutant peptides. The UVRR and simulation results are in quantitative agreement in terms of the change in the population of non-PPII conformations involving Leu26 upon mutation of Pro27 to serine. Finally, our simulations reveal that the MDM2-bound conformation of the peptide is significantly populated in both the wild-type and mutant isolated peptide ensembles in their unbound states, suggesting that MDM2 binding of the p53 peptides may involve conformational selection. PMID:21528875

Cofactor-binding sites in proteins of deviating sequence: comparative analysis and clustering in torsion angle, cavity, and fold space.

PubMed

Stegemann, Björn; Klebe, Gerhard

2012-02-01

Small molecules are recognized in protein-binding pockets through surface-exposed physicochemical properties. To optimize binding, they have to adopt a conformation corresponding to a local energy minimum within the formed protein-ligand complex. However, their conformational flexibility makes them competent to bind not only to homologous proteins of the same family but also to proteins of remote similarity with respect to the shape of the binding pockets and folding pattern. Considering drug action, such observations can give rise to unexpected and undesired cross reactivity. In this study, datasets of six different cofactors (ADP, ATP, NAD(P)(H), FAD, and acetyl CoA, sharing an adenosine diphosphate moiety as common substructure), observed in multiple crystal structures of protein-cofactor complexes exhibiting sequence identity below 25%, have been analyzed for the conformational properties of the bound ligands, the distribution of physicochemical properties in the accommodating protein-binding pockets, and the local folding patterns next to the cofactor-binding site. State-of-the-art clustering techniques have been applied to group the different protein-cofactor complexes in the different spaces. Interestingly, clustering in cavity (Cavbase) and fold space (DALI) reveals virtually the same data structuring. Remarkable relationships can be found among the different spaces. They provide information on how conformations are conserved across the host proteins and which distinct local cavity and fold motifs recognize the different portions of the cofactors. In those cases, where different cofactors are found to be accommodated in a similar fashion to the same fold motifs, only a commonly shared substructure of the cofactors is used for the recognition process. Copyright © 2011 Wiley Periodicals, Inc.

Correlated motion of protein subdomains and large-scale conformational flexibility of RecA protein filament

NASA Astrophysics Data System (ADS)

Yu, Garmay; A, Shvetsov; D, Karelov; D, Lebedev; A, Radulescu; M, Petukhov; V, Isaev-Ivanov

2012-02-01

Based on X-ray crystallographic data available at Protein Data Bank, we have built molecular dynamics (MD) models of homologous recombinases RecA from E. coli and D. radiodurans. Functional form of RecA enzyme, which is known to be a long helical filament, was approximated by a trimer, simulated in periodic water box. The MD trajectories were analyzed in terms of large-scale conformational motions that could be detectable by neutron and X-ray scattering techniques. The analysis revealed that large-scale RecA monomer dynamics can be described in terms of relative motions of 7 subdomains. Motion of C-terminal domain was the major contributor to the overall dynamics of protein. Principal component analysis (PCA) of the MD trajectories in the atom coordinate space showed that rotation of C-domain is correlated with the conformational changes in the central domain and N-terminal domain, that forms the monomer-monomer interface. Thus, even though C-terminal domain is relatively far from the interface, its orientation is correlated with large-scale filament conformation. PCA of the trajectories in the main chain dihedral angle coordinate space implicates a co-existence of a several different large-scale conformations of the modeled trimer. In order to clarify the relationship of independent domain orientation with large-scale filament conformation, we have performed analysis of independent domain motion and its implications on the filament geometry.

A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor

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

Datta, Supratim; Koutmos, Markos; Pattridge, Katherine A.

2008-07-08

B{sub 12}-dependent methionine synthase (MetH) from Escherichia coli is a large modular protein that is alternately methylated by methyltetrahydrofolate to form methylcobalamin and demethylated by homocysteine to form cob(I)alamin. Major domain rearrangements are required to allow cobalamin to react with three different substrates: homocysteine, methyltetrahydrofolate, and S-adenosyl-l-methionine (AdoMet). These same rearrangements appear to preclude crystallization of the wild-type enzyme. Disulfide cross-linking was used to lock a C-terminal fragment of the enzyme into a unique conformation. Cysteine point mutations were introduced at Ile-690 and Gly-743. These cysteine residues span the cap and the cobalamin-binding module and form a cross-link that reducesmore » the conformational space accessed by the enzyme, facilitating protein crystallization. Here, we describe an x-ray structure of the mutant fragment in the reactivation conformation; this conformation enables the transfer of a methyl group from AdoMet to the cobalamin cofactor. In the structure, the axial ligand to the cobalamin, His-759, dissociates from the cobalamin and forms intermodular contacts with residues in the AdoMet-binding module. This unanticipated intermodular interaction is expected to play a major role in controlling the distribution of conformers required for the catalytic and the reactivation cycles of the enzyme.« less

High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner*

PubMed Central

Gagnon, Susannah M. L.; Meloncelli, Peter J.; Zheng, Ruixiang B.; Haji-Ghassemi, Omid; Johal, Asha R.; Borisova, Svetlana N.; Lowary, Todd L.; Evans, Stephen V.

2015-01-01

Homologous glycosyltransferases α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and α-(1→3)-galactosyltransferase (GTB) catalyze the final step in ABO(H) blood group A and B antigen synthesis through sugar transfer from activated donor to the H antigen acceptor. These enzymes have a GT-A fold type with characteristic mobile polypeptide loops that cover the active site upon substrate binding and, despite intense investigation, many aspects of substrate specificity and catalysis remain unclear. The structures of GTA, GTB, and their chimeras have been determined to between 1.55 and 1.39 Å resolution in complex with natural donors UDP-Gal, UDP-Glc and, in an attempt to overcome one of the common problems associated with three-dimensional studies, the non-hydrolyzable donor analog UDP-phosphono-galactose (UDP-C-Gal). Whereas the uracil moieties of the donors are observed to maintain a constant location, the sugar moieties lie in four distinct conformations, varying from extended to the “tucked under” conformation associated with catalysis, each stabilized by different hydrogen bonding partners with the enzyme. Further, several structures show clear evidence that the donor sugar is disordered over two of the observed conformations and so provide evidence for stepwise insertion into the active site. Although the natural donors can both assume the tucked under conformation in complex with enzyme, UDP-C-Gal cannot. Whereas UDP-C-Gal was designed to be “isosteric” with natural donor, the small differences in structure imposed by changing the epimeric oxygen atom to carbon appear to render the enzyme incapable of binding the analog in the active conformation and so preclude its use as a substrate mimic in GTA and GTB. PMID:26374898

Molecular dynamics correctly models the unusual major conformation of the GAGU RNA internal loop and with NMR reveals an unusual minor conformation.

PubMed

Spasic, Aleksandar; Kennedy, Scott D; Needham, Laura; Manoharan, Muthiah; Kierzek, Ryszard; Turner, Douglas H; Mathews, David H

2018-05-01

The RNA "GAGU" duplex, (5'GAC GAGU GUCA) 2 , contains the internal loop (5'-GAGU-3') 2 , which has two conformations in solution as determined by NMR spectroscopy. The major conformation has a loop structure consisting of trans -Watson-Crick/Hoogsteen GG pairs, A residues stacked on each other, U residues bulged outside the helix, and all sugars with a C2'- endo conformation. This differs markedly from the internal loops, (5'-G AG C-3') 2 , (5'-A AG U-3') 2 , and (5'-UAGG-3') 2 , which all have cis -Watson-Crick/Watson-Crick AG "imino" pairs flanked by cis -Watson-Crick/Watson-Crick canonical pairs resulting in maximal hydrogen bonding. Here, molecular dynamics was used to test whether the Amber force field (ff99 + bsc0 + OL3) approximates molecular interactions well enough to keep stable the unexpected conformation of the GAGU major duplex structure and the NMR structures of the duplexes containing (5'-G AG C-3') 2 , (5'-A AG U-3') 2 , and (5'-U AG G-3') 2 internal loops. One-microsecond simulations were repeated four times for each of the duplexes starting in their NMR conformations. With the exception of (5'-UAGG-3') 2 , equivalent simulations were also run starting with alternative conformations. Results indicate that the Amber force field keeps the NMR conformations of the duplexes stable for at least 1 µsec. They also demonstrate an unexpected minor conformation for the (5'-GAGU-3') 2 loop that is consistent with newly measured NMR spectra of duplexes with natural and modified nucleotides. Thus, unrestrained simulations led to the determination of the previously unknown minor conformation. The stability of the native (5'-GAGU-3') 2 internal loop as compared to other loops can be explained by changes in hydrogen bonding and stacking as the flanking bases are changed. © 2018 Spasic et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Regulation of Response Regulator Autophosphorylation through Interdomain Contacts*♦

PubMed Central

Barbieri, Christopher M.; Mack, Timothy R.; Robinson, Victoria L.; Miller, Matthew T.; Stock, Ann M.

2010-01-01

DNA-binding response regulators (RRs) of the OmpR/PhoB subfamily alternate between inactive and active conformational states, with the latter having enhanced DNA-binding affinity. Phosphorylation of an aspartate residue in the receiver domain, usually via phosphotransfer from a cognate histidine kinase, stabilizes the active conformation. Many of the available structures of inactive OmpR/PhoB family proteins exhibit extensive interfaces between the N-terminal receiver and C-terminal DNA-binding domains. These interfaces invariably involve the α4-β5-α5 face of the receiver domain, the locus of the largest differences between inactive and active conformations and the surface that mediates dimerization of receiver domains in the active state. Structures of receiver domain dimers of DrrB, DrrD, and MtrA have been determined, and phosphorylation kinetics were analyzed. Analysis of phosphotransfer from small molecule phosphodonors has revealed large differences in autophosphorylation rates among OmpR/PhoB RRs. RRs with substantial domain interfaces exhibit slow rates of phosphorylation. Rates are greatly increased in isolated receiver domain constructs. Such differences are not observed between autophosphorylation rates of full-length and isolated receiver domains of a RR that lacks interdomain interfaces, and they are not observed in histidine kinase-mediated phosphotransfer. These findings suggest that domain interfaces restrict receiver domain conformational dynamics, stabilizing an inactive conformation that is catalytically incompetent for phosphotransfer from small molecule phosphodonors. Inhibition of phosphotransfer by domain interfaces provides an explanation for the observation that some RRs cannot be phosphorylated by small molecule phosphodonors in vitro and provides a potential mechanism for insulating some RRs from small molecule-mediated phosphorylation in vivo. PMID:20702407

Conformational variability of the stationary phase survival protein E from Xylella fastidiosa revealed by X-ray crystallography, small-angle X-ray scattering studies, and normal mode analysis.

PubMed

Machado, Agnes Thiane Pereira; Fonseca, Emanuella Maria Barreto; Reis, Marcelo Augusto Dos; Saraiva, Antonio Marcos; Santos, Clelton Aparecido Dos; de Toledo, Marcelo Augusto Szymanski; Polikarpov, Igor; de Souza, Anete Pereira; Aparicio, Ricardo; Iulek, Jorge

2017-10-01

Xylella fastidiosa is a xylem-limited bacterium that infects a wide variety of plants. Stationary phase survival protein E is classified as a nucleotidase, which is expressed when bacterial cells are in the stationary growth phase and subjected to environmental stresses. Here, we report four refined X-ray structures of this protein from X. fastidiosa in four different crystal forms in the presence and/or absence of the substrate 3'-AMP. In all chains, the conserved loop verified in family members assumes a closed conformation in either condition. Therefore, the enzymatic mechanism for the target protein might be different of its homologs. Two crystal forms exhibit two monomers whereas the other two show four monomers in the asymmetric unit. While the biological unit has been characterized as a tetramer, differences of their sizes and symmetry are remarkable. Four conformers identified by Small-Angle X-ray Scattering (SAXS) in a ligand-free solution are related to the low frequency normal modes of the crystallographic structures associated with rigid body-like protomer arrangements responsible for the longitudinal and symmetric adjustments between tetramers. When the substrate is present in solution, only two conformers are selected. The most prominent conformer for each case is associated to a normal mode able to elongate the protein by moving apart two dimers. To our knowledge, this work was the first investigation based on the normal modes that analyzed the quaternary structure variability for an enzyme of the SurE family followed by crystallography and SAXS validation. The combined results raise new directions to study allosteric features of XfSurE protein. © 2017 Wiley Periodicals, Inc.

Analysis of cytochrome P450 CYP119 ligand-dependent conformational dynamics by two-dimensional NMR and X-ray crystallography.

PubMed

Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; Lampe, Jed N; Nishida, Clinton R; de Montellano, Paul R Ortiz

2015-04-17

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional (1)H,(15)N HSQC chemical shift perturbation mapping of (15)N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography

DOE PAGES

Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; ...

2015-02-10

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. In this paper, we used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop withmore » various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. Finally, the results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.« less

Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography

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

Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. In this paper, we used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop withmore » various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. Finally, the results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.« less

Factors affecting the use of 13Cα chemical shifts to determine, refine, and validate protein structures

PubMed Central

Vila, Jorge A.; Scheraga, Harold A.

2008-01-01

Interest centers here on the analysis of two different, but related, phenomena that affect side-chain conformations and consequently 13Cα chemical shifts and their applications to determine, refine, and validate protein structures. The first is whether 13Cα chemical shifts, computed at the DFT level of approximation with charged residues is a better approximation of observed 13Cα chemical shifts than those computed with neutral residues for proteins in solution. Accurate computation of 13Cα chemical shifts requires a proper representation of the charges, which might not take on integral values. For this analysis, the charges for 139 conformations of the protein ubiquitin were determined by explicit consideration of protein binding equilibria, at a given pH, that is, by exploring the 2ξ possible ionization states of the whole molecule, with ξ being the number of ionizable groups. The results of this analysis, as revealed by the shielding/deshield-ing of the 13Cα nucleus, indicated that: (i) there is a significant difference in the computed 13Cα chemical shifts, between basic and acidic groups, as a function of the degree of charge of the side chain; (ii) this difference is attributed to the distance between the ionizable groups and the 13Cα nucleus, which is shorter for the acidic Asp and Glu groups as compared with that for the basic Lys and Arg groups; and (iii) the use of neutral, rather than charged, basic and acidic groups is a better approximation of the observed 13Cα chemical shifts of a protein in solution. The second is how side-chain flexibility influences computed 13Cα chemical shifts in an additional set of ubiquitin conformations, in which the side chains are generated from an NMR-derived structure with the backbone conformation assumed to be fixed. The 13Cα chemical shift of a given amino acid residue in a protein is determined, mainly, by its own backbone and side-chain torsional angles, independent of the neighboring residues; the conformation of a given residue itself, however, depends on the environment of this residue and, hence, on the whole protein structure. As a consequence, this analysis reveals the role and impact of an accurate side-chain computation in the determination and refinement of protein conformation. The results of this analysis are: (i) a lower error between computed and observed 13Cα chemical shifts (by up to 3.7 ppm), was found for ~68% and ~63% of all ionizable residues and all non-Ala/Pro/Gly residues, respectively, in the additional set of conformations, compared with results for the model from which the set was derived; and (ii) all the additional conformations exhibit a lower root-mean-square-deviation (1.97 ppm ≤ rmsd ≤ 2.13 ppm), between computed and observed 13Cα chemical shifts, than the rmsd (2.32 ppm) computed for the starting conformation from which this additional set was derived. As a validation test, an analysis of the additional set of ubiquitin conformations, comparing computed and observed values of both 13Cα chemical shifts and χ1 torsional angles (given by the vicinal coupling constants, 3JN–Cγ and 3JC′–Cγ, is discussed. PMID:17975838

Ferrocene Orientation Determined Intramolecular Interactions Using Energy Decomposition Analysis.

PubMed

Wang, Feng; Islam, Shawkat; Vasilyev, Vladislav

2015-11-16

Two very different quantum mechanically based energy decomposition analyses (EDA) schemes are employed to study the dominant energy differences between the eclipsed and staggered ferrocene conformers. One is the extended transition state (ETS) based on the Amsterdam Density Functional (ADF) package and the other is natural EDA (NEDA) based in the General Atomic and Molecular Electronic Structure System (GAMESS) package. It reveals that in addition to the model (theory and basis set), the fragmentation channels more significantly affect the interaction energy terms (Δ E ) between the conformers. It is discovered that such an interaction energy can be absorbed into the pre-partitioned fragment channels so that to affect the interaction energies in a particular conformer of Fc. To avoid this, the present study employs a complete fragment channel-the fragments of ferrocene are individual neutral atoms. It therefore discovers that the major difference between the ferrocene conformers is due to the quantum mechanical Pauli repulsive energy and orbital attractive energy, leading to the eclipsed ferrocene the energy preferred structure. The NEDA scheme further indicates that the sum of attractive (negative) polarization (POL) and charge transfer (CL) energies prefers the eclipsed ferrocene. The repulsive (positive) deformation (DEF) energy, which is dominated by the cyclopentadienyle (Cp) rings, prefers the staggered ferrocene. Again, the cancellation results in a small energy residue in favour of the eclipsed ferrocene, in agreement with the ETS scheme. Further Natural Bond Orbital (NBO) analysis indicates that all NBO energies, total Lewis (no Fe) and lone pair (LP) deletion all prefer the eclipsed Fc conformer. The most significant energy preferring the eclipsed ferrocene without cancellation is the interactions between the donor lone pairs (LP) of the Fe atom and the acceptor antibond (BD*) NBOs of all C-C and C-H bonds in the ligand, LP(Fe)-BD*(C-C & C-H), which strongly stabilizes the eclipsed (D 5h ) conformation by -457.6 kcal·mol -1 .

Mutational scanning of the human serotonin transporter reveals fast translocating serotonin transporter mutants.

PubMed

Kristensen, Anders S; Larsen, Mads B; Johnsen, Laust B; Wiborg, Ove

2004-03-01

The serotonin transporter (SERT) belongs to a family of sodium-chloride-dependent transporters responsible for uptake of amino acids and biogenic amines from the extracellular space. SERT represents a major pharmacological target in the treatment of several clinical conditions, including depression and anxiety. In the present study we have undertaken a mutational scanning of human SERT in order to identify residues that are responsible for individual differences among related monoamine transporters. One mutant, G100A, was inactive in transport. However, ligand binding affinity was similar to wild-type, suggesting that G100A amongst different possible SERT conformations is restrained to a binding conformation. We suggest that the main role of glycine-100 is to confer structural flexibility during substrate translocation. For the two single mutants, T178A and F263C, uptake rates and K(m) values were both several-fold higher than wild-type while binding affinities and inhibitory potencies decreased considerably for several drugs. Ion dependency increased and only at hyperosmotic concentrations were K(m) values partly restored. For the double mutant, T178A/F263C, shifts in uptake kinetics and ligand affinities, as well as ion dependencies, were drastic. Effects were synergistic compared to the corresponding single mutants. In conclusion, we suggest that mutating threonine-178 to an alanine and phenylalanine-263 to a cysteine mainly alter the overall uptake kinetics of SERT by affecting the conformational equilibrium of different transporter conformations.

Ab initio conformational analysis of N-formyl ?-alanine amide including electron correlation

NASA Astrophysics Data System (ADS)

Yu, Ching-Hsing; Norman, Mya A.; Schäfer, Lothar; Ramek, Michael; Peeters, Anik; van Alsenoy, Christian

2001-06-01

The conformational properties of N-formyl L-alanine amide (ALA) were investigated using RMP2/6-311G∗∗ ab initio gradient geometry optimization. One hundred forty four structures of ALA were optimized at 30° grid points in its φ(N-C(α)), ψ(C(α)-C‧) conformational space. Using cubic spline functions, the grid structures were then used to construct analytical representations of complete surfaces, in φ,ψ-space, of bond lengths, bond angles, torsional sensitivity and electrostatic atomic charges. Analyses show that, in agreement with previous studies, the right-handed helical conformation, αR, is not a local energy minimum of the potential energy surface of ALA. Comparisons with protein crystallographic data show that the characteristic differences between geometrical trends in dipeptides and proteins, previously found for ab initio dipeptide structures obtained without electron correlation, are also found in the electron-correlated geometries. In contrast to generally accepted features of force fields used in empirical molecular modeling, partial atomic charges obtained by the CHELPG method are found to be not constant, but to vary significantly throughout the φ,ψ-space. By comparing RHF and MP2 structures, the effects of dispersion forces on ALA were studied, revealing molecular contractions for those conformations, in which small adjustments of torsional angles entail large changes in non-bonded distances.

Phenotypic Characterization and Multivariate Analysis to Explain Body Conformation in Lesser Known Buffalo (Bubalus bubalis) from North India

PubMed Central

Vohra, V.; Niranjan, S. K.; Mishra, A. K.; Jamuna, V.; Chopra, A.; Sharma, Neelesh; Jeong, Dong Kee

2015-01-01

Phenotypic characterization and body biometric in 13 traits (height at withers, body length, chest girth, paunch girth, ear length, tail length, length of tail up to switch, face length, face width, horn length, circumference of horn at base, distances between pin bone and hip bone) were recorded in 233 adult Gojri buffaloes from Punjab and Himachal Pradesh states of India. Traits were analysed by using varimax rotated principal component analysis (PCA) with Kaiser Normalization to explain body conformation. PCA revealed four components which explained about 70.9% of the total variation. First component described the general body conformation and explained 31.5% of total variation. It was represented by significant positive high loading of height at wither, body length, heart girth, face length and face width. The communality ranged from 0.83 (hip bone distance) to 0.45 (horn length) and unique factors ranged from 0.16 to 0.55 for all these 13 different biometric traits. Present study suggests that first principal component can be used in the evaluation and comparison of body conformation in buffaloes and thus provides an opportunity to distinguish between early and late maturing to adult, based on a small group of biometric traits to explain body conformation in adult buffaloes. PMID:25656215

Capturing Chromosome Conformation

NASA Astrophysics Data System (ADS)

Dekker, Job; Rippe, Karsten; Dekker, Martijn; Kleckner, Nancy

2002-02-01

We describe an approach to detect the frequency of interaction between any two genomic loci. Generation of a matrix of interaction frequencies between sites on the same or different chromosomes reveals their relative spatial disposition and provides information about the physical properties of the chromatin fiber. This methodology can be applied to the spatial organization of entire genomes in organisms from bacteria to human. Using the yeast Saccharomyces cerevisiae, we could confirm known qualitative features of chromosome organization within the nucleus and dynamic changes in that organization during meiosis. We also analyzed yeast chromosome III at the G1 stage of the cell cycle. We found that chromatin is highly flexible throughout. Furthermore, functionally distinct AT- and GC-rich domains were found to exhibit different conformations, and a population-average 3D model of chromosome III could be determined. Chromosome III emerges as a contorted ring.

GLUT-1 GLUCOSE TRANSPORTERS IN THE BLOOD-BRAIN BARRIER: DIFFERENTIAL PHOSPHORYLATION

PubMed Central

Devraj, Kavi; Klinger, Marianne E.; Myers, Roland L.; Mokashi, Ashwini; Hawkins, Richard A.; Simpson, Ian A.

2013-01-01

Glucose is the primary metabolic fuel for the mammalian brain and a continuous supply is required to maintain normal CNS function. The transport of glucose across the blood-brain barrier (BBB) into the brain is mediated by the facilitative glucose transporter GLUT-1. Prior studies (Simpson et al. 2001) had revealed that the conformations of the GLUT-1 transporter were different in luminal (blood facing) and abluminal (brain facing) membranes of bovine cerebral endothelial cells, based on differential antibody recognition. In this study we have extended these observations and using a combination of 2D-PAGE/Western blotting and immunogold electron microscopy we determined that these different conformations are exhibited in vivo and arise from differential phosphorylation of GLUT-1 and not from alternative splicing or altered O- or N-linked glycosylation. PMID:21910135

The Volumetric Diversity of Misfolded Prion Protein Oligomers Revealed by Pressure Dissociation*

PubMed Central

Torrent, Joan; Lange, Reinhard; Rezaei, Human

2015-01-01

Protein oligomerization has been associated with a wide range of diseases. High pressure approaches offer a powerful tool for deciphering the underlying molecular mechanisms by revealing volume changes associated with the misfolding and assembly reactions. We applied high pressure to induce conformational changes in three distinct β-sheet-rich oligomers of the prion protein PrP, a protein characterized by a variety of infectious quaternary structures that can propagate stably and faithfully and cause diseases with specific phenotypic traits. We show that pressure induces dissociation of the oligomers and leads to a lower volume monomeric PrP state that refolds into the native conformation after pressure release. By measuring the different pressure and temperature sensitivity of the tested PrP oligomers, we demonstrate significantly different void volumes in their quaternary structure. In addition, by focusing on the kinetic and energetic behavior of the pressure-induced dissociation of one specific PrP oligomer, we reveal a large negative activation volume and an increase in both apparent activation enthalpy and entropy. This suggests a transition state ensemble that is less structured and significantly more hydrated than the oligomeric state. Finally, we found that site-specific fluorescent labeling allows monitoring of the transient population of a kinetic intermediate in the dissociation reaction. Our results indicate that defects in atomic packing may deserve consideration as a new factor that influences differences between PrP assemblies and that could be relevant also for explaining the origin of prion strains. PMID:26126829

Revealing time bunching effect in single-molecule enzyme conformational dynamics.

PubMed

Lu, H Peter

2011-04-21

In this perspective, we focus our discussion on how the single-molecule spectroscopy and statistical analysis are able to reveal enzyme hidden properties, taking the study of T4 lysozyme as an example. Protein conformational fluctuations and dynamics play a crucial role in biomolecular functions, such as in enzymatic reactions. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structure-function mechanisms. Using single-molecule fluorescence spectroscopy, we have probed T4 lysozyme conformational motions under the hydrolysis reaction of a polysaccharide of E. coli B cell walls by monitoring the fluorescence resonant energy transfer (FRET) between a donor-acceptor probe pair tethered to T4 lysozyme domains involving open-close hinge-bending motions. Based on the single-molecule spectroscopic results, molecular dynamics simulation, a random walk model analysis, and a novel 2D statistical correlation analysis, we have revealed a time bunching effect in protein conformational motion dynamics that is critical to enzymatic functions. Bunching effect implies that conformational motion times tend to bunch in a finite and narrow time window. We show that convoluted multiple Poisson rate processes give rise to the bunching effect in the enzymatic reaction dynamics. Evidently, the bunching effect is likely common in protein conformational dynamics involving in conformation-gated protein functions. In this perspective, we will also discuss a new approach of 2D regional correlation analysis capable of analyzing fluctuation dynamics of complex multiple correlated and anti-correlated fluctuations under a non-correlated noise background. Using this new method, we are able to map out any defined segments along the fluctuation trajectories and determine whether they are correlated, anti-correlated, or non-correlated; after which, a cross correlation analysis can be applied for each specific segment to obtain a detailed fluctuation dynamics analysis.

A molecular mechanism of P-loop pliability of Rho-kinase investigated by molecular dynamic simulation

NASA Astrophysics Data System (ADS)

Gohda, Keigo; Hakoshima, Toshio

2008-11-01

Rho-kinase is a leading player in the regulation of cytoskeletal events involving smooth muscle contraction and neurite growth-cone collapse and retraction, and is a promising drug target in the treatment of both vascular and neurological disorders. Recent crystal structure of Rho-kinase complexed with a small-molecule inhibitor fasudil has revealed structural details of the ATP-binding site, which represents the target site for the inhibitor, and showed that the conserved phenylalanine on the P-loop occupies the pocket, resulting in an increase of protein-ligand contacts. Thus, the P-loop pliability is considered to play an important role in inhibitor binding affinity and specificity. In this study, we carried out a molecular dynamic simulation for Rho-kinase-fasudil complexes with two different P-loop conformations, i.e., the extended and folded conformations, in order to understand the P-loop pliability and dynamics at atomic level. A PKA-fasudil complex was also used for comparison. In the MD simulation, the flip-flop movement of the P-loop conformation starting either from the extended or folded conformation was not able to be observed. However, a significant conformational change in a long loop region covering over the P-loop, and also alteration of ionic interaction-manner of fasudil with acidic residues in the ATP binding site were shown only in the Rho-kinase-fasudil complex with the extended P-loop conformation, while Rho-kinase with the folded P-loop conformation and PKA complexes did not show large fluctuations, suggesting that the Rho-kinase-fasudil complex with the extended P-loop conformation represents a meta-stable state. The information of the P-loop pliability at atomic level obtained in this study could provide valuable clues to designing potent and/or selective inhibitors for Rho-kinase.

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

Sparse networks of directly coupled, polymorphic, and functional side chains in allosteric proteins.

PubMed

Soltan Ghoraie, Laleh; Burkowski, Forbes; Zhu, Mu

2015-03-01

Recent studies have highlighted the role of coupled side-chain fluctuations alone in the allosteric behavior of proteins. Moreover, examination of X-ray crystallography data has recently revealed new information about the prevalence of alternate side-chain conformations (conformational polymorphism), and attempts have been made to uncover the hidden alternate conformations from X-ray data. Hence, new computational approaches are required that consider the polymorphic nature of the side chains, and incorporate the effects of this phenomenon in the study of information transmission and functional interactions of residues in a molecule. These studies can provide a more accurate understanding of the allosteric behavior. In this article, we first present a novel approach to generate an ensemble of conformations and an efficient computational method to extract direct couplings of side chains in allosteric proteins, and provide sparse network representations of the couplings. We take the side-chain conformational polymorphism into account, and show that by studying the intrinsic dynamics of an inactive structure, we are able to construct a network of functionally crucial residues. Second, we show that the proposed method is capable of providing a magnified view of the coupled and conformationally polymorphic residues. This model reveals couplings between the alternate conformations of a coupled residue pair. To the best of our knowledge, this is the first computational method for extracting networks of side chains' alternate conformations. Such networks help in providing a detailed image of side-chain dynamics in functionally important and conformationally polymorphic sites, such as binding and/or allosteric sites. © 2014 Wiley Periodicals, Inc.

Survey of conformational isomerism in (E)-2-[(4-bromophenylimino)methyl]-5-(diethylamino)phenol compound from structural and thermochemical points of view.

PubMed

Albayrak, Çiğdem; Kaştaş, Gökhan; Odabaşoğlu, Mustafa; Frank, René

2012-09-01

In this study, (E)-2-[(4-bromophenylimino)methyl]-5-(diethylamino)phenol compound was investigated by mainly focusing on conformational isomerism. For this purpose, molecular structure and spectroscopic properties of the compound were experimentally characterized by X-ray diffraction, FT-IR and UV-Vis spectroscopic techniques, and computationally by DFT method. The X-ray diffraction analysis of the compound shows the formation of two conformers (anti and eclipsed) related to the ethyl groups of the compound. The two conformers are connected to each other by non-covalent C-H⋯Br and C-H⋯π interactions. The combination of these interactions is resulted in fused R(2)(2)(10) and R(2)(4)(20) synthons which are responsible for the tape structure of crystal packing arrangement. The X-ray diffraction and FT-IR analyses also reveal the existence of enol form in the solid state. From thermochemical point of view, the computational investigation of isomerism includes three studies: the calculation of (a) the rate constants for transmission from anti or eclipsed conformations to transition state by using Eyring equation, (b) the activation energy needed for isomerism by using Arrhenius equation, (c) the equilibrium constant from anti conformer to eclipsed conformer by using the equation including the change in Gibbs free energy. The dependence of tautomerism on solvent types was studied on the basis of UV-Vis spectra recorded in different organic solvents. The results showed that the compound exists in enol form in all solvents except ethyl alcohol. Copyright © 2012 Elsevier B.V. All rights reserved.

Computational study of the fibril organization of polyglutamine repeats reveals a common motif identified in beta-helices.

PubMed

Zanuy, David; Gunasekaran, Kannan; Lesk, Arthur M; Nussinov, Ruth

2006-04-21

The formation of fibril aggregates by long polyglutamine sequences is assumed to play a major role in neurodegenerative diseases such as Huntington. Here, we model peptides rich in glutamine, through a series of molecular dynamics simulations. Starting from a rigid nanotube-like conformation, we have obtained a new conformational template that shares structural features of a tubular helix and of a beta-helix conformational organization. Our new model can be described as a super-helical arrangement of flat beta-sheet segments linked by planar turns or bends. Interestingly, our comprehensive analysis of the Protein Data Bank reveals that this is a common motif in beta-helices (termed beta-bend), although it has not been identified so far. The motif is based on the alternation of beta-sheet and helical conformation as the protein sequence is followed from the N to the C termini (beta-alpha(R)-beta-polyPro-beta). We further identify this motif in the ssNMR structure of the protofibril of the amyloidogenic peptide Abeta(1-40). The recurrence of the beta-bend suggests a general mode of connecting long parallel beta-sheet segments that would allow the growth of partially ordered fibril structures. The design allows the peptide backbone to change direction with a minimal loss of main chain hydrogen bonds. The identification of a coherent organization beyond that of the beta-sheet segments in different folds rich in parallel beta-sheets suggests a higher degree of ordered structure in protein fibrils, in agreement with their low solubility and dense molecular packing.

Comprehensive theoretical study towards the accurate proton affinity values of naturally occurring amino acids

NASA Astrophysics Data System (ADS)

Dinadayalane, T. C.; Sastry, G. Narahari; Leszczynski, Jerzy

Systematic quantum chemical studies of Hartree-Fock (HF) and second-order Møller-Plesset (MP2) methods, and B3LYP functional, with a range of basis sets were employed to evaluate proton affinity values of all naturally occurring amino acids. The B3LYP and MP2 in conjunction with 6-311+G(d,p) basis set provide the proton affinity values that are in very good agreement with the experimental results, with an average deviation of ?1 kcal/mol. The number and the relative strength of intramolecular hydrogen bonding play a key role in the proton affinities of amino acids. The computational exploration of the conformers reveals that the global minima conformations of the neutral and protonated amino acids are different in eight cases. The present study reveals that B3LYP/6-311+G(d,p) is a very good choice of technique to evaluate the proton affinities of amino acids and the compounds derived from them reliably and economically.

Force-Manipulation Single-Molecule Spectroscopy Studies of Enzymatic Dynamics

NASA Astrophysics Data System (ADS)

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

2014-03-01

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

Structure-based design of Aurora A & B inhibitors

NASA Astrophysics Data System (ADS)

Poulsen, Anders; William, Anthony; Lee, Angeline; Blanchard, Stéphanie; Teo, Eeling; Deng, Weiping; Tu, Noah; Tan, Evelyn; Sun, Eric; Goh, Kay Lin; Ong, Wai Chung; Ng, Chee Pang; Goh, Kee Chuan; Bonday, Zahid

2008-12-01

The Aurora family of serine/threonine kinases are mitotic regulators involved in centrosome duplication, formation of the bipolar mitotic spindle and the alignment of the chromosomes along the spindle. These proteins are frequently overexpressed in tumor cells as compared to normal cells and are therefore potential therapeutic oncology targets. An Aurora A high throughput screen revealed a promising sub-micromolar indazole-benzimidazole lead. Modification of the benzimidazole portion of the lead to a C2 linker with a phenyl ring was proposed to achieve novelty. Docking revealed that a conjugated linker was optimal and the resulting compounds were equipotent with the lead. Further structure-guided optimization of substituents on the 5 & 6 position of the indazole led to single digit nanomolar potency. The homology between the Aurora A & Aurora B kinase domains is 71% but their binding sites only differ at residues 212 & 217 (Aurora A numbering). However interactions with only the latter residue may be used for obtaining selectivity. An analysis of published Aurora A and Aurora B X-ray structures reveals subtle differences in the shape of the binding sites. This was exploited by introduction of appropriately sized substituents in the 4 & 6 position of the indazole leading to Aurora B selective inhibitors. Finally we calculate the conformational energy penalty of the putative bioactive conformation of our inhibitors and show that this property correlates well with the Aurora A binding affinity.

Time-Resolved Small-Angle X-ray Scattering Reveals Millisecond Transitions of a DNA Origami Switch.

PubMed

Bruetzel, Linda K; Walker, Philipp U; Gerling, Thomas; Dietz, Hendrik; Lipfert, Jan

2018-04-11

Self-assembled DNA structures enable creation of specific shapes at the nanometer-micrometer scale with molecular resolution. The construction of functional DNA assemblies will likely require dynamic structures that can undergo controllable conformational changes. DNA devices based on shape complementary stacking interactions have been demonstrated to undergo reversible conformational changes triggered by changes in ionic environment or temperature. An experimentally unexplored aspect is how quickly conformational transitions of large synthetic DNA origami structures can actually occur. Here, we use time-resolved small-angle X-ray scattering to monitor large-scale conformational transitions of a two-state DNA origami switch in free solution. We show that the DNA device switches from its open to its closed conformation upon addition of MgCl 2 in milliseconds, which is close to the theoretical diffusive speed limit. In contrast, measurements of the dimerization of DNA origami bricks reveal much slower and concentration-dependent assembly kinetics. DNA brick dimerization occurs on a time scale of minutes to hours suggesting that the kinetics depend on local concentration and molecular alignment.

Kinetics of protein unfolding at interfaces

NASA Astrophysics Data System (ADS)

Yano, Yohko F.

2012-12-01

The conformation of protein molecules is determined by a balance of various forces, including van der Waals attraction, electrostatic interaction, hydrogen bonding, and conformational entropy. When protein molecules encounter an interface, they are often adsorbed on the interface. The conformation of an adsorbed protein molecule strongly depends on the interaction between the protein and the interface. Recent time-resolved investigations have revealed that protein conformation changes during the adsorption process due to the protein-protein interaction increasing with increasing interface coverage. External conditions also affect the protein conformation. This review considers recent dynamic observations of protein adsorption at various interfaces and their implications for the kinetics of protein unfolding at interfaces.

Multiple conformations are a conserved and regulatory feature of the RB1 5′ UTR

PubMed Central

Kutchko, Katrina M.; Sanders, Wes; Ziehr, Ben; Phillips, Gabriela; Solem, Amanda; Halvorsen, Matthew; Weeks, Kevin M.; Moorman, Nathaniel

2015-01-01

Folding to a well-defined conformation is essential for the function of structured ribonucleic acids (RNAs) like the ribosome and tRNA. Structured elements in the untranslated regions (UTRs) of specific messenger RNAs (mRNAs) are known to control expression. The importance of unstructured regions adopting multiple conformations, however, is still poorly understood. High-resolution SHAPE-directed Boltzmann suboptimal sampling of the Homo sapiens Retinoblastoma 1 (RB1) 5′ UTR yields three distinct conformations compatible with the experimental data. Private single nucleotide variants (SNVs) identified in two patients with retinoblastoma each collapse the structural ensemble to a single but distinct well-defined conformation. The RB1 5′ UTRs from Bos taurus (cow) and Trichechus manatus latirostris (manatee) are divergent in sequence from H. sapiens (human) yet maintain structural compatibility with high-probability base pairs. SHAPE chemical probing of the cow and manatee RB1 5′ UTRs reveals that they also adopt multiple conformations. Luciferase reporter assays reveal that 5′ UTR mutations alter RB1 expression. In a traditional model of disease, causative SNVs disrupt a key structural element in the RNA. For the subset of patients with heritable retinoblastoma-associated SNVs in the RB1 5′ UTR, the absence of multiple structures is likely causative of the cancer. Our data therefore suggest that selective pressure will favor multiple conformations in eukaryotic UTRs to regulate expression. PMID:25999316

Microsecond-Scale MD Simulations of HIV-1 DIS Kissing-Loop Complexes Predict Bulged-In Conformation of the Bulged Bases and Reveal Interesting Differences between Available Variants of the AMBER RNA Force Fields.

PubMed

Havrila, Marek; Zgarbová, Marie; Jurečka, Petr; Banáš, Pavel; Krepl, Miroslav; Otyepka, Michal; Šponer, Jiří

2015-12-10

We report an extensive set of explicit solvent molecular dynamics (MD) simulations (∼25 μs of accumulated simulation time) of the RNA kissing-loop complex of the HIV-1 virus initiation dimerization site. Despite many structural investigations by X-ray, NMR, and MD techniques, the position of the bulged purines of the kissing complex has not been unambiguously resolved. The X-ray structures consistently show bulged-out positions of the unpaired bases, while several NMR studies show bulged-in conformations. The NMR studies are, however, mutually inconsistent regarding the exact orientations of the bases. The earlier simulation studies predicted the bulged-out conformation; however, this finding could have been biased by the short simulation time scales. Our microsecond-long simulations reveal that all unpaired bases of the kissing-loop complex stay preferably in the interior of the kissing-loop complex. The MD results are discussed in the context of the available experimental data and we suggest that both conformations are biochemically relevant. We also show that MD provides a quite satisfactory description of this RNA system, contrasting recent reports of unsatisfactory performance of the RNA force fields for smaller systems such as tetranucleotides and tetraloops. We explain this by the fact that the kissing complex is primarily stabilized by an extensive network of Watson-Crick interactions which are rather well described by the force fields. We tested several different sets of water/ion parameters but they all lead to consistent results. However, we demonstrate that a recently suggested modification of van der Waals interactions of the Cornell et al. force field deteriorates the description of the kissing complex by the loss of key stacking interactions stabilizing the interhelical junction and excessive hydrogen-bonding interactions.

Separate and simultaneous binding effects through a non-cooperative behavior between cyclophosphamide hydrochloride and fluoxymesterone upon interaction with human serum albumin: Multi-spectroscopic and molecular modeling approaches

NASA Astrophysics Data System (ADS)

Zohoorian-Abootorabi, Toktam; Sanee, Hamideh; Iranfar, Hediyeh; Saberi, Mohammad Reza; Chamani, Jamshidkhan

2012-03-01

This study was designed to examine the interaction of two anti-breast cancer drugs, i.e., fluoxymesterone (FLU) and cyclophosphamide (CYC), with human serum albumin (HSA) using different kinds of spectroscopic, zeta potential and molecular modeling techniques under imitated physiological conditions. The RLS technique was utilized to investigate the effect of the two anticancer drugs on changes of the protein conformation, both separately and simultaneously. Our study suggested that the enhancement in RLS intensity was attributed to the formation of a new complex between the two drugs and the protein. Both drugs demonstrated a powerful ability to quench the fluorescence of HSA, and the fluorescence quenching action was much stronger when the two drugs coexisted. The quenching mechanism was suggested to be static as confirmed by time-resolved fluorescence spectroscopy results. The effect of both drugs on the conformation of HSA was analyzed using synchronous fluorescence spectroscopy. Our results revealed that the fluorescence quenching of HSA originated from the Trp and Tyr residues, and demonstrated a conformational change of HSA with the addition of both drugs. The binding distances between HSA and the drugs were estimated by the Förster theory, and it was revealed that nonradiative energy transfer from HSA to both drugs occurred with a high probability. According to CD measurements, the influence of both drugs on the secondary structure of HSA in aqueous solutions was also investigated and illustrated that the α-helix content of HSA decreased with increasing drug concentration in both systems. Moreover, the zeta-potential experiments revealed that both drugs induced conformational changes on HSA. Docking studies were also performed and demonstrated that a reduction of the binding affinity between the drugs and HSA occurred in the presence of both drugs.

Global brain dynamics during social exclusion predict subsequent behavioral conformity

PubMed Central

Wasylyshyn, Nick; Hemenway Falk, Brett; Garcia, Javier O; Cascio, Christopher N; O’Donnell, Matthew Brook; Bingham, C Raymond; Simons-Morton, Bruce; Vettel, Jean M; Falk, Emily B

2018-01-01

Abstract Individuals react differently to social experiences; for example, people who are more sensitive to negative social experiences, such as being excluded, may be more likely to adapt their behavior to fit in with others. We examined whether functional brain connectivity during social exclusion in the fMRI scanner can be used to predict subsequent conformity to peer norms. Adolescent males (n = 57) completed a two-part study on teen driving risk: a social exclusion task (Cyberball) during an fMRI session and a subsequent driving simulator session in which they drove alone and in the presence of a peer who expressed risk-averse or risk-accepting driving norms. We computed the difference in functional connectivity between social exclusion and social inclusion from each node in the brain to nodes in two brain networks, one previously associated with mentalizing (medial prefrontal cortex, temporoparietal junction, precuneus, temporal poles) and another with social pain (dorsal anterior cingulate cortex, anterior insula). Using predictive modeling, this measure of global connectivity during exclusion predicted the extent of conformity to peer pressure during driving in the subsequent experimental session. These findings extend our understanding of how global neural dynamics guide social behavior, revealing functional network activity that captures individual differences. PMID:29529310

Visualizing Key Hinges and a Potential Major Source of Compliance in the Lever Arm of Myosin

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

J Brown; V Senthil Kumar; E ONeall-Hennessey

2011-12-31

We have determined the 2.3-{angstrom}-resolution crystal structure of a myosin light chain domain, corresponding to one type found in sea scallop catch ('smooth') muscle. This structure reveals hinges that may function in the 'on' and 'off' states of myosin. The molecule adopts two different conformations about the heavy chain 'hook' and regulatory light chain (RLC) helix D. This conformational change results in extended and compressed forms of the lever arm whose lengths differ by 10 {angstrom}. The heavy chain hook and RLC helix D hinges could thus serve as a potential major and localized source of cross-bridge compliance during themore » contractile cycle. In addition, in one of the molecules of the crystal, part of the RLC N-terminal extension is seen in atomic detail and forms a one-turn alpha-helix that interacts with RLC helix D. This extension, whose sequence is highly variable in different myosins, may thus modulate the flexibility of the lever arm. Moreover, the relative proximity of the phosphorylation site to the helix D hinge suggests a potential role for conformational changes about this hinge in the transition between the on and off states of regulated myosins.« less

Visualizing key hinges and a potential major source of compliance in the lever arm of myosin

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

Brown, J.H.; Robinson, H.; Senthil Kumar, V. S.

2011-01-04

We have determined the 2.3-{angstrom}-resolution crystal structure of a myosin light chain domain, corresponding to one type found in sea scallop catch ('smooth') muscle. This structure reveals hinges that may function in the 'on' and 'off' states of myosin. The molecule adopts two different conformations about the heavy chain 'hook' and regulatory light chain (RLC) helix D. This conformational change results in extended and compressed forms of the lever arm whose lengths differ by 10 {angstrom}. The heavy chain hook and RLC helix D hinges could thus serve as a potential major and localized source of cross-bridge compliance during themore » contractile cycle. In addition, in one of the molecules of the crystal, part of the RLC N-terminal extension is seen in atomic detail and forms a one-turn alpha-helix that interacts with RLC helix D. This extension, whose sequence is highly variable in different myosins, may thus modulate the flexibility of the lever arm. Moreover, the relative proximity of the phosphorylation site to the helix D hinge suggests a potential role for conformational changes about this hinge in the transition between the on and off states of regulated myosins.« less

[Analysis of Conformational Features of Watson-Crick Duplex Fragments by Molecular Mechanics and Quantum Mechanics Methods].

PubMed

Poltev, V I; Anisimov, V M; Sanchez, C; Deriabina, A; Gonzalez, E; Garcia, D; Rivas, F; Polteva, N A

2016-01-01

It is generally accepted that the important characteristic features of the Watson-Crick duplex originate from the molecular structure of its subunits. However, it still remains to elucidate what properties of each subunit are responsible for the significant characteristic features of the DNA structure. The computations of desoxydinucleoside monophosphates complexes with Na-ions using density functional theory revealed a pivotal role of DNA conformational properties of single-chain minimal fragments in the development of unique features of the Watson-Crick duplex. We found that directionality of the sugar-phosphate backbone and the preferable ranges of its torsion angles, combined with the difference between purines and pyrimidines. in ring bases, define the dependence of three-dimensional structure of the Watson-Crick duplex on nucleotide base sequence. In this work, we extended these density functional theory computations to the minimal' fragments of DNA duplex, complementary desoxydinucleoside monophosphates complexes with Na-ions. Using several computational methods and various functionals, we performed a search for energy minima of BI-conformation for complementary desoxydinucleoside monophosphates complexes with different nucleoside sequences. Two sequences are optimized using ab initio method at the MP2/6-31++G** level of theory. The analysis of torsion angles, sugar ring puckering and mutual base positions of optimized structures demonstrates that the conformational characteristic features of complementary desoxydinucleoside monophosphates complexes with Na-ions remain within BI ranges and become closer to the corresponding characteristic features of the Watson-Crick duplex crystals. Qualitatively, the main characteristic features of each studied complementary desoxydinucleoside monophosphates complex remain invariant when different computational methods are used, although the quantitative values of some conformational parameters could vary lying within the limits typical for the corresponding family. We observe that popular functionals in density functional theory calculations lead to the overestimated distances between base pairs, while MP2 computations and the newer complex functionals produce the structures that have too close atom-atom contacts. A detailed study of some complementary desoxydinucleoside monophosphate complexes with Na-ions highlights the existence of several energy minima corresponding to BI-conformations, in other words, the complexity of the relief pattern of the potential energy surface of complementary desoxydinucleoside monophosphate complexes. This accounts for variability of conformational parameters of duplex fragments with the same base sequence. Popular molecular mechanics force fields AMBER and CHARMM reproduce most of the conformational characteristics of desoxydinucleoside monophosphates and their complementary complexes with Na-ions but fail to reproduce some details of the dependence of the Watson-Crick duplex conformation on the nucleotide sequence.

Stereochemical and conformational study on fenoterol by ECD spectroscopy and TD-DFT calculations.

PubMed

Tedesco, Daniele; Zanasi, Riccardo; Wainer, Irving W; Bertucci, Carlo

2014-03-01

Fenoterol and its derivatives are selective β2-adrenergic receptor (β2-AR) agonists whose stereoselective biological activities have been extensively investigated in the past decade; a complete stereochemical characterization of fenoterol derivatives is therefore crucial for a better understanding of the effects of stereochemistry on β2-AR binding. In the present project, the relationship between chiroptical properties and absolute stereochemistry of the stereoisomers of fenoterol (1) was investigated by experimental ECD spectroscopy and time-dependent density functional theory (TD-DFT). DFT geometry optimizations were carried out at the RI-B97D/TZVP/IEFPCM(MeOH) level and subsequent TD-DFT calculations were performed using the PBE0 hybrid functional. Despite the large pool of equilibrium conformers found for the investigated compounds and the known limitations of the level of theory employed, the computational protocol was able to reproduce the experimental ECD spectra of the stereoisomers of 1. The main contribution to the overall chiroptical properties was found to arise from the absolute configuration of the chiral center in α-position to the resorcinol moiety. Based on this evidence, a thorough conformational analysis was performed on the optimized DFT conformers, which revealed the occurrence of a different equilibrium between conformational patterns for the diastereomers of fenoterol: the (R,R')/(S,S') enantiomeric pair showed a higher population of folded conformations than the (R,S')/(S,R') pair. Copyright © 2013 Elsevier B.V. All rights reserved.

High-speed atomic force microscopy reveals structural dynamics of α -synuclein monomers and dimers

NASA Astrophysics Data System (ADS)

Zhang, Yuliang; Hashemi, Mohtadin; Lv, Zhengjian; Williams, Benfeard; Popov, Konstantin I.; Dokholyan, Nikolay V.; Lyubchenko, Yuri L.

2018-03-01

α-Synuclein (α-syn) is the major component of the intraneuronal inclusions called Lewy bodies, which are the pathological hallmark of Parkinson's disease. α-Syn is capable of self-assembly into many different species, such as soluble oligomers and fibrils. Even though attempts to resolve the structures of the protein have been made, detailed understanding about the structures and their relationship with the different aggregation steps is lacking, which is of interest to provide insights into the pathogenic mechanism of Parkinson's disease. Here we report the structural flexibility of α-syn monomers and dimers in an aqueous solution environment as probed by single-molecule time-lapse high-speed AFM. In addition, we present the molecular basis for the structural transitions using discrete molecular dynamics (DMD) simulations. α-Syn monomers assume a globular conformation, which is capable of forming tail-like protrusions over dozens of seconds. Importantly, a globular monomer can adopt fully extended conformations. Dimers, on the other hand, are less dynamic and show a dumbbell conformation that experiences morphological changes over time. DMD simulations revealed that the α-syn monomer consists of several tightly packed small helices. The tail-like protrusions are also helical with a small β-sheet, acting as a "hinge". Monomers within dimers have a large interfacial interaction area and are stabilized by interactions in the non-amyloid central (NAC) regions. Furthermore, the dimer NAC-region of each α-syn monomer forms a β-rich segment. Moreover, NAC-regions are located in the hydrophobic core of the dimer.

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

Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

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

Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs

The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. colimore » α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.« less

Probing Structural Changes among Analogous Inhibitor-Bound Forms of HIV-1 Protease and a Drug-Resistant Mutant in Solution by Nuclear Magnetic Resonance.

PubMed

Khan, Shahid N; Persons, John D; Paulsen, Janet L; Guerrero, Michel; Schiffer, Celia A; Kurt-Yilmaz, Nese; Ishima, Rieko

2018-03-13

In the era of state-of-the-art inhibitor design and high-resolution structural studies, detection of significant but small protein structural differences in the inhibitor-bound forms is critical to further developing the inhibitor. Here, we probed differences in HIV-1 protease (PR) conformation among darunavir and four analogous inhibitor-bound forms and compared them with a drug-resistant mutant using nuclear magnetic resonance chemical shifts. Changes in amide chemical shifts of wild-type (WT) PR among these inhibitor-bound forms, ΔCSP, were subtle but detectable and extended >10 Å from the inhibitor-binding site, asymmetrically between the two subunits of PR. Molecular dynamics simulations revealed differential local hydrogen bonding as the molecular basis of this remote asymmetric change. Inhibitor-bound forms of the drug-resistant mutant also showed a similar long-range ΔCSP pattern. Differences in ΔCSP values of the WT and the mutant (ΔΔCSPs) were observed at the inhibitor-binding site and in the surrounding region. Comparing chemical shift changes among highly analogous inhibitors and ΔΔCSPs effectively eliminated local environmental effects stemming from different chemical groups and enabled exploitation of these sensitive parameters to detect subtle protein conformational changes and to elucidate asymmetric and remote conformational effects upon inhibitor interaction.

Elucidation of Ligand-Dependent Modulation of Disorder-Order Transitions in the Oncoprotein MDM2.

PubMed

Bueren-Calabuig, Juan A; Michel, Julien

2015-06-01

Numerous biomolecular interactions involve unstructured protein regions, but how to exploit such interactions to enhance the affinity of a lead molecule in the context of rational drug design remains uncertain. Here clarification was sought for cases where interactions of different ligands with the same disordered protein region yield qualitatively different results. Specifically, conformational ensembles for the disordered lid region of the N-terminal domain of the oncoprotein MDM2 in the presence of different ligands were computed by means of a novel combination of accelerated molecular dynamics, umbrella sampling, and variational free energy profile methodologies. The resulting conformational ensembles for MDM2, free and bound to p53 TAD (17-29) peptide identify lid states compatible with previous NMR measurements. Remarkably, the MDM2 lid region is shown to adopt distinct conformational states in the presence of different small-molecule ligands. Detailed analyses of small-molecule bound ensembles reveal that the ca. 25-fold affinity improvement of the piperidinone family of inhibitors for MDM2 constructs that include the full lid correlates with interactions between ligand hydrophobic groups and the C-terminal lid region that is already partially ordered in apo MDM2. By contrast, Nutlin or benzodiazepinedione inhibitors, that bind with similar affinity to full lid and lid-truncated MDM2 constructs, interact additionally through their solubilizing groups with N-terminal lid residues that are more disordered in apo MDM2.

Selenoglycosides in silico: ab initio-derived reparameterization of MM4, conformational analysis using histo-blood group ABH antigens and lectin docking as indication for potential of bioactivity

NASA Astrophysics Data System (ADS)

Strino, Francesco; Lii, Jenn-Huei; Koppisetty, Chaitanya A. K.; Nyholm, Per-Georg; Gabius, Hans-Joachim

2010-12-01

The identification of glycan epitopes such as the histo-blood group ABH determinants as docking sites for bacterial/viral infections and signals in growth regulation fuels the interest to develop non-hydrolysable mimetics for therapeutic applications. Inevitably, the required substitution of the linkage oxygen atom will alter the derivative's topology. Our study addresses the question of the impact of substitution of oxygen by selenium. In order to characterize spatial parameters and flexibility of selenoglycosides, we first performed ab initio calculations on model compounds to refine the MM4 force field. The following application of the resulting MM4R version appears to reduce the difference to ab initio data when compared to using the MM4 estimator. Systematic conformational searches on the derivatives of histo-blood group ABH antigens revealed increased flexibility with acquisition of additional low-energy conformer(s), akin to the behavior of S-glycosides. Docking analysis using the Glide program for eight test cases indicated potential for bioactivity, giving further experimental investigation a clear direction to testing Se-glycosides as lectin ligands.

Facet-selective nucleation and conformal epitaxy of Ge shells on Si nanowires

DOE PAGES

Nguyen, Binh -Minh; Swartzentruber, Brian; Ro, Yun Goo; ...

2015-10-08

Knowledge of nanoscale heteroepitaxy is continually evolving as advances in material synthesis reveal new mechanisms that have not been theoretically predicted and are different than what is known about planar structures. In addition to a wide range of potential applications, core/shell nanowire structures offer a useful template to investigate heteroepitaxy at the atomistic scale. We show that the growth of a Ge shell on a Si core can be tuned from the theoretically predicted island growth mode to a conformal, crystalline, and smooth shell by careful adjustment of growth parameters in a narrow growth window that has not been exploredmore » before. In the latter growth mode, Ge adatoms preferentially nucleate islands on the {113} facets of the Si core, which outgrow over the {220} facets. Islands on the low-energy {111} facets appear to have a nucleation delay compared to the {113} islands; however, they eventually coalesce to form a crystalline conformal shell. As a result, synthesis of epitaxial and conformal Si/Ge/Si core/multishell structures enables us to fabricate unique cylindrical ring nanowire field-effect transistors, which we demonstrate to have steeper on/off characteristics than conventional core/shell nanowire transistors.« less

Unravelling the Conformational Landscape of Nicotinoids: the Structure of Cotinine by Broadband Rotational Spectroscopy

NASA Astrophysics Data System (ADS)

Uriarte, Iciar; Ecija, Patricia; Cocinero, Emilio J.; Perez, Cristobal; Caballero-Mancebo, Elena; Lesarri, Alberto

2015-06-01

Alkaloids such as nicotine, cotinine or anabasine share a common floppy structural motif consisting of a two-ring assembly with a 3-pyridil methylamine skeleton. In order to investigate the structure-activity relationship of these biomolecules, structural studies with rotational resolution have been carried out for nicotine and anabasine in the gas phase, where these molecules can be probed in an "interaction-free" environment (no solvent or crystal-packing interactions). We hereby present a structural investigation of cotinine in a jet expansion using the chirped-pulse Fourier-transform microwave (CP-FTMW) spectrometer recently built at the University of the Basque Country (UPV-EHU). The rotational spectrum (6-18 GHz) reveals the presence of two different conformations. The conformational preferences of cotinine originate from the internal rotation of the two ring moieties, the detected species differing in a near 180° rotation of pyridine. The final structure is modulated by steric effects. J.-U. Grabow, S. Mata, J. L. Alonso, I. Peña, S. Blanco, J. C. López, C. Cabezas, Phys. Chem. Chem. Phys. 2011, 13, 21063. A. Lesarri, E. J. Cocinero, L. Evangelisti, R. D. Suenram, W. Caminati, J.-U. Grabow, Chem. Eur. J. 2010, 16, 10214.

Photodissociation of conformer-selected ubiquitin ions reveals site-specific cis/trans isomerization of proline peptide bonds.

PubMed

Warnke, Stephan; Baldauf, Carsten; Bowers, Michael T; Pagel, Kevin; von Helden, Gert

2014-07-23

Ultraviolet photodissociation (UVPD) of gas-phase proteins has attracted increased attention in recent years. This growing interest is largely based on the fact that, in contrast to slow heating techniques such as collision induced dissociation (CID), the cleavage propensity after absorption of UV light is distributed over the entire protein sequence, which can lead to a very high sequence coverage as required in typical top-down proteomics applications. However, in the gas phase, proteins can adopt a multitude of distinct and sometimes coexisting conformations, and it is not clear how this three-dimensional structure affects the UVPD fragmentation behavior. Using ion mobility-UVPD-mass spectrometry in conjunction with molecular dynamics simulations, we provide the first experimental evidence that UVPD is sensitive to the higher order structure of gas-phase proteins. Distinct UVPD spectra were obtained for different extended conformations of 11(+) ubiquitin ions. Assignment of the fragments showed that the majority of differences arise from cis/trans isomerization of one particular proline peptide bond. Seen from a broader perspective, these data highlight the potential of UVPD to be used for the structural analysis of proteins in the gas phase.

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

PubMed

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

2009-10-22

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

CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations

PubMed Central

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

2017-01-01

CRISPR-Cas9 has become a facile genome editing technology, yet the structural and mechanistic features underlying its function are unclear. Here, we perform extensive molecular simulations in an enhanced sampling regime, using a Gaussian-accelerated molecular dynamics (GaMD) methodology, which probes displacements over hundreds of microseconds to milliseconds, to reveal the conformational dynamics of the endonuclease Cas9 during its activation toward catalysis. We disclose the conformational transition of Cas9 from its apo form to the RNA-bound form, suggesting a mechanism for RNA recruitment in which the domain relocations cause the formation of a positively charged cavity for nucleic acid binding. GaMD also reveals the conformation of a catalytically competent Cas9, which is prone for catalysis and whose experimental characterization is still limited. We show that, upon DNA binding, the conformational dynamics of the HNH domain triggers the formation of the active state, explaining how the HNH domain exerts a conformational control domain over DNA cleavage [Sternberg SH et al. (2015) Nature, 527, 110–113]. These results provide atomic-level information on the molecular mechanism of CRISPR-Cas9 that will inspire future experimental investigations aimed at fully clarifying the biophysics of this unique genome editing machinery and at developing new tools for nucleic acid manipulation based on CRISPR-Cas9. PMID:28652374

CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations.

PubMed

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

2017-07-11

CRISPR-Cas9 has become a facile genome editing technology, yet the structural and mechanistic features underlying its function are unclear. Here, we perform extensive molecular simulations in an enhanced sampling regime, using a Gaussian-accelerated molecular dynamics (GaMD) methodology, which probes displacements over hundreds of microseconds to milliseconds, to reveal the conformational dynamics of the endonuclease Cas9 during its activation toward catalysis. We disclose the conformational transition of Cas9 from its apo form to the RNA-bound form, suggesting a mechanism for RNA recruitment in which the domain relocations cause the formation of a positively charged cavity for nucleic acid binding. GaMD also reveals the conformation of a catalytically competent Cas9, which is prone for catalysis and whose experimental characterization is still limited. We show that, upon DNA binding, the conformational dynamics of the HNH domain triggers the formation of the active state, explaining how the HNH domain exerts a conformational control domain over DNA cleavage [Sternberg SH et al. (2015) Nature , 527 , 110-113]. These results provide atomic-level information on the molecular mechanism of CRISPR-Cas9 that will inspire future experimental investigations aimed at fully clarifying the biophysics of this unique genome editing machinery and at developing new tools for nucleic acid manipulation based on CRISPR-Cas9.

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

PubMed Central

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

2016-01-01

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

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

DOE PAGES

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

2016-11-03

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

Experimental and theoretical IR study of methyl thioglycolate, CH3OC(O)CH2SH, in different phases: Evidence of a dimer formation

NASA Astrophysics Data System (ADS)

Bava, Yanina B.; Tamone, Luciana M.; Juncal, Luciana C.; Seng, Samantha; Tobón, Yeny A.; Sobanska, Sophie; Picone, A. Lorena; Romano, Rosana M.

2017-07-01

The IR spectrum of methyl thioglycolate (MTG) was studied in three different phases, and interpreted with the aid of DFT calculations. The gas phase IR spectrum was explainable by the presence of the most stable conformer (syn-gauche-(-)gauche) only, while the IR spectrum of the liquid reveals strong intermolecular interactions, coincident with the formation of a dimeric form. The matrix-isolated spectra allow the identification of the second conformer (syn-gauche-gauche), in addition to the most stable form. The MTG dimer was also isolated by increasing the proportion of MTG in the matrix. The theoretical most stable structure of the dimer, which calculated IR spectrum agrees very well with the experimental one, is stabilized by a double interaction of the lone pair of the O atom of each of the Cdbnd O groups with the antibonding orbitals σ* (Ssbnd H).

Role of gender norms and group identification on hypothetical and experimental pain tolerance.

PubMed

Pool, Gregory J; Schwegler, Andria F; Theodore, Brian R; Fuchs, Perry N

2007-05-01

Previous research indicates that men typically tolerate more pain in experimental settings than women. One likely explanation for these group differences in pain tolerance is conformity to traditional, gender group social norms (i.e., the ideal man is masculine and tolerates more pain; the ideal woman is feminine and tolerates less pain). According to self-categorization theory, norms guide behavior to the degree that group members adopt the group identity. Therefore, high-identifying men are expected to conform to gender norms and tolerate more pain than high-identifying women who conform to different gender norms as a guide for their behavior. We conducted two studies to investigate whether gender group identification moderates individuals' conformity to pain tolerance and reporting norms. In the first study, participants indicated their gender identification and expected tolerance of a hypothetical painful stimulus. As anticipated, high-identifying men reported significantly greater pain tolerance than high-identifying women. No differences existed between low-identifying men and women. To determine if self-reported pain tolerance in a role-playing scenario corresponds to actual pain tolerance in an experimental setting, the second study examined pain tolerance to a noxious stimulus induced by electrical stimulation of the index finger. The experimental outcome revealed that high-identifying men tolerated more painful stimulation than high-identifying women. Further, high-identifying men tolerated more pain than low-identifying men. These results highlight the influence of social norms on behavior and suggest the need to further explore the role of norms in pain reporting behaviors.

Arabidopsis thaliana dehydroascorbate reductase 2: Conformational flexibility during catalysis

NASA Astrophysics Data System (ADS)

Bodra, Nandita; Young, David; Astolfi Rosado, Leonardo; Pallo, Anna; Wahni, Khadija; de Proft, Frank; Huang, Jingjing; van Breusegem, Frank; Messens, Joris

2017-02-01

Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational differences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release.

Arabidopsis thaliana dehydroascorbate reductase 2: Conformational flexibility during catalysis

PubMed Central

Bodra, Nandita; Young, David; Astolfi Rosado, Leonardo; Pallo, Anna; Wahni, Khadija; De Proft, Frank; Huang, Jingjing; Van Breusegem, Frank; Messens, Joris

2017-01-01

Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational differences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release. PMID:28195196

Free-energy simulations reveal molecular mechanism for functional switch of a DNA helicase

PubMed Central

Ma, Wen; Whitley, Kevin D; Schulten, Klaus

2018-01-01

Helicases play key roles in genome maintenance, yet it remains elusive how these enzymes change conformations and how transitions between different conformational states regulate nucleic acid reshaping. Here, we developed a computational technique combining structural bioinformatics approaches and atomic-level free-energy simulations to characterize how the Escherichia coli DNA repair enzyme UvrD changes its conformation at the fork junction to switch its function from unwinding to rezipping DNA. The lowest free-energy path shows that UvrD opens the interface between two domains, allowing the bound ssDNA to escape. The simulation results predict a key metastable 'tilted' state during ssDNA strand switching. By simulating FRET distributions with fluorophores attached to UvrD, we show that the new state is supported quantitatively by single-molecule measurements. The present study deciphers key elements for the 'hyper-helicase' behavior of a mutant and provides an effective framework to characterize directly structure-function relationships in molecular machines. PMID:29664402

Free-energy simulations reveal molecular mechanism for functional switch of a DNA helicase.

PubMed

Ma, Wen; Whitley, Kevin D; Chemla, Yann R; Luthey-Schulten, Zaida; Schulten, Klaus

2018-04-17

Helicases play key roles in genome maintenance, yet it remains elusive how these enzymes change conformations and how transitions between different conformational states regulate nucleic acid reshaping. Here, we developed a computational technique combining structural bioinformatics approaches and atomic-level free-energy simulations to characterize how the Escherichia coli DNA repair enzyme UvrD changes its conformation at the fork junction to switch its function from unwinding to rezipping DNA. The lowest free-energy path shows that UvrD opens the interface between two domains, allowing the bound ssDNA to escape. The simulation results predict a key metastable 'tilted' state during ssDNA strand switching. By simulating FRET distributions with fluorophores attached to UvrD, we show that the new state is supported quantitatively by single-molecule measurements. The present study deciphers key elements for the 'hyper-helicase' behavior of a mutant and provides an effective framework to characterize directly structure-function relationships in molecular machines. © 2018, Ma et al.

Pyruvate Dehydrogenase Kinase-4 Structures Reveal a Metastable Open Conformation Fostering Robust Core-free Basal Activity

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

Wynn, R. Max; Kato, Masato; Chuang, Jacinta L.

2008-10-21

Human pyruvate dehydrogenase complex (PDC) is down-regulated by pyruvate dehydrogenase kinase (PDK) isoforms 1-4. PDK4 is overexpressed in skeletal muscle in type 2 diabetes, resulting in impaired glucose utilization. Here we show that human PDK4 has robust core-free basal activity, which is considerably higher than activity levels of other PDK isoforms stimulated by the PDC core. PDK4 binds the L3 lipoyl domain, but its activity is not significantly stimulated by any individual lipoyl domains or the core of PDC. The 2.0-{angstrom} crystal structures of the PDK4 dimer with bound ADP reveal an open conformation with a wider active-site cleft, comparedmore » with that in the closed conformation epitomized by the PDK2-ADP structure. The open conformation in PDK4 shows partially ordered C-terminal cross-tails, in which the conserved DW (Asp{sup 394}-Trp{sup 395}) motif from one subunit anchors to the N-terminal domain of the other subunit. The open conformation fosters a reduced binding affinity for ADP, facilitating the efficient removal of product inhibition by this nucleotide. Alteration or deletion of the DW-motif disrupts the C-terminal cross-tail anchor, resulting in the closed conformation and the nearly complete inactivation of PDK4. Fluorescence quenching and enzyme activity data suggest that compounds AZD7545 and dichloroacetate lock PDK4 in the open and the closed conformational states, respectively. We propose that PDK4 with bound ADP exists in equilibrium between the open and the closed conformations. The favored metastable open conformation is responsible for the robust basal activity of PDK4 in the absence of the PDC core.« less

Ligand binding turns moth pheromone-binding protein into a pH sensor: effect on the Antheraea polyphemus PBP1 conformation.

PubMed

Katre, Uma V; Mazumder, Suman; Prusti, Rabi K; Mohanty, Smita

2009-11-13

In moths, pheromone-binding proteins (PBPs) are responsible for the transport of the hydrophobic pheromones to the membrane-bound receptors across the aqueous sensillar lymph. We report here that recombinant Antheraea polyphemus PBP1 (ApolPBP1) picks up hydrophobic molecule(s) endogenous to the Escherichia coli expression host that keeps the protein in the "open" (bound) conformation at high pH but switches to the "closed" (free) conformation at low pH. This finding has bearing on the solution structures of undelipidated lepidopteran moth PBPs determined thus far. Picking up a hydrophobic molecule from the host expression system could be a common feature for lipid-binding proteins. Thus, delipidation is critical for bacterially expressed lipid-binding proteins. We have shown for the first time that the delipidated ApolPBP1 exists primarily in the closed form at all pH levels. Thus, current views on the pH-induced conformational switch of PBPs hold true only for the ligand-bound open conformation of the protein. Binding of various ligands to delipidated ApolPBP1 studied by solution NMR revealed that the protein in the closed conformation switches to the open conformation only at or above pH 6.0 with a protein to ligand stoichiometry of approximately 1:1. Mutation of His(70) and His(95) to alanine drives the equilibrium toward the open conformation even at low pH for the ligand-bound protein by eliminating the histidine-dependent pH-induced conformational switch. Thus, the delipidated double mutant can bind ligand even at low pH in contrast to the wild type protein as revealed by fluorescence competitive displacement assay using 1-aminoanthracene and solution NMR.

Pyruvate dehydrogenase kinase-4 structures reveal a metastable open conformation fostering robust core-free basal activity.

PubMed

Wynn, R Max; Kato, Masato; Chuang, Jacinta L; Tso, Shih-Chia; Li, Jun; Chuang, David T

2008-09-12

Human pyruvate dehydrogenase complex (PDC) is down-regulated by pyruvate dehydrogenase kinase (PDK) isoforms 1-4. PDK4 is overexpressed in skeletal muscle in type 2 diabetes, resulting in impaired glucose utilization. Here we show that human PDK4 has robust core-free basal activity, which is considerably higher than activity levels of other PDK isoforms stimulated by the PDC core. PDK4 binds the L3 lipoyl domain, but its activity is not significantly stimulated by any individual lipoyl domains or the core of PDC. The 2.0-A crystal structures of the PDK4 dimer with bound ADP reveal an open conformation with a wider active-site cleft, compared with that in the closed conformation epitomized by the PDK2-ADP structure. The open conformation in PDK4 shows partially ordered C-terminal cross-tails, in which the conserved DW (Asp(394)-Trp(395)) motif from one subunit anchors to the N-terminal domain of the other subunit. The open conformation fosters a reduced binding affinity for ADP, facilitating the efficient removal of product inhibition by this nucleotide. Alteration or deletion of the DW-motif disrupts the C-terminal cross-tail anchor, resulting in the closed conformation and the nearly complete inactivation of PDK4. Fluorescence quenching and enzyme activity data suggest that compounds AZD7545 and dichloroacetate lock PDK4 in the open and the closed conformational states, respectively. We propose that PDK4 with bound ADP exists in equilibrium between the open and the closed conformations. The favored metastable open conformation is responsible for the robust basal activity of PDK4 in the absence of the PDC core.

A Conformance Test Suite for Arden Syntax Compilers and Interpreters.

PubMed

Wolf, Klaus-Hendrik; Klimek, Mike

2016-01-01

The Arden Syntax for Medical Logic Modules is a standardized and well-established programming language to represent medical knowledge. To test the compliance level of existing compilers and interpreters no public test suite exists. This paper presents the research to transform the specification into a set of unit tests, represented in JUnit. It further reports on the utilization of the test suite testing four different Arden Syntax processors. The presented and compared results reveal the status conformance of the tested processors. How test driven development of Arden Syntax processors can help increasing the compliance with the standard is described with two examples. In the end some considerations how an open source test suite can improve the development and distribution of the Arden Syntax are presented.

The Conformational Flexibility of the Acyltransferase from the Disorazole Polyketide Synthase Is Revealed by an X-ray Free-Electron Laser Using a Room-Temperature Sample Delivery Method for Serial Crystallography

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

Mathews, Irimpan I.; Allison, Kim; Robbins, Thomas

The crystal structure of the trans-acyltransferase (AT) from the disorazole polyketide synthase (PKS) was determined at room temperature to a resolution of 2.5 Å using a new method for sample delivery directly into an X-ray free-electron laser. A novel sample extractor efficiently delivered limited quantities of microcrystals directly from the native crystallization solution into the X-ray beam at room temperature. The AT structure revealed important catalytic features of this core PKS enzyme, including the occurrence of conformational changes around the active site. The implications of these conformational changes on polyketide synthase reaction dynamics are discussed.

The Conformational Flexibility of the Acyltransferase from the Disorazole Polyketide Synthase Is Revealed by an X-ray Free-Electron Laser Using a Room-Temperature Sample Delivery Method for Serial Crystallography

DOE PAGES

Mathews, Irimpan I.; Allison, Kim; Robbins, Thomas; ...

2017-08-23

The crystal structure of the trans-acyltransferase (AT) from the disorazole polyketide synthase (PKS) was determined at room temperature to a resolution of 2.5 Å using a new method for sample delivery directly into an X-ray free-electron laser. A novel sample extractor efficiently delivered limited quantities of microcrystals directly from the native crystallization solution into the X-ray beam at room temperature. The AT structure revealed important catalytic features of this core PKS enzyme, including the occurrence of conformational changes around the active site. The implications of these conformational changes on polyketide synthase reaction dynamics are discussed.

First-principles anharmonic quantum calculations for peptide spectroscopy: VSCF calculations and comparison with experiments.

PubMed

Roy, Tapta Kanchan; Sharma, Rahul; Gerber, R Benny

2016-01-21

First-principles quantum calculations for anharmonic vibrational spectroscopy of three protected dipeptides are carried out and compared with experimental data. Using hybrid HF/MP2 potentials, the Vibrational Self-Consistent Field with Second-Order Perturbation Correction (VSCF-PT2) algorithm is used to compute the spectra without any ad hoc scaling or fitting. All of the vibrational modes (135 for the largest system) are treated quantum mechanically and anharmonically using full pair-wise coupling potentials to represent the interaction between different modes. In the hybrid potential scheme the MP2 method is used for the harmonic part of the potential and a modified HF method is used for the anharmonic part. The overall agreement between computed spectra and experiment is very good and reveals different signatures for different conformers. This study shows that first-principles spectroscopic calculations of good accuracy are possible for dipeptides hence it opens possibilities for determination of dipeptide conformer structures by comparison of spectroscopic calculations with experiment.

Structure of the vacuolar H+-ATPase rotary motor reveals new mechanistic insights.

PubMed

Rawson, Shaun; Phillips, Clair; Huss, Markus; Tiburcy, Felix; Wieczorek, Helmut; Trinick, John; Harrison, Michael A; Muench, Stephen P

2015-03-03

Vacuolar H(+)-ATPases are multisubunit complexes that operate with rotary mechanics and are essential for membrane proton transport throughout eukaryotes. Here we report a ∼ 1 nm resolution reconstruction of a V-ATPase in a different conformational state from that previously reported for a lower-resolution yeast model. The stator network of the V-ATPase (and by implication that of other rotary ATPases) does not change conformation in different catalytic states, and hence must be relatively rigid. We also demonstrate that a conserved bearing in the catalytic domain is electrostatic, contributing to the extraordinarily high efficiency of rotary ATPases. Analysis of the rotor axle/membrane pump interface suggests how rotary ATPases accommodate different c ring stoichiometries while maintaining high efficiency. The model provides evidence for a half channel in the proton pump, supporting theoretical models of ion translocation. Our refined model therefore provides new insights into the structure and mechanics of the V-ATPases. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Structural Basis of Immune Evasion at the Site of CD4 Attachment on HIV-1 gp120

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

Chen, Lei; Kwon, Young Do; Zhou, Tongqing

2010-01-13

The site on HIV-1 gp120 that binds to the CD4 receptor is vulnerable to antibodies. However, most antibodies that interact with this site cannot neutralize HIV-1. To understand the basis of this resistance, we determined co-crystal structures for two poorly neutralizing, CD4-binding site (CD4BS) antibodies, F105 and b13, in complexes with gp120. Both antibodies exhibited approach angles to gp120 similar to those of CD4 and a rare, broadly neutralizing CD4BS antibody, b12. Slight differences in recognition, however, resulted in substantial differences in F105- and b13-bound conformations relative to b12-bound gp120. Modeling and binding experiments revealed these conformations to be poorlymore » compatible with the viral spike. This incompatibility, the consequence of slight differences in CD4BS recognition, renders HIV-1 resistant to all but the most accurately targeted antibodies.« less

"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.

Conversion of Natively Unstructured α-Synuclein to Its α-Helical Conformation Significantly Attenuates Production of Reactive Oxygen Species

PubMed Central

Zhou, Binbin; Hao, Yuanqiang; Wang, Chengshan; Li, Ding; Liu, You-Nian; Zhou, Feimeng

2012-01-01

The intracellular α-synuclein (α-syn) protein, whose conformational change and aggregation have been closely linked to the pathology of Parkingson’s disease (PD), is highly populated at the presynaptic termini and remains there in the α-helical conformation. In this study, circular dichroism confirmed that natively unstructured α-syn in aqueous solution was transformed to its α-helical conformation upon addition of trifluoroethanol (TFE). Electrochemical and UV–visible spectroscopic experiments reveal that both Cu(I) and Cu(II) are stabilized, with the former being stabilized by about two orders of magnitude. Compared to unstructured α-syn (Binolfi et al., J. Am. Chem. Soc. 133 (2011) 194–196), α-helical α-syn stabilizes Cu(I) by more than three orders of magnitude. Through the measurements of H2O2 and hydroxyl radicals (OH•) in solutions containing different forms of Cu(II) (free and complexed by unstructured or α-helical α-syn), we demonstrate that the significantly enhanced Cu(I) binding affinity helps inhibit the production of highly toxic reactive oxygen species, especially the hydroxyl radicals. Our study provides strong evidence that, as a possible means to prevent neuronal cell damage, conversion of the natively unstructured α-syn to its α-helical conformation in vivo could significantly attenuate the copper-modulated ROS production. PMID:23123341

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

PubMed

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

2012-01-01

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

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

PubMed

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

2016-10-26

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

Nearly reversible conformational change of amyloid fibrils as revealed by pH-jump experiments.

PubMed

Yamaguchi, Kei-ichi; Kamatari, Yuji O; Fukuoka, Mayuko; Miyaji, Reiji; Kuwata, Kazuo

2013-10-01

pH-jump induced conformational transitions between substates of preformed amyloid fibrils made by a fragmented peptide of helix 2 (H2 peptide) of MoPrP were detected, and their kinetics were analyzed using a novel pH-jump apparatus specially designed for observing amyloids. Previously, we reported that H2 peptide formed ordered fibrils with a minimum at 207 nm on CD spectra at pH 2.9 (named pH 2.9 fibrils), but formed aggregate-like fibrils with a minimum at 220 nm at pH 7.5 (named pH 7.5 fibrils). When pH-jump from 2.9 to 7.5 was performed, the CD spectrum changed instantly, but the finally observed ellipticities were clearly distinct from those of pH 7.5 fibrils. Thus, the finally observed state is termed 'pH 7.5-like fibrils'. However, pH 7.5-like fibrils reverted to the conformation very similar to that of the pH 2.9 fibrils when the pH of the solution was restored to 2.9. Then, we examined the kinetics of the nearly reversible conformational changes between pH 2.9 fibrils and pH 7.5-like fibrils using ANS fluorescence stopped-flow, and we observed relatively fast phases (0.7-18 s(-1)). In contrast, the conversion between pH 7.5-like fibrils and pH 7.5 fibrils never occurred (<0.2 day(-1)). Thus, H2 fibrils can be switched readily between distinct conformations separated by a low energy barrier, while a large energy barrier clearly separated the different conformations. These conformational varieties of amyloid fibrils may explain the physical basis of the diversity in prion.

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.

Evidence of native α-synuclein conformers in the human brain.

PubMed

Gould, Neal; Mor, Danielle E; Lightfoot, Richard; Malkus, Kristen; Giasson, Benoit; Ischiropoulos, Harry

2014-03-14

α-Synuclein aggregation is central to the pathogenesis of several brain disorders. However, the native conformations and functions of this protein in the human brain are not precisely known. The native state of α-synuclein was probed by gel filtration coupled with native gradient gel separation, an array of antibodies with non-overlapping epitopes, and mass spectrometry. The existence of metastable conformers and stable monomer was revealed in the human brain.

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

PubMed

Desdouits, Nathan; Nilges, Michael; Blondel, Arnaud

2015-02-01

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

Distribution, transition and thermodynamic stability of protein conformations in the denaturant-induced unfolding of proteins.

PubMed

Bian, Liujiao; Ji, Xu

2014-01-01

Extensive and intensive studies on the unfolding of proteins require appropriate theoretical model and parameter to clearly illustrate the feature and characteristic of the unfolding system. Over the past several decades, four approaches have been proposed to describe the interaction between proteins and denaturants, but some ambiguity and deviations usually occur in the explanation of the experimental data. In this work, a theoretical model was presented to show the dependency of the residual activity ratio of the proteins on the molar denaturant concentration. Through the characteristic unfolding parameters ki and Δmi in this model, the distribution, transition and thermodynamic stability of protein conformations during the unfolding process can be quantitatively described. This model was tested with the two-state unfolding of bovine heart cytochrome c and the three-state unfolding of hen egg white lysozyme induced by both guanidine hydrochloride and urea, the four-state unfolding of bovine carbonic anhydrase b induced by guanidine hydrochloride and the unfolding of some other proteins induced by denaturants. The results illustrated that this model could be used accurately to reveal the distribution and transition of protein conformations in the presence of different concentrations of denaturants and to evaluate the unfolding tendency and thermodynamic stability of different conformations. In most denaturant-induced unfolding of proteins, the unfolding became increasingly hard in next transition step and the proteins became more unstable as they attained next successive stable conformation. This work presents a useful method for people to study the unfolding of proteins and may be used to describe the unfolding and refolding of other biopolymers induced by denaturants, inducers, etc.

Nucleic Acid-Dependent Conformational Changes in CRISPR-Cas9 Revealed by Site-Directed Spin Labeling.

PubMed

Vazquez Reyes, Carolina; Tangprasertchai, Narin S; Yogesha, S D; Nguyen, Richard H; Zhang, Xiaojun; Rajan, Rakhi; Qin, Peter Z

2017-06-01

In a type II clustered regularly interspaced short palindromic repeats (CRISPR) system, RNAs that are encoded at the CRISPR locus complex with the CRISPR-associated (Cas) protein Cas9 to form an RNA-guided nuclease that cleaves double-stranded DNAs at specific sites. In recent years, the CRISPR-Cas9 system has been successfully adapted for genome engineering in a wide range of organisms. Studies have indicated that a series of conformational changes in Cas9, coordinated by the RNA and the target DNA, direct the protein into its active conformation, yet details on these conformational changes, as well as their roles in the mechanism of function of Cas9, remain to be elucidated. Here, nucleic acid-dependent conformational changes in Streptococcus pyogenes Cas9 (SpyCas9) were investigated using the method of site-directed spin labeling (SDSL). Single nitroxide spin labels were attached, one at a time, at one of the two native cysteine residues (Cys80 and Cys574) of SpyCas9, and the spin-labeled proteins were shown to maintain their function. X-band continuous-wave electron paramagnetic resonance spectra of the nitroxide attached at Cys80 revealed conformational changes of SpyCas9 that are consistent with a large-scale domain re-arrangement upon binding to its RNA partner. The results demonstrate the use of SDSL to monitor conformational changes in CRISPR-Cas9, which will provide key information for understanding the mechanism of CRISPR function.

Revision of orthovoltage chest wall treatment using Monte Carlo simulations.

PubMed

Zeinali-Rafsanjani, B; Faghihi, R; Mosleh-Shirazi, M A; Mosalaei, A; Hadad, K

2017-01-01

Given the high local control rates observed in breast cancer patients undergoing chest wall irradiation by kilovoltage x-rays, we aimed to revisit this treatment modality by accurate calculation of dose distributions using Monte Carlo simulation. The machine components were simulated using the MCNPX code. This model was used to assess the dose distribution of chest wall kilovoltage treatment in different chest wall thicknesses and larger contour or fat patients in standard and mid sternum treatment plans. Assessments were performed at 50 and 100 cm focus surface distance (FSD) and different irradiation angles. In order to evaluate different plans, indices like homogeneity index, conformity index, the average dose of heart, lung, left anterior descending artery (LAD) and percentage target coverage (PTC) were used. Finally, the results were compared with the indices provided by electron therapy which is a more routine treatment of chest wall. These indices in a medium chest wall thickness in standard treatment plan at 50 cm FSD and 15 degrees tube angle was as follows: homogeneity index 2.57, conformity index 7.31, average target dose 27.43 Gy, average dose of heart, lung and LAD, 1.03, 2.08 and 1.60 Gy respectively and PTC 11.19%. Assessments revealed that dose homogeneity in planning target volume (PTV) and conformity between the high dose region and PTV was poor. To improve the treatment indices, the reference point was transferred from the chest wall skin surface to the center of PTV. The indices changed as follows: conformity index 7.31, average target dose 60.19 Gy, the average dose of heart, lung and LAD, 3.57, 6.38 and 5.05 Gy respectively and PTC 55.24%. Coverage index of electron therapy was 89% while it was 22.74% in the old orthovoltage method and also the average dose of the target was about 50 Gy but in the given method it was almost 30 Gy. The results of the treatment study show that the optimized standard and mid sternum treatment for different chest wall thicknesses is with 50 cm FSD and zero (vertical) tube angle, while in large contour patients, it is with 100 cm FSD and zero tube angle. Finally, chest wall kilovoltage and electron therapies were compared, which revealed that electron therapy produces a better dose distribution than kilovoltage therapy.

Evolutionary conservation of the polyproline II conformation surrounding intrinsically disordered phosphorylation sites.

PubMed

Elam, W Austin; Schrank, Travis P; Campagnolo, Andrew J; Hilser, Vincent J

2013-04-01

Intrinsically disordered (ID) proteins function in the absence of a unique stable structure and appear to challenge the classic structure-function paradigm. The extent to which ID proteins take advantage of subtle conformational biases to perform functions, and whether signals for such mechanism can be identified in proteome-wide studies is not well understood. Of particular interest is the polyproline II (PII) conformation, suggested to be highly populated in unfolded proteins. We experimentally determine a complete calorimetric propensity scale for the PII conformation. Projection of the scale into representative eukaryotic proteomes reveals significant PII bias in regions coding for ID proteins. Importantly, enrichment of PII in ID proteins, or protein segments, is also captured by other PII scales, indicating that this enrichment is robustly encoded and universally detectable regardless of the method of PII propensity determination. Gene ontology (GO) terms obtained using our PII scale and other scales demonstrate a consensus for molecular functions performed by high PII proteins across the proteome. Perhaps the most striking result of the GO analysis is conserved enrichment (P < 10(-8) ) of phosphorylation sites in high PII regions found by all PII scales. Subsequent conformational analysis reveals a phosphorylation-dependent modulation of PII, suggestive of a conserved "tunability" within these regions. In summary, the application of an experimentally determined polyproline II (PII) propensity scale to proteome-wide sequence analysis and gene ontology reveals an enrichment of PII bias near disordered phosphorylation sites that is conserved throughout eukaryotes. Copyright © 2013 The Protein Society.

Political Implications of Heavy Television Viewing.

ERIC Educational Resources Information Center

Jackson-Beeck, Marilyn

This paper empirically evaluates the proposition that political conformism, specifically structural, passive, psychological, and defensive conformism, is a function of exposure to mass media. Secondary analysis of data from the National Opinion Research Center's 1975 General Social Survey revealed a significant relationship between TV viewing and…

NMR Structural Profiling of Transcriptional Intermediates Reveals Riboswitch Regulation by Metastable RNA Conformations.

PubMed

Helmling, Christina; Wacker, Anna; Wolfinger, Michael T; Hofacker, Ivo L; Hengesbach, Martin; Fürtig, Boris; Schwalbe, Harald

2017-02-22

Gene repression induced by the formation of transcriptional terminators represents a prime example for the coupling of RNA synthesis, folding, and regulation. In this context, mapping the changes in available conformational space of transcription intermediates during RNA synthesis is important to understand riboswitch function. A majority of riboswitches, an important class of small metabolite-sensing regulatory RNAs, act as transcriptional regulators, but the dependence of ligand binding and the subsequent allosteric conformational switch on mRNA transcript length has not yet been investigated. We show a strict fine-tuning of binding and sequence-dependent alterations of conformational space by structural analysis of all relevant transcription intermediates at single-nucleotide resolution for the I-A type 2'dG-sensing riboswitch from Mesoplasma florum by NMR spectroscopy. Our results provide a general framework to dissect the coupling of synthesis and folding essential for riboswitch function, revealing the importance of metastable states for RNA-based gene regulation.

Non-destructive analysis of the conformational differences among feedstock sources and their corresponding co-products from bioethanol production with molecular spectroscopy.

PubMed

Gamage, I H; Jonker, A; Zhang, X; Yu, P

2014-01-24

The objective of this study was to determine the possibility of using molecular spectroscopy with multivariate technique as a fast method to detect the source effects among original feedstock sources of wheat and their corresponding co-products, wheat DDGS, from bioethanol production. Different sources of the bioethanol feedstock and their corresponding bioethanol co-products, three samples per source, were collected from the same newly-built bioethanol plant with current bioethanol processing technology. Multivariate molecular spectral analyses were carried out using agglomerative hierarchical cluster analysis (AHCA) and principal component analysis (PCA). The molecular spectral data of different feedstock sources and their corresponding co-products were compared at four different regions of ca. 1800-1725 cm(-1) (carbonyl CO ester, mainly related to lipid structure conformation), ca. 1725-1482 cm(-1) (amide I and amide II region mainly related to protein structure conformation), ca. 1482-1180 cm(-1) (mainly associated with structural carbohydrate) and ca. 1180-800 cm(-1) (mainly related to carbohydrates) in complex plant-based system. The results showed that the molecular spectroscopy with multivariate technique could reveal the structural differences among the bioethanol feedstock sources and among their corresponding co-products. The AHCA and PCA analyses were able to distinguish the molecular structure differences associated with chemical functional groups among the different sources of the feedstock and their corresponding co-products. The molecular spectral differences indicated the differences in functional, biomolecular and biopolymer groups which were confirmed by wet chemical analysis. These biomolecular and biopolymer structural differences were associated with chemical and nutrient profiles and nutrient utilization and availability. Molecular spectral analyses had the potential to identify molecular structure difference among bioethanol feedstock sources and their corresponding co-products. Copyright © 2013 Elsevier B.V. All rights reserved.

Non-destructive analysis of the conformational differences among feedstock sources and their corresponding co-products from bioethanol production with molecular spectroscopy

NASA Astrophysics Data System (ADS)

Gamage, I. H.; Jonker, A.; Zhang, X.; Yu, P.

2014-01-01

The objective of this study was to determine the possibility of using molecular spectroscopy with multivariate technique as a fast method to detect the source effects among original feedstock sources of wheat and their corresponding co-products, wheat DDGS, from bioethanol production. Different sources of the bioethanol feedstock and their corresponding bioethanol co-products, three samples per source, were collected from the same newly-built bioethanol plant with current bioethanol processing technology. Multivariate molecular spectral analyses were carried out using agglomerative hierarchical cluster analysis (AHCA) and principal component analysis (PCA). The molecular spectral data of different feedstock sources and their corresponding co-products were compared at four different regions of ca. 1800-1725 cm-1 (carbonyl Cdbnd O ester, mainly related to lipid structure conformation), ca. 1725-1482 cm-1 (amide I and amide II region mainly related to protein structure conformation), ca. 1482-1180 cm-1 (mainly associated with structural carbohydrate) and ca. 1180-800 cm-1 (mainly related to carbohydrates) in complex plant-based system. The results showed that the molecular spectroscopy with multivariate technique could reveal the structural differences among the bioethanol feedstock sources and among their corresponding co-products. The AHCA and PCA analyses were able to distinguish the molecular structure differences associated with chemical functional groups among the different sources of the feedstock and their corresponding co-products. The molecular spectral differences indicated the differences in functional, biomolecular and biopolymer groups which were confirmed by wet chemical analysis. These biomolecular and biopolymer structural differences were associated with chemical and nutrient profiles and nutrient utilization and availability. Molecular spectral analyses had the potential to identify molecular structure difference among bioethanol feedstock sources and their corresponding co-products.

A Triad of Molecular Regions Contribute to the Formation of Two Distinct MHC Class II Conformers

PubMed Central

Drake, Lisa A.; Drake, James R.

2016-01-01

MHC class II molecules present antigen-derived peptides to CD4 T cells to drive the adaptive immune response. Previous work has established that class II αβ dimers can adopt two distinct conformations, driven by the differential pairing of transmembrane domain GxxxG dimerization motifs. These class II conformers differ in their ability to be loaded with antigen-derived peptide and to effectively engage CD4 T cells. Motif 1 (M1) paired I-Ak class II molecules are efficiently loaded with peptides derived from the processing of B cell receptor-bound antigen, have unique B cell signaling properties and high T cell stimulation activity. The 11-5.2 mAb selectively binds M1 paired I-Ak class II molecules. However, the molecular determinants of 11-5.2 binding are currently unclear. Here, we report the ability of a human class II transmembrane domain to drive both M1 and M2 class II conformer formation. Protease sensitivity analysis further strengthens the idea that there are conformational differences between the extracellular domains of M1 and M2 paired class II. Finally, MHC class II chain alignments and site directed mutagenesis reveals a triad of molecular regions that contributes to 11-5.2 mAb binding. In addition to transmembrane GxxxG motif domain pairing, 11-5.2 binding is influenced directly by α chain residue Glu-71 and indirectly by the region around the inter-chain salt bridge formed by α chain Arg-52 and β chain Glu-86. These findings provide insight into the complexity of 11-5.2 mAb recognition of the M1 paired I-Ak class II conformer and further highlight the molecular heterogeneity of peptide-MHC class II complexes that drive T cell antigen recognition. PMID:27148821

An Accurate Method for Measuring Airplane-Borne Conformal Antenna's Radar Cross Section

NASA Astrophysics Data System (ADS)

Guo, Shuxia; Zhang, Lei; Wang, Yafeng; Hu, Chufeng

2016-09-01

The airplane-borne conformal antenna attaches itself tightly with the airplane skin, so the conventional measurement method cannot determine the contribution of the airplane-borne conformal antenna to its radar cross section (RCS). This paper uses the 2D microwave imaging to isolate and extract the distribution of the reflectivity of the airplane-borne conformal antenna. It obtains the 2D spatial spectra of the conformal antenna through the wave spectral transform between the 2D spatial image and the 2D spatial spectrum. After the interpolation from the rectangular coordinate domain to the polar coordinate domain, the spectral domain data for the variation of the scatter of the conformal antenna with frequency and angle is obtained. The experimental results show that the measurement method proposed in this paper greatly enhances the airplane-borne conformal antenna's RCS measurement accuracy, essentially eliminates the influences caused by the airplane skin and more accurately reveals the airplane-borne conformal antenna's RCS scatter properties.

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

Spectral analysis and quantum chemical studies of chair and twist-boat conformers of cycloheximide in gas and solution phases

NASA Astrophysics Data System (ADS)

Tokatli, A.; Ucun, F.; Sütçü, K.; Osmanoğlu, Y. E.; Osmanoğlu, Ş.

2018-02-01

In this study the conformational behavior of cycloheximide in the gas and solution (CHCl3) phases has theoretically been investigated by spectroscopic and quantum chemical properties using density functional theory (wB97X-D) method with 6-31++G(d,p) basis set, for the first time. The calculated IR results reveal that in the ground state the molecule exits as a mixture of the chair and twist-boat conformers in the gas phase, while the calculated NMR results reveal that it only exits as the chair conformer in the solution phase. In order to obtain the contributions coming from intramolecular interactions to the stability of the conformers in the gas and solution phases, the quantum theory of atoms in molecules (QTAIM), noncovalent interactions (NCI) method, and natural bond orbital analysis (NBO) have been employed. The QTAIM and NCI methods indicated that by intramolecular interactions with bond critical point (BCP) the twist-boat conformer is more stabilized than the chair conformer, while by steric interactions it is more destabilized. Considering that these interactions balance each other, the stabilities of the conformers are understood to be dictated by the van der Waals interactions. The NBO analyses show that the hyperconjugative and steric effects play an important role in the stabilization in the gas and solution phases. Furthermore, to get a better understanding of the chemical behavior of this important antibiotic drug we have evaluated and, commented the global and local reactivity descriptors of the both conformers. Finally, the EPR analysis of γ-irradiated cycloheximide has been done. The comparison of the experimental and calculated data have showed the inducement of a radical structure of (CH2)2ĊCH2 in the molecule. The experimental EPR spectrum has also confirmed that the molecule simultaneously exists in the chair and twist-boat conformers in the solid phase.

[High frequency of ancestral allele of the TJP1 polymorphism rs2291166 in Mexican population, conformational effect and applications in surgery and medicine].

PubMed

Ramirez-Garcia, Sergio Alberto; Flores-Alvarado, Luis Javier; Topete-González, Luz Rosalba; Charles-Niño, Claudia; Mazariegos-Rubi, Manuel; Dávalos-Rodríguez, Nory Omayra

2016-01-01

TJP1 gene encodes a ZO-1 protein that is required for the recruitment of occludins and claudins in tight junction, and is involved in cell polarisation. It has different variations, the frequency of which has been studied in different populations. In Mexico there are no studies of this gene. These are required because their polymorphisms can be used in studies associated with medicine and surgery. Therefore, the aim of this study was to estimate the frequency of alleles and genotypes of rs2291166 gene polymorphism TJP1 in Mexico Mestizos population, and to estimate the conformational effect of an amino acid change. A total of 473 individuals were included. The rs2291166 polymorphism was identified PASA PCR-7% PAGE, and stained with silver nitrate. The conformational effect of amino acid change was performed in silico, and was carried out with servers ProtPraram Tool and Search Database with Fasta. The most frequent allele in the two populations is the ancestral allele (T). A genotype distribution similar to other populations was found. The polymorphism is in Hardy-Weinberg, p>0.05. Changing aspartate to alanine produced a conformational change. The study reveals a high frequency of the ancestral allele at rs2291166 polymorphism in the Mexican population. Copyright © 2015 Academia Mexicana de Cirugía A.C. Published by Masson Doyma México S.A. All rights reserved.

Myotonic Dystrophy Type 1 RNA Crystal Structures Reveal Heterogeneous 1×1 Nucleotide UU Internal Loop Conformations⊥

PubMed Central

Kumar, Amit; Park, HaJeung; Fang, Pengfei; Parkesh, Raman; Guo, Min; Nettles, Kendall W.; Disney, Matthew D.

2011-01-01

RNA internal loops often display a variety of conformations in solution. Herein, we visualize conformational heterogeneity in the context of the 5′CUG/3′GUC repeat motif present in the RNA that causes myotonic dystrophy type 1 (DM1). Specifically, two crystal structures are disclosed of a model DM1 triplet repeating construct, 5′r(UUGGGC(CUG)3GUCC)2, refined to 2.20 Å and 1.52 Å resolution. Here, differences in orientation of the 5′ dangling UU end between the two structures induce changes in the backbone groove width, which reveals that non-canonical 1×1 nucleotide UU internal loops can display an ensemble of pairing conformations. In the 2.20 Å structure, CUGa, the 5′UU forms one hydrogen-bonded pairs with a 5′UU of a neighboring helix in the unit cell to form a pseudo-infinite helix. The central 1×1 nucleotide UU internal loop has no hydrogen bonds, while the terminal 1×1 nucleotide UU internal loops each form a one hydrogen-bonded pair. In the 1.52 Å structure, CUGb, the 5′ UU dangling end is tucked into the major groove of the duplex. While the canonical paired bases show no change in base pairing, in CUGb the terminal 1×1 nucleotide UU internal loops form now two hydrogen-bonded pairs. Thus, the shift in major groove induced by the 5′UU dangling end alters non-canonical base patterns. Collectively, these structures indicate that 1×1 nucleotide UU internal loops in DM1 may sample multiple conformations in vivo. This observation has implications for the recognition of this RNA, and other repeating transcripts, by protein and small molecule ligands. PMID:21988728

Aggregation-primed molten globule conformers of the p53 core domain provide potential tools for studying p53C aggregation in cancer.

PubMed

Pedrote, Murilo M; de Oliveira, Guilherme A P; Felix, Adriani L; Mota, Michelle F; Marques, Mayra de A; Soares, Iaci N; Iqbal, Anwar; Norberto, Douglas R; Gomes, Andre M O; Gratton, Enrico; Cino, Elio A; Silva, Jerson L

2018-05-31

The functionality of the tumor suppressor p53 is altered in more than 50% of human cancers, and many individuals with cancer exhibit amyloid-like buildups of aggregated p53. An understanding of what triggers the pathogenic amyloid conversion of p53 is required for the further development of cancer therapies. Here, perturbation of the p53 core domain (p53C) with sub-denaturing concentrations of guanidine hydrochloride and high hydrostatic pressure revealed native-like molten globule (MG) states, a subset of which were highly prone to amyloidogenic aggregation. We found that MG conformers of p53C, likely representing population-weighted averages of multiple states, have different volumetric properties, as determined by pressure perturbation and size-exclusion chromatography. We also found that they bind the fluorescent dye 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and have a native-like tertiary structure that occludes the single Trp residue in p53. Fluorescence experiments revealed conformational changes of the single Trp and Tyr residues before p53 unfolding and the presence of MG conformers, some of which were highly prone to aggregation. P53C exhibited marginal unfolding cooperativity, which could be modulated from unfolding to aggregation pathways with chemical or physical forces. We conclude that trapping amyloid precursor states in solution is a promising approach for understanding p53 aggregation in cancer. Our findings support the use of single-Trp fluorescence as a probe for evaluating p53 stability, effects of mutations, and the efficacy of therapeutics designed to stabilize p53. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

The evaluation of hospital laboratory information management systems based on the standards of the American National Standard Institute

PubMed Central

Isfahani, Sakineh Saghaeiannejad; Khajouei, Reza; Jahanbakhsh, Maryan; Mirmohamadi, Mahboubeh

2014-01-01

Introduction: Nowadays, modern laboratories are faced with a huge volume of information. One of the goals of the Laboratory Information Management System (LIMS) is to assist in the management of the information generated in the laboratory. This study intends to evaluate the LIMS based on the standards of the American National Standard Institute (ANSI). Materials and Methods: This research is a descriptive–analytical study, which had been conducted in 2011, on the LIMSs in use, in the teaching and private hospitals in Isfahan. The data collecting instrument was a checklist, which was made by evaluating three groups of information components namely: ‘System capabilities’, ‘work list functions,’ and ‘reporting’ based on LIS8-A. Data were analyzed using the SPSS 20. Data were analyzed using (relative) frequency, percentage. To compare the data the following statistical tests were used: Leven test, t-test, and Analysis of Variance (ANOVA). Results: The results of the study indicated that the LIMS had a low conformity (30%) with LIS8-A (P = 0.001), with no difference between teaching and private hospitals (P = 0.806). The ANOVA revealed that in terms of conformity with the LIS8-A standard, there was a significant difference between the systems produced by different vendors (P = 0.023). According to the results, a Kowsar system with more than %57 conformity in the three groups of information components had a better conformity to the standard, compared to the other systems. Conclusions: This study indicated that none of the LIMSs had a good conformity to the standard. It seems that system providers did not pay sufficient attention to many of the information components required by the standards when designing and developing their systems. It was suggested that standards from certified organizations and institutions be followed in the design and development process of health information systems. PMID:25077154

The evaluation of hospital laboratory information management systems based on the standards of the American National Standard Institute.

PubMed

Isfahani, Sakineh Saghaeiannejad; Khajouei, Reza; Jahanbakhsh, Maryan; Mirmohamadi, Mahboubeh

2014-01-01

Nowadays, modern laboratories are faced with a huge volume of information. One of the goals of the Laboratory Information Management System (LIMS) is to assist in the management of the information generated in the laboratory. This study intends to evaluate the LIMS based on the standards of the American National Standard Institute (ANSI). This research is a descriptive-analytical study, which had been conducted in 2011, on the LIMSs in use, in the teaching and private hospitals in Isfahan. The data collecting instrument was a checklist, which was made by evaluating three groups of information components namely: 'System capabilities', 'work list functions,' and 'reporting' based on LIS8-A. Data were analyzed using the SPSS 20. Data were analyzed using (relative) frequency, percentage. To compare the data the following statistical tests were used: Leven test, t-test, and Analysis of Variance (ANOVA). The results of the study indicated that the LIMS had a low conformity (30%) with LIS8-A (P = 0.001), with no difference between teaching and private hospitals (P = 0.806). The ANOVA revealed that in terms of conformity with the LIS8-A standard, there was a significant difference between the systems produced by different vendors (P = 0.023). According to the results, a Kowsar system with more than %57 conformity in the three groups of information components had a better conformity to the standard, compared to the other systems. This study indicated that none of the LIMSs had a good conformity to the standard. It seems that system providers did not pay sufficient attention to many of the information components required by the standards when designing and developing their systems. It was suggested that standards from certified organizations and institutions be followed in the design and development process of health information systems.

Axial coordination and conformational heterogeneity of nickel(II) tetraphenylprophyrin complexes with nitrogenous bases

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

Jia, S.L.; Song, X.Z.; Ma, J.G.

1998-08-24

Axial ligation of nickel(II) 5,10,15,20-tetraphenylporphyrin (NiTPP) with pyrrolidine or piperidine has been investigated using X-ray crystallography, UV-visible spectroscopy, resonance Raman spectroscopy, and molecular mechanics (MM) calculations. Distinct v{sub 4} Raman lines are found for the 4-, 5-, and 6-coordinate species of NiTPP. The equilibrium constants for addition of the first and second pyrrolidine axial ligands are 1.1 and 3.8 M{sup {minus}1}, respectively. The differences in the calculated energies of the conformers having different ligand rotational angles are small so they may coexist in solution. Because of the similarity in macrocyclic structural parameters of these conformers and the free rotation ofmore » the axial ligands, narrow and symmetric v{sub 2} and v{sub 8} Raman lines are observed. Nonetheless, the normal-coordinate structural-decomposition analysis of the nonplanar distortions of the calculated structures and the crystal structure of the bis(piperidine) complex reveals a relationship between the orientations of axial ligand(s) and the macrocyclic distortions. For the 5-coordinate complex with the plane of the axial ligand bisecting the Ni-N{sub pyrrole} bonds, a primarily ruffled deformation results. With the ligand plane eclipsing the Ni-N{sub pyrrole} bonds, a mainly saddled deformation occurs. With the addition of the second axial ligand, the small doming of the 5-coordinate complexes disappears, and ruffling or saddling deformations change depending on the relative orientation of the two axial ligands. The crystal structure of the NiTPP bis(piperidine) complex shows a macrocycle distortion composed of wav(x) and wav(y) symmetric deformations, but no ruffling, saddling, or doming. The difference in the calculated and observed distortions results partly from the phenyl group orientation imposed by crystal packing forces. MM calculations predict three stable conformers (ruf, sad, and planar) for 4-coordinate NiTPP, and resonance Raman evidence for these conformers was given previously.« less

The nucleotide-free state of heterotrimeric G proteins α-subunit adopts a highly stable conformation.

PubMed

Andhirka, Sai Krishna; Vignesh, Ravichandran; Aradhyam, Gopala Krishna

2017-08-01

Deciphering the mechanism of activation of heterotrimeric G proteins by their cognate receptors continues to be an intriguing area of research. The recently solved crystal structure of the ternary complex captured the receptor-bound α-subunit in an open conformation, without bound nucleotide has improved our understanding of the activation process. Despite these advancements, the mechanism by which the receptor causes GDP release from the α-subunit remains elusive. To elucidate the mechanism of activation, we studied guanine nucleotide-induced structural stability of the α-subunit (in response to thermal/chaotrope-mediated stress). Inherent stabilities of the inactive (GDP-bound) and active (GTP-bound) forms contribute antagonistically to the difference in conformational stability whereas the GDP-bound protein is able to switch to a stable intermediate state, GTP-bound protein loses this ability. Partial perturbation of the protein fold reveals the underlying influence of the bound nucleotide providing an insight into the mechanism of activation. An extra stable, pretransition intermediate, 'empty pocket' state (conformationally active-state like) in the unfolding pathway of GDP-bound protein mimics a gating system - the activation process having to overcome this stable intermediate state. We demonstrate that a relatively more complex conformational fold of the GDP-bound protein is at the core of the gating system. We report capturing this threshold, 'metastable empty pocket' conformation (the gate) of α-subunit of G protein and hypothesize that the receptor activates the G protein by enabling it to achieve this structure through mild structural perturbation. © 2017 Federation of European Biochemical Societies.

1,3-syn-Diaxial Repulsion of Typical Protecting Groups Used in Carbohydrate Chemistry in 3-O-Substituted Derivatives of Isopropyl d-Idopyranosides.

PubMed

Komarova, Bozhena S; Gerbst, Alexey G; Finogenova, Anastasiia M; Dmitrenok, Andrey S; Tsvetkov, Yury E; Nifantiev, Nikolay E

2017-09-01

The strength of 1,3-syn-diaxial repulsion was evaluated for main types of protecting groups (alkyl, silyl, and acyl) usually used in carbohydrate chemistry. As molecular probes for this study, derivatives of isopropyl 2-O-benzyl-4,6-O-benzylidene-α-d-idopyranoside bearing allyl, acetyl, and tert-butyldiphenylsilyl (TBDPS) protecting groups at O-3 were prepared from p-methoxyphenyl d-galactopyranoside. The equilibrium between O S 2 and 4 C 1 conformations in these compounds was investigated using 3 J H,H and 3 J C,H coupling constants that were determined from 1D 1 H NMR and 2D J-resolved HMBC spectra in various solvents. The analysis of the corresponding coupling constants calculated using DFT/B3LYP/pcJ-1 approximation applied to conformations optimized at DFT/B3LYP/6-311++G** level supported the investigation. Proportions of conformers in the equilibrium revealed the highest repulsion between the 3-allyloxy group and the isopropoxy aglycon and its dependence on the solvent polarity. Differences in the conformational behavior of 3-O-allyl and 3-O-acetyl-α-d-idopyranoside derivatives complied with the notion that higher electron density on O-3 increased 1,3-syn-diaxial repulsion. 3-O-TBDPS derivative existed mainly in 4 C 1 conformation. The attenuation of the 1,3-syn-diaxial repulsive interaction indicates that TBDPS has stereoelectronic properties that may have significance in context of fixing unnatural pyranoside conformation with the help of silyl groups but have been disregarded until now.

REVIEW: High pressure NMR study of proteins - seeking roots for function, evolution, disease and food applications

NASA Astrophysics Data System (ADS)

Akasaka, Kazuyuki

2010-12-01

NMR experiments at variable pressure reveal a wide range of conformation of a globular protein spanning from within the folded ensemble to the fully unfolded ensemble, herewith collectively called "high-energy conformers". The observation of "high-energy conformers" in a wide variety of globular proteins has led to the "volume theorem": the partial molar volume of a protein decreases with the decrease in its conformational order. Since "high-energy conformers" are intrinsically more reactive than the basic folded conformer, they could play decisive roles in all phenomena of proteins, namely function, environmental adaptation and misfolding. Based on the information on high-energy conformers and the rules on their partial volume in its monomeric state and amyloidosis, one may have a general view on what is happening on proteins under pressure. Moreover, one may even choose a high-energy conformer of a protein with pressure as variable for a particular purpose. Bridging "high-energy conformers" to macroscopic pressure effects could be a key to success in pressure application to biology, medicine, food technology and industry in the near future.

Elucidating the ensemble of functionally-relevant transitions in protein systems with a robotics-inspired method.

PubMed

Molloy, Kevin; Shehu, Amarda

2013-01-01

Many proteins tune their biological function by transitioning between different functional states, effectively acting as dynamic molecular machines. Detailed structural characterization of transition trajectories is central to understanding the relationship between protein dynamics and function. Computational approaches that build on the Molecular Dynamics framework are in principle able to model transition trajectories at great detail but also at considerable computational cost. Methods that delay consideration of dynamics and focus instead on elucidating energetically-credible conformational paths connecting two functionally-relevant structures provide a complementary approach. Effective sampling-based path planning methods originating in robotics have been recently proposed to produce conformational paths. These methods largely model short peptides or address large proteins by simplifying conformational space. We propose a robotics-inspired method that connects two given structures of a protein by sampling conformational paths. The method focuses on small- to medium-size proteins, efficiently modeling structural deformations through the use of the molecular fragment replacement technique. In particular, the method grows a tree in conformational space rooted at the start structure, steering the tree to a goal region defined around the goal structure. We investigate various bias schemes over a progress coordinate for balance between coverage of conformational space and progress towards the goal. A geometric projection layer promotes path diversity. A reactive temperature scheme allows sampling of rare paths that cross energy barriers. Experiments are conducted on small- to medium-size proteins of length up to 214 amino acids and with multiple known functionally-relevant states, some of which are more than 13Å apart of each-other. Analysis reveals that the method effectively obtains conformational paths connecting structural states that are significantly different. A detailed analysis on the depth and breadth of the tree suggests that a soft global bias over the progress coordinate enhances sampling and results in higher path diversity. The explicit geometric projection layer that biases the exploration away from over-sampled regions further increases coverage, often improving proximity to the goal by forcing the exploration to find new paths. The reactive temperature scheme is shown effective in increasing path diversity, particularly in difficult structural transitions with known high-energy barriers.

Trickle-Down Preferences: Preferential Conformity to High Status Peers in Fashion Choices.

PubMed

Galak, Jeff; Gray, Kurt; Elbert, Igor; Strohminger, Nina

2016-01-01

How much do our choices represent stable inner preferences versus social conformity? We examine conformity and consistency in sartorial choices surrounding a common life event of new norm exposure: relocation. A large-scale dataset of individual purchases of women's shoes (16,236 transactions) across five years and 2,007 women reveals a balance of conformity and consistency, moderated by changes in location socioeconomic status. Women conform to new local norms (i.e., average heel size) when moving to relatively higher status locations, but mostly ignore new local norms when moving to relatively lower status locations. In short, at periods of transition, it is the fashion norms of the rich that trickle down to consumers. These analyses provide the first naturalistic large-scale demonstration of the tension between psychological conformity and consistency, with real decisions in a highly visible context.

Structure and Uncoating of Immature Adenovirus

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

Perez-Berna, A.J.; Mangel, W.; Marabini, R.

2009-09-18

Maturation via proteolytic processing is a common trait in the viral world and is often accompanied by large conformational changes and rearrangements in the capsid. The adenovirus protease has been shown to play a dual role in the viral infectious cycle: (a) in maturation, as viral assembly starts with precursors to several of the structural proteins but ends with proteolytically processed versions in the mature virion, and (b) in entry, because protease-impaired viruses have difficulties in endosome escape and uncoating. Indeed, viruses that have not undergone proteolytic processing are not infectious. We studied the three-dimensional structure of immature adenovirus particlesmore » as represented by the adenovirus type 2 thermosensitive mutant ts1 grown under non-permissive conditions and compared it with the mature capsid. Our three-dimensional electron microscopy maps at subnanometer resolution indicate that adenovirus maturation does not involve large-scale conformational changes in the capsid. Difference maps reveal the locations of unprocessed peptides pIIIa and pVI and help define their role in capsid assembly and maturation. An intriguing difference appears in the core, indicating a more compact organization and increased stability of the immature cores. We have further investigated these properties by in vitro disassembly assays. Fluorescence and electron microscopy experiments reveal differences in the stability and uncoating of immature viruses, both at the capsid and core levels, as well as disassembly intermediates not previously imaged.« less

Different Vaccine Vectors Delivering the Same Antigen Elicit CD8plus T Cell Responses with Distinct Clonotype and Epitope Specificity

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

M Honda; R Wang; W Kong

Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8{sup +} cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8{sup +} T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2D{sup d} better than the native sequences, and clones with distinct specificities were elicited by alternativemore » vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8{sup +} T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.« less

Different Vaccine Vectors Delivering the Same Antigen Elicit CD8+ T Cell Responses with Distinct Clonotype and Epitope Specificity

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

Honda, M.; Robinson, H.; Wang, R.

Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8{sup +} cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8{sup +} T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2D{sup d} better than the native sequences, and clones with distinct specificities were elicited by alternativemore » vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8{sup +} T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.« less

Simultaneous use of solution NMR and X-ray data in REFMAC5 for joint refinement/detection of structural differences.

PubMed

Rinaldelli, Mauro; Ravera, Enrico; Calderone, Vito; Parigi, Giacomo; Murshudov, Garib N; Luchinat, Claudio

2014-04-01

The program REFMAC5 from CCP4 was modified to allow the simultaneous use of X-ray crystallographic data and paramagnetic NMR data (pseudocontact shifts and self-orientation residual dipolar couplings) and/or diamagnetic residual dipolar couplings. Incorporation of these long-range NMR restraints in REFMAC5 can reveal differences between solid-state and solution conformations of molecules or, in their absence, can be used together with X-ray crystallographic data for structural refinement. Since NMR and X-ray data are complementary, when a single structure is consistent with both sets of data and still maintains reasonably `ideal' geometries, the reliability of the derived atomic model is expected to increase. The program was tested on five different proteins: the catalytic domain of matrix metalloproteinase 1, GB3, ubiquitin, free calmodulin and calmodulin complexed with a peptide. In some cases the joint refinement produced a single model consistent with both sets of observations, while in other cases it indicated, outside the experimental uncertainty, the presence of different protein conformations in solution and in the solid state.

The Conformational Flexibility of the Acyltransferase from the Disorazole Polyketide Synthase Is Revealed by an X-ray Free-Electron Laser Using a Room-Temperature Sample Delivery Method for Serial Crystallography

PubMed Central

Allison, Kim; Robbins, Thomas; Lyubimov, Artem Y.; Uervirojnangkoorn, Monarin; Brunger, Axel T.; Khosla, Chaitan; DeMirci, Hasan; McPhillips, Scott E.; Hollenbeck, Michael; Soltis, Michael; Cohen, Aina E.

2017-01-01

The crystal structure of the trans-acyltrans-ferase (AT) from the disorazole polyketide synthase (PKS) was determined at room temperature to a resolution of 2.5 Å using a new method for the direct delivery of the sample into an X-ray free-electron laser. A novel sample extractor efficiently delivered limited quantities of microcrystals directly from the native crystallization solution into the X-ray beam at room temperature. The AT structure revealed important catalytic features of this core PKS enzyme, including the occurrence of conformational changes around the active site. The implications of these conformational changes for polyketide synthase reaction dynamics are discussed. PMID:28832129

Molecular mechanisms of conformational specificity: A study of Hox in vivo target DNA binding specificities and the structure of a Ure2p mutation that affects fibril formation rates

NASA Astrophysics Data System (ADS)

Bauer, William Joseph, Jr.

The fate of an individual cell, or even an entire organism, is often determined by minute, yet very specific differences in the conformation of a single protein species. Very often, proteins take on alternate folds or even side chain conformations to deal with different situations present within the cell. These differences can be as large as a whole domain or as subtle as the alteration of a single amino acid side chain. Yet, even these seemingly minor side chain conformational differences can determine the development of a cell type during differentiation or even dictate whether a cell will live or die. Two examples of situations where minor conformational differences within a specific protein could lead to major differences in the life cycle of a cell are described herein. The first example describes the variations seen in DNA conformations which can lead to slightly different Hox protein binding conformations responsible for recognizing biologically relevant regulatory sites. These specific differences occur in the minor groove of the bound DNA and are limited to the conformation of only two side chains. The conformation of the bound DNA, however, is not solely determined by the sequence of the DNA, as multiple sequences can result in the same DNA conformation. The second example takes place in the context of a yeast prion protein which contains a mutation that decreases the frequency at which fibrils form. While the specific interactions leading to this physiological change were not directly detected, it can be ascertained from the crystal structure that the structural changes are subtle and most likely involve another binding partner. In both cases, these conformational changes are very slight but have a profound effect on the downstream processes.

An investigation into Ca-DNA conformation as a function of relative humidity

NASA Astrophysics Data System (ADS)

Smith, Megan Schwenker

Raman spectroscopy experiments on CaDNA free-standing, highly-ordered wet-spun films containing various concentrations of CaCl2 show that CaDNA does not adopt the A conformation and reveals a maximum of DNA in the B conformation at 80% relative humidity. Swelling experiments on these same films give information as to the intermolecular spacing between molecules. Finally, a proof of principle measurement of the activation enthalpy of guanosine is also given.

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

PubMed

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

2016-12-16

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

3D printed conformal microfluidics for isolation and profiling of biomarkers from whole organs.

PubMed

Singh, Manjot; Tong, Yuxin; Webster, Kelly; Cesewski, Ellen; Haring, Alexander P; Laheri, Sahil; Carswell, Bill; O'Brien, Timothy J; Aardema, Charles H; Senger, Ryan S; Robertson, John L; Johnson, Blake N

2017-07-25

The ability to interface microfluidic devices with native complex biological architectures, such as whole organs, has the potential to shift the paradigm for the study and analysis of biological tissue. Here, we show 3D printing can be used to fabricate bio-inspired conformal microfluidic devices that directly interface with the surface of whole organs. Structured-light scanning techniques enabled the 3D topographical matching of microfluidic device geometry to porcine kidney anatomy. Our studies show molecular species are spontaneously transferred from the organ cortex to the conformal microfluidic device in the presence of fluid flow through the organ-conforming microchannel. Large animal studies using porcine kidneys (n = 32 organs) revealed the profile of molecular species in the organ-conforming microfluidic stream was dependent on the organ preservation conditions. Enzyme-linked immunosorbent assay (ELISA) studies revealed conformal microfluidic devices isolate clinically relevant metabolic and pathophysiological biomarkers from whole organs, including heat shock protein 70 (HSP-70) and kidney injury molecule-1 (KIM-1), which were detected in the microfluidic device as high as 409 and 12 pg mL -1 , respectively. Overall, these results show conformal microfluidic devices enable a novel minimally invasive 'microfluidic biopsy' technique for isolation and profiling of biomarkers from whole organs within a clinically relevant interval. This achievement could shift the paradigm for whole organ preservation and assessment, thereby helping to relieve the organ shortage crisis through increased availability and quality of donor organs. Ultimately, this work provides a major advance in microfluidics through the design and manufacturing of organ-conforming microfluidic devices and a novel technique for microfluidic-based analysis of whole organs.

Spectroscopic study of jet-cooled indole-3-carbinol by laser desorption technique: Franck-Condon simulations and anharmonic calculations

NASA Astrophysics Data System (ADS)

Ahn, Ahreum; Min, Ahreum; Moon, Cheol Joo; Lee, Ji Hoon; Lee, Seung Jun; Warashina, Taichi; Ishiuchi, Shun-ichi; Fujii, Masaaki; Choi, Myong Yong

2015-10-01

The conformational structure of indole-3-carbinol (I3C) has been investigated in the gas phase for the first time using a laser desorption technique. A UV-UV hole-burning technique revealed the presence of a single conformer of I3C in the mass-selected resonant two-photon ionization spectrum. The assignment of the observed IR spectrum of I3C is inconclusive due to almost identically predicted IR frequencies of the two lowest energy conformers from harmonic calculations. A conclusive assignment for the conformer of I3C has been reported with an aid of performing anharmonic calculations and Franck-Condon simulations on the two lowest-energy conformers.

Aqueous ionic liquids and their influence on peptide conformations: denaturation and dehydration mechanisms.

PubMed

Diddens, Diddo; Lesch, Volker; Heuer, Andreas; Smiatek, Jens

2017-08-09

Low concentrated aqueous ionic liquids (ILs) and their influence on protein structures have attracted a lot of interest over the last few years. This can be mostly attributed to the fact that aqueous ILs, depending on the ion species involved, can be used as protein protectants or protein denaturants. Atomistic molecular dynamics (MD) simulations are performed in order to study the influence of different aprotic ILs on the properties of a short hairpin peptide. Our results reveal distinct binding and denaturation effects for 1-ethyl-3-methylimidazolium (EMIM) in combination with different anions, namely, chloride (CL), tetrafluoroborate (BF4) and acetate (ACE). The simulation outcomes demonstrate that the studied ILs with larger anions reveal a more pronounced accumulation behavior of the individual ion species around the peptide, which is accomplished by a stronger dehydration effect. We can relate these findings to the implications of the Kirkwood-Buff theory, which provides a thermodynamic explanation for the denaturation strength in terms of the IL accumulation behavior. The results for the spatial distribution functions, the binding energies and the local/bulk partition coefficients are in good agreement with metadynamics simulations in order to determine the energetically most stable peptide conformations. The free energy landscapes indicate a decrease of the denaturation strength in the order EMIM/ACE, EMIM/BF4 and EMIM/CL, which coincides with a decreasing size of the anion species. An analysis of the potential binding energies reveals that this effect is mainly of enthalpic nature.

A folded viral noncoding RNA blocks host cell exoribonucleases through a conformationally dynamic RNA structure.

PubMed

Steckelberg, Anna-Lena; Akiyama, Benjamin M; Costantino, David A; Sit, Tim L; Nix, Jay C; Kieft, Jeffrey S

2018-06-19

Folded RNA elements that block processive 5' → 3' cellular exoribonucleases (xrRNAs) to produce biologically active viral noncoding RNAs have been discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, whether this RNA structure-dependent mechanism exists elsewhere and, if so, whether a singular RNA fold is required, have been unclear. Here we demonstrate the existence of authentic RNA structure-dependent xrRNAs in dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These xrRNAs have no sequence similarity to known xrRNAs; thus, we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping exoribonucleases. By solving the structure of a dianthovirus xrRNA by X-ray crystallography, we reveal a complex fold that is very different from that of the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature, a pseudoknot that creates a protective ring around the 5' end of the RNA structure; this may be a defining structural feature of xrRNAs. Single-molecule FRET experiments reveal that the dianthovirus xrRNAs undergo conformational changes and can use "codegradational remodeling," exploiting the exoribonucleases' degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.

Exploration of nucleoprotein α-MoRE and XD interactions of Nipah and Hendra viruses.

PubMed

Shang, Xu; Chu, Wenting; Chu, Xiakun; Xu, Liufang; Longhi, Sonia; Wang, Jin

2018-04-24

Henipavirus, including Hendra virus (HeV) and Nipah virus (NiV), is a newly discovered human pathogen genus. The nucleoprotein of Henipavirus contains an α-helical molecular recognition element (α-MoRE) that folds upon binding to the X domain (XD) of the phosphoprotein (P). In order to explore the conformational dynamics of free α-MoREs and the underlying binding-folding mechanism with XD, atomic force field-based and hybrid structure-based MD simulations were carried out. In our empirical force field-based simulations, characteristic structures and helicities of α-MoREs reveal the co-existence of partially structured and disordered conformations, as in the case of the well characterized cognate measles virus (MeV) α-MoRE. In spite of their overall similarity, the two α-MoREs display subtle helicity differences in their C-terminal region, but much different from that of MeV. For the α-MoRE/XD complexes, the results of our hybrid structure-based simulations provide the coupled binding-folding landscapes, and unveil a wide conformational selection mechanism at early binding stages, followed by a final induce-fit mechanism selection process. However, the HeV and NiV complexes have a lower binding barrier compared to that of MeV. Moreover, the HeV α-MoRE/XD complex shows much less coupling effects between binding and folding compared to that from both NiV and MeV. Our analysis revealed that contrary to NiV and MeV, the N- and C-terminal regions of the HeV α-MoRE maintains a low helicity also in the bound form.

Molecular dynamics studies of the conformation of sorbitol

PubMed Central

Lerbret, A.; Mason, P.E.; Venable, R.M.; Cesàro, A.; Saboungi, M.-L.; Pastor, R.W.; Brady, J.W.

2009-01-01

Molecular dynamics simulations of a 3 m aqueous solution of D-sorbitol (also called D-glucitol) have been performed at 300 K, as well as at two elevated temperatures to promote conformational transitions. In principle, sorbitol is more flexible than glucose since it does not contain a constraining ring. However, a conformational analysis revealed that the sorbitol chain remains extended in solution, in contrast to the bent conformation found experimentally in the crystalline form. While there are 243 staggered conformations of the backbone possible for this open-chain polyol, only a very limited number were found to be stable in the simulations. Although many conformers were briefly sampled, only eight were significantly populated in the simulation. The carbon backbones of all but two of these eight conformers were completely extended, unlike the bent crystal conformation. These extended conformers were stabilized by a quite persistent intramolecular hydrogen bond between the hydroxyl groups of carbon C-2 and C-4. The conformational populations were found to be in good agreement with the limited available NMR data except for the C-2–C-3 torsion (spanned by the O-2–O-4 hydrogen bond), where the NMR data supports a more bent structure. PMID:19744646

Making the Grade? Classroom Climate for LGBTQ Students across Gender Conformity

ERIC Educational Resources Information Center

Garvey, Jason C.; Rankin, Susan R.

2015-01-01

Using data from the "2010 State of Higher Education for LGBT People" (Rankin, Weber, Blumenfeld, & Frazer), this study examines campus climate perceptions for LGBTQ undergraduate students across gender conformity and the extent to which relevant variables influence perceptions of classroom climate. Findings reveal more positive…

Structural Basis for Parathyroid Hormone-related Protein Binding to the Parathyroid Hormone Receptor and Design of Conformation-selective Peptides

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

Pioszak, Augen A.; Parker, Naomi R.; Gardella, Thomas J.

2009-12-01

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are two related peptides that control calcium/phosphate homeostasis and bone development, respectively, through activation of the PTH/PTHrP receptor (PTH1R), a class B G protein-coupled receptor. Both peptides hold clinical interest for their capacities to stimulate bone formation. PTH and PTHrP display different selectivity for two distinct PTH1R conformations, but how their binding to the receptor differs is unclear. The high resolution crystal structure of PTHrP bound to the extracellular domain (ECD) of PTH1R reveals that PTHrP binds as an amphipathic {alpha}-helix to the same hydrophobic groove in the ECD as occupied by PTH,more » but in contrast to a straight, continuous PTH helix, the PTHrP helix is gently curved and C-terminally 'unwound.' The receptor accommodates the altered binding modes by shifting the side chain conformations of two residues within the binding groove: Leu-41 and Ile-115, the former acting as a rotamer toggle switch to accommodate PTH/PTHrP sequence divergence, and the latter adapting to the PTHrP curvature. Binding studies performed with PTH/PTHrP hybrid ligands having reciprocal exchanges of residues involved in different contacts confirmed functional consequences for the altered interactions and enabled the design of altered PTH and PTHrP peptides that adopt the ECD-binding mode of the opposite peptide. Hybrid peptides that bound the ECD poorly were selective for the G protein-coupled PTH1R conformation. These results establish a molecular model for better understanding of how two biologically distinct ligands can act through a single receptor and provide a template for designing better PTH/PTHrP therapeutics.« less

Protein adsorption on tailored substrates: long-range forces and conformational changes

NASA Astrophysics Data System (ADS)

Bellion, M.; Santen, L.; Mantz, H.; Hähl, H.; Quinn, A.; Nagel, A.; Gilow, C.; Weitenberg, C.; Schmitt, Y.; Jacobs, K.

2008-10-01

Adsorption of proteins onto solid surfaces is an everyday phenomenon that is not yet fully understood. To further the current understanding, we have performed in situ ellipsometry studies to reveal the adsorption kinetics of three different proteins, lysozyme, α-amylase and bovine serum albumin. As substrates we offer Si wafers with a controlled Si oxide layer thickness and a hydrophilic or hydrophobic surface functionalization, allowing the tailoring of the influence of short- and long-range interactions. Our studies show that not only the surface chemistry determines the properties of an adsorbed protein layer but also the van der Waals contributions of a composite substrate. We compare the experimental findings to results of a colloidal Monte Carlo approach that includes conformational changes of the adsorbed proteins induced by density fluctuations.

Loop Electrostatics Asymmetry Modulates the Preexisting Conformational Equilibrium in Thrombin.

PubMed

Pozzi, Nicola; Zerbetto, Mirco; Acquasaliente, Laura; Tescari, Simone; Frezzato, Diego; Polimeno, Antonino; Gohara, David W; Di Cera, Enrico; De Filippis, Vincenzo

2016-07-19

Thrombin exists as an ensemble of active (E) and inactive (E*) conformations that differ in their accessibility to the active site. Here we show that redistribution of the E*-E equilibrium can be achieved by perturbing the electrostatic properties of the enzyme. Removal of the negative charge of the catalytic Asp102 or Asp189 in the primary specificity site destabilizes the E form and causes a shift in the 215-217 segment that compromises substrate entrance. Solution studies and existing structures of D102N document stabilization of the E* form. A new high-resolution structure of D189A also reveals the mutant in the collapsed E* form. These findings establish a new paradigm for the control of the E*-E equilibrium in the trypsin fold.

Strain-engineering of Janus SiC monolayer functionalized with H and F atoms

NASA Astrophysics Data System (ADS)

Drissi, L. B.; Sadki, K.; Kourra, M.-H.; Bousmina, M.

2018-05-01

Based on ab initio density functional theory calculations, the structural, electronic, mechanical, acoustic, thermodynamic, and piezoelectric properties of (F,H) Janus SiC monolayers are studied. The new set of derivatives shows buckled structures and different band gap values. Under strain, the buckling changes and the structures pass from semiconducting to metallic. The elastic limits and the metastable regions are determined. The Young's modulus and Poisson ratio reveal stronger behavior for the modified conformers with respect to graphene. The values of the Debye temperature make the new materials suitable for thermal application. Moreover, all the conformers show in-plane and out-of-plane piezoelectric responses comparable with known two-dimensional materials. If engineered, such piezoelectric Janus structures may be promising materials for various nanoelectromechanical applications.

The neural basis for novel semantic categorization.

PubMed

Koenig, Phyllis; Smith, Edward E; Glosser, Guila; DeVita, Chris; Moore, Peachie; McMillan, Corey; Gee, Jim; Grossman, Murray

2005-01-15

We monitored regional cerebral activity with BOLD fMRI during acquisition of a novel semantic category and subsequent categorization of test stimuli by a rule-based strategy or a similarity-based strategy. We observed different patterns of activation in direct comparisons of rule- and similarity-based categorization. During rule-based category acquisition, subjects recruited anterior cingulate, thalamic, and parietal regions to support selective attention to perceptual features, and left inferior frontal cortex to helps maintain rules in working memory. Subsequent rule-based categorization revealed anterior cingulate and parietal activation while judging stimuli whose conformity with the rules was readily apparent, and left inferior frontal recruitment during judgments of stimuli whose conformity was less apparent. By comparison, similarity-based category acquisition showed recruitment of anterior prefrontal and posterior cingulate regions, presumably to support successful retrieval of previously encountered exemplars from long-term memory, and bilateral temporal-parietal activation for perceptual feature integration. Subsequent similarity-based categorization revealed temporal-parietal, posterior cingulate, and anterior prefrontal activation. These findings suggest that large-scale networks support relatively distinct categorization processes during the acquisition and judgment of semantic category knowledge.

The role of the DNA sliding clamp in Okazaki fragment maturation in archaea and eukaryotes.

PubMed

Beattie, Thomas R; Bell, Stephen D

2011-01-01

Efficient processing of Okazaki fragments generated during discontinuous lagging-strand DNA replication is critical for the maintenance of genome integrity. In eukaryotes, a number of enzymes co-ordinate to ensure the removal of initiating primers from the 5'-end of each fragment and the generation of a covalently linked daughter strand. Studies in eukaryotic systems have revealed that the co-ordination of DNA polymerase δ and FEN-1 (Flap Endonuclease 1) is sufficient to remove the majority of primers. Other pathways such as that involving Dna2 also operate under certain conditions, although, notably, Dna2 is not universally conserved between eukaryotes and archaea, unlike the other core factors. In addition to the catalytic components, the DNA sliding clamp, PCNA (proliferating-cell nuclear antigen), plays a pivotal role in binding and co-ordinating these enzymes at sites of lagging-strand replication. Structural studies in eukaryotic and archaeal systems have revealed that PCNA-binding proteins can adopt different conformations when binding PCNA. This conformational malleability may be key to the co-ordination of these enzymes' activities.

Crystal Structure of West Nile Virus Envelope Glycoprotein Reveals Viral Surface Epitopes

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

Kanai,R.; Kar, K.; Anthony, K.

2006-01-01

West Nile virus, a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specificmore » antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.« less

Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex

PubMed Central

Chakraborty, Sagnik; Steinbach, Peter J; Paul, Debamita; Mu, Hong; Broyde, Suse

2018-01-01

Abstract Rad4/XPC recognizes diverse DNA lesions including ultraviolet-photolesions and carcinogen-DNA adducts, initiating nucleotide excision repair. Studies have suggested that Rad4/XPC senses lesion-induced helix-destabilization to flip out nucleotides from damaged DNA sites. However, characterizing how DNA deformability and/or distortions impact recognition has been challenging. Here, using fluorescence lifetime measurements empowered by a maximum entropy algorithm, we mapped the conformational heterogeneities of artificially destabilized mismatched DNA substrates of varying Rad4-binding specificities. The conformational distributions, as probed by FRET between a cytosine-analog pair exquisitely sensitive to DNA twisting/bending, reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity CCC/CCC mismatch, free in solution, sampled a strikingly broad range of conformations from B-DNA-like to highly distorted conformations that resembled those observed with Rad4 bound; the extent of these distortions increased with bound Rad4 and with temperature. Conversely, the non-specific TAT/TAT mismatch had a homogeneous, B-DNA-like conformation. Molecular dynamics simulations also revealed a wide distribution of conformations for CCC/CCC, complementing experimental findings. We propose that intrinsic deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating ‘conformational capture’ of pre-distorted damaged sites. Surprisingly, even mismatched DNA specifically bound to Rad4 remains highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions. PMID:29267981

Conformational analysis and circular dichroism of bilirubin, the yellow pigment of jaundice

NASA Astrophysics Data System (ADS)

Lightner, David A.; Person, Richard; Peterson, Blake; Puzicha, Gisbert; Pu, Yu-Ming; Bojadziev, Stefan

1991-06-01

Conformational analysis of (4Z, 15Z)-bilirubin-IX(alpha) by molecular mechanics computations reveals a global energy minimum folded conformation. Powerful added stabilization is achieved through intramolecular hydrogen bonding. Theoretical treatment of bilirubin as a molecular exciton predicts an intense bisignate circular dichroism spectrum for the folded conformation: (Delta) (epsilon) is congruent to 270 L (DOT) mole-1 (DOT) cm-1 for the $OM450 nm electronic transition(s). Synthesis of bilirubin analogs with propionic acid groups methylated at the (alpha) or (beta) position introduces an allosteric effect that allows for an optical resolution of the pigments, with enantiomers exhibiting the theoretically predicted circular dichroism.

Non-invasive SFG spectroscopy: a tool to reveal the conformational change of grafted chains due to bacterial adhesion

NASA Astrophysics Data System (ADS)

Bulard, Emilie; Dubost, Henri; Fontaine-Aupart, Marie-Pierre; Zheng, Wanquan; Herry, Jean-Marie; Bellon-Fontaine, Marie-No"lle; Briandet, Romain; Bourguignon, Bernard

2011-07-01

In many fields such as biomedical or food industry, surface colonization by micro-organisms leads to biofilms formation that are tridimentional biostructures highly resistant to the action of antimicrobials, by mechanisms still unclear. In order to deepen our understanding of the initial interaction of bacteria cells with a solid surface, we analyze by in situ vibrational Sum Frequency Generation (SFG) spectroscopy the effect of the adhesion of hydrophilic Lactoccocus lactis bacteria and its hydrophobic mutants in distilled water on a self-assembled monolayer (SAM) of octadecanethiol (ODT) on a gold film. When a homogeneous bacterial monolayer is deposited on this ordered surface, SFG spectrum of the ODT SAM shows significant intensity changes from that in air or in water. Its modelling as a function of conformation allows to distinguish optical effects due to the water solution surrounding bacteria from conformational changes of the ODT SAM due to the presence of the bacteria cells. Futhermore, bacterial adhesion induces different measurable effects on the ODT SAM conformation, depending on the hydrophobic / hydrophilic character of the bacterial surface. Such a result deserves to be taken into account for the design of new materials with improved properties or to control biofilm formation.

EGCG in Green Tea Induces Aggregation of HMGB1 Protein through Large Conformational Changes with Polarized Charge Redistribution

NASA Astrophysics Data System (ADS)

Meng, Xuan-Yu; Li, Baoyu; Liu, Shengtang; Kang, Hongsuk; Zhao, Lin; Zhou, Ruhong

2016-02-01

As a major effective component in green tea, (-)-epigallocatechin-3-gallate (EGCG)’s potential benefits to human health have been widely investigated. Recent experimental evidences indicate that EGCG can induce the aggregation of HMGB1 protein, a late mediator of inflammation, which subsequently stimulates the autophagic degradation and thus provides protection from lethal endotoxemia and sepsis. In this study, we use molecular dynamics (MD) simulations to explore the underlying molecular mechanism of this aggregation of HMGB1 facilitated by EGCG. Our simulation results reveal that EGCG firmly binds to HMGB1 near Cys106, which supports previous preliminary experimental evidence. A large HMGB1 conformational change is observed, where Box A and Box B, two homogenous domains of HMGB1, are repositioned and packed together by EGCG. This new HMGB1 conformation has large molecular polarity and distinctive electrostatic potential surface. We suggest that the highly polarized charge distribution leads to the aggregation of HMGB1, which differs from the previous hypothesis that two HMGB1 monomers are linked by the dimer of EGCG. Possible aggregating modes have also been investigated with potential of mean force (PMF) calculations. Finally, we conclude that the conformation induced by EGCG is more aggregation-prone with higher binding free energies as compared to those without EGCG.

Impact of graphene-based nanomaterials (GBNMs) on the structural and functional conformations of hepcidin peptide

NASA Astrophysics Data System (ADS)

Singh, Krishna P.; Baweja, Lokesh; Wolkenhauer, Olaf; Rahman, Qamar; Gupta, Shailendra K.

2018-03-01

Graphene-based nanomaterials (GBNMs) are widely used in various industrial and biomedical applications. GBNMs of different compositions, size and shapes are being introduced without thorough toxicity evaluation due to the unavailability of regulatory guidelines. Computational toxicity prediction methods are used by regulatory bodies to quickly assess health hazards caused by newer materials. Due to increasing demand of GBNMs in various size and functional groups in industrial and consumer based applications, rapid and reliable computational toxicity assessment methods are urgently needed. In the present work, we investigate the impact of graphene and graphene oxide nanomaterials on the structural conformations of small hepcidin peptide and compare the materials for their structural and conformational changes. Our molecular dynamics simulation studies revealed conformational changes in hepcidin due to its interaction with GBMNs, which results in a loss of its functional properties. Our results indicate that hepcidin peptide undergo severe structural deformations when superimposed on the graphene sheet in comparison to graphene oxide sheet. These observations suggest that graphene is more toxic than a graphene oxide nanosheet of similar area. Overall, this study indicates that computational methods based on structural deformation, using molecular dynamics (MD) simulations, can be used for the early evaluation of toxicity potential of novel nanomaterials.

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

PubMed

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

2010-03-17

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

Raman scattering-based multiconformational analysis for probing the structural differences between acetylcholine and acetylthiocholine.

PubMed

Hernández, Belén; Houzé, Pascal; Pflüger, Fernando; Kruglik, Sergei G; Ghomi, Mahmoud

2017-05-10

Acetylcholine is the first discovered neurotransmitter that has received a great attention regarding its capability of binding to several cellular targets. The chemical composition of acetylcholine, including a positively charged trimethylammonium and a carbonyl group, as well as its conformational flexibility was pointed out as the key factors in the stabilization of its interactions. Here, the possibilities offered by a Raman scattering-based multiconformatioal analysis to access the most stable conformers of acetylcholine, is discussed. To control the validity of this protocol, acetylcholine and one of its closely structured analogues, acetylthiocholine, were simultaneously analyzed. Solution Raman spectra revealed distinct and well resolved strong markers for each molecule. Density functional theory calculations were consistent with the fact that the energy order of the low energy conformers is considerably affected by the acyloxy oxygen→sulfur atom substitution. Raman spectra were calculated on the basis of the thermal average of the spectra arising from the low energy conformers. It has been evidenced that the carbonyl and trimethylammonium groups are the most favorable hydration sites in aqueous environment. Taking into account the large gap between the carbonyl bond-stretch and aliphatic bending bands, Raman spectra also allowed separation of the HOH bending vibrations arising from the bound and bulk water molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

Kinetics, Assembling and Conformation Control of L-Cysteine Adsorption on Pt by In Situ FTIR Spectroscopy and QCM-D.

PubMed

Cordoba de Torresi, Susana Ines; Dourado, Andre H B; Silva, Rubens A; Torresi, Roberto M; Sumodjo, Paulo T A; Arenz, Matthias

2018-06-05

A quartz crystal microbalance method with dissipation (QCM-D) and attenuated total reflection infrared (ATR-FTIRS) spectroscopy were used to study the adsorption of L-cysteine (L-Cys) on Pt. Using QCM-D, it was possible to verify that the viscoelastic properties of the adsorbed species play an important role in the adsorption, rendering Sauerbrey's equation inapplicable. The modelling of QCM-D data exposed two different processes for the adsorption reaction. The first one had an activation time and is fast, whereas the second is slow. These processes were also resolved by ATR-FTIRS identified to be water and anion adsorption preceded by L-Cys adsorption. Both techniques reveal that the degree of surface coverage is pH dependent. Spectroscopic data indicate that the conformation of L-Cys(ads) changes with pH and that the structures do not fully agree with those proposed in literature for other metallic surfaces. The assembling of the adsorbed monolayer appeared to be very fast, and it was not possible to determine or quantify this kinetics. The conformation is also controlled by applied potential, and the anion adsorption and interfacial water depends on the conformation of the adsorbed molecules. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Conformational Properties of Seven Toac-Labeled Angiotensin I Analogues Correlate with Their Muscle Contraction Activity and Their Ability to Act as ACE Substrates.

PubMed

Teixeira, Luis Gustavo D; Malavolta, Luciana; Bersanetti, Patrícia A; Schreier, Shirley; Carmona, Adriana K; Nakaie, Clovis R

2015-01-01

Conformational properties of the angiotensin II precursor, angiotensin I (AngI) and analogues containing the paramagnetic amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 1, 3, 5, 8, 9, and 10, were examined by EPR, CD, and fluorescence. The conformational data were correlated to their activity in muscle contraction experiments and to their properties as substrates of the angiotensin I-converting enzyme (ACE). Biological activity studies indicated that TOAC0-AngI and TOAC1-AngI maintained partial potency in guinea pig ileum and rat uterus. Kinetic parameters revealed that only derivatives labeled closer to the N-terminus (positions 0, 1, 3, and 5) were hydrolyzed by ACE, indicating that peptides bearing the TOAC moiety far from the ACE cleavage site (Phe8-His9 peptide bond) were susceptible to hydrolysis, albeit less effectively than the parent compound. CD spectra indicated that AngI exhibited a flexible structure resulting from equilibrium between different conformers. While the conformation of N-terminally-labeled derivatives was similar to that of the native peptide, a greater propensity to acquire folded structures was observed for internally-labeled, as well as C-terminally labeled, analogues. These structures were stabilized in secondary structure-inducing agent, TFE. Different analogues gave rise to different β-turns. EPR spectra in aqueous solution also distinguished between N-terminally, internally-, and C-terminally labeled peptides, yielding narrower lines, indicative of greater mobility for the former. Interestingly, the spectra of peptides labeled at, or close, to the C-terminus, showed that the motion in this part of the peptides was intermediate between that of N-terminally and internally-labeled peptides, in agreement with the suggestion of turn formation provided by the CD spectra. Quenching of the Tyr4 fluorescence by the differently positioned TOAC residues corroborated the data obtained by the other spectroscopic techniques. Lastly, we demonstrated the feasibility of monitoring the progress of ACE-catalyzed hydrolysis of TOAC-labeled peptides by following time-dependent changes in their EPR spectra.

Conformational Properties of Seven Toac-Labeled Angiotensin I Analogues Correlate with Their Muscle Contraction Activity and Their Ability to Act as ACE Substrates

PubMed Central

Teixeira, Luis Gustavo D.; Malavolta, Luciana; Bersanetti, Patrícia A.; Schreier, Shirley; Carmona, Adriana K.; Nakaie, Clovis R.

2015-01-01

Conformational properties of the angiotensin II precursor, angiotensin I (AngI) and analogues containing the paramagnetic amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 1, 3, 5, 8, 9, and 10, were examined by EPR, CD, and fluorescence. The conformational data were correlated to their activity in muscle contraction experiments and to their properties as substrates of the angiotensin I-converting enzyme (ACE). Biological activity studies indicated that TOAC0-AngI and TOAC1-AngI maintained partial potency in guinea pig ileum and rat uterus. Kinetic parameters revealed that only derivatives labeled closer to the N-terminus (positions 0, 1, 3, and 5) were hydrolyzed by ACE, indicating that peptides bearing the TOAC moiety far from the ACE cleavage site (Phe8-His9 peptide bond) were susceptible to hydrolysis, albeit less effectively than the parent compound. CD spectra indicated that AngI exhibited a flexible structure resulting from equilibrium between different conformers. While the conformation of N-terminally-labeled derivatives was similar to that of the native peptide, a greater propensity to acquire folded structures was observed for internally-labeled, as well as C-terminally labeled, analogues. These structures were stabilized in secondary structure-inducing agent, TFE. Different analogues gave rise to different β-turns. EPR spectra in aqueous solution also distinguished between N-terminally, internally-, and C-terminally labeled peptides, yielding narrower lines, indicative of greater mobility for the former. Interestingly, the spectra of peptides labeled at, or close, to the C-terminus, showed that the motion in this part of the peptides was intermediate between that of N-terminally and internally-labeled peptides, in agreement with the suggestion of turn formation provided by the CD spectra. Quenching of the Tyr4 fluorescence by the differently positioned TOAC residues corroborated the data obtained by the other spectroscopic techniques. Lastly, we demonstrated the feasibility of monitoring the progress of ACE-catalyzed hydrolysis of TOAC-labeled peptides by following time-dependent changes in their EPR spectra. PMID:26317625

Crystal structure and MD simulation of mouse EndoV reveal wedge motif plasticity in this inosine-specific endonuclease

NASA Astrophysics Data System (ADS)

Nawaz, Meh Sameen; Vik, Erik Sebastian; Ronander, Mia Elise; Solvoll, Anne Marthe; Blicher, Pernille; Bjørås, Magnar; Alseth, Ingrun; Dalhus, Bjørn

2016-04-01

Endonuclease V (EndoV) is an enzyme with specificity for deaminated adenosine (inosine) in nucleic acids. EndoV from Escherichia coli (EcEndoV) acts both on inosines in DNA and RNA, whereas the human homolog cleaves only at inosines in RNA. Inosines in DNA are mutagenic and the role of EndoV in DNA repair is well established. In contrast, the biological function of EndoV in RNA processing is largely unexplored. Here we have characterized a second mammalian EndoV homolog, mouse EndoV (mEndoV), and show that mEndoV shares the same RNA selectivity as human EndoV (hEndoV). Mouse EndoV cleaves the same inosine-containing substrates as hEndoV, but with reduced efficiencies. The crystal structure of mEndoV reveals a conformation different from the hEndoV and prokaryotic EndoV structures, particularly for the conserved tyrosine in the wedge motif, suggesting that this strand separating element has some flexibility. Molecular dynamics simulations of mouse and human EndoV reveal alternative conformations for the invariant tyrosine. The configuration of the active site, on the other hand, is very similar between the prokaryotic and mammalian versions of EndoV.

Trickle-Down Preferences: Preferential Conformity to High Status Peers in Fashion Choices

PubMed Central

Galak, Jeff; Gray, Kurt; Elbert, Igor; Strohminger, Nina

2016-01-01

How much do our choices represent stable inner preferences versus social conformity? We examine conformity and consistency in sartorial choices surrounding a common life event of new norm exposure: relocation. A large-scale dataset of individual purchases of women’s shoes (16,236 transactions) across five years and 2,007 women reveals a balance of conformity and consistency, moderated by changes in location socioeconomic status. Women conform to new local norms (i.e., average heel size) when moving to relatively higher status locations, but mostly ignore new local norms when moving to relatively lower status locations. In short, at periods of transition, it is the fashion norms of the rich that trickle down to consumers. These analyses provide the first naturalistic large-scale demonstration of the tension between psychological conformity and consistency, with real decisions in a highly visible context. PMID:27144595

Conformational Aspects of the O-acetylation of C-tetra(phenyl)calixpyrogallol[4]arene.

PubMed

Casas-Hinestroza, José Luis; Maldonado, Mauricio

2018-05-20

Reaction between pyrogallol and benzaldehyde results in a conformational mixture of C- tetra(phenyl)pyrogallol[4]arene (crown and chair). The conformer mixture was separated using crystallization procedures and the structures were determined using FTIR, ¹H-NMR, and 13 C-NMR. O -acetylation of C- tetra(phenyl)pyrogallol[4]arene (chair) with acetic anhydride, in pyridine results in the formation of dodecaacetyl-tetra(phenyl)pyrogallol[4]arene. The structure was determined using ¹H-NMR and 13 C-NMR finding that the product maintains the conformation of the starting conformer. On the other hand, the O -acetylation reaction of C- tetra(phenyl)pirogallol[4]arene (crown) under same conditions proceeded efficiently, and its structure was determined using ¹H-NMR and 13 C-NMR. Dynamic ¹H-NMR of acetylated pyrogallolarene was studied by means of variable temperature in DMSO- d ₆ solution, and it revealed that two conformers are formed in the solution. Boat conformations for acetylated pyrogallolarene showed a slow interconversion at room temperature.

Acid-enhanced conformation changes of yeast cytochrome c coated onto gold nanoparticles, a FT-IR spectroscopic analysis.

PubMed

Dong, Aichun; Brown, Corina; Bai, Shufeng; Dong, Jian

2018-06-01

Under conditions with or without linker molecules, the effects of acidic pH on the conformation of yeast iso-1-cytochrome c coated onto gold nanoparticles (AuNPs) in correlation with color changes of a Cyt c-coated AuNPs solution/suspension were examined by Fourier transform infrared (FT-IR) spectroscopy and correlated to color change. The results of detailed secondary structural analysis revealed that although the color changes coincide with acid-induced conformational changes in Cyt c coated onto AuNPs, the pH-related conformational unfolding of Cyt c coated onto AuNPs differed dramatically from that of its counterpart in solution. For Cyt c free in solution, the acid-induced unfolding did not occur until the pH was below 3.0, whereas for Cyt c coated onto AuNPs via C102 coordination near the C-terminal, a partial unfolding was observed even at near neutral pH which continuously intensified as pH decreased. Insertion of a short alkanethiol (3-mercaptoproprionic acid, 3-MPA) molecule between Cyt c and AuNP, which changes the interaction mode from a thiol coordination between Cyt c and AuNP to an electrostatic interaction between Cyt c and 3-MPA, which stabilized the conformation of Cyt c significantly, but did not prevent the acid-induced aggregation of Cyt c-3MPA-AuNPs. Copyright © 2018 Elsevier B.V. All rights reserved.

Improved 68 Ga-labeling method using ethanol addition: Application to the α-helical peptide DOTA-FAMP.

PubMed

Hasegawa, Koki; Kawachi, Emi; Uehara, Yoshinari; Yoshida, Tsuyoshi; Imaizumi, Satoshi; Ogawa, Masahiro; Miura, Shin-Ichiro; Saku, Keijiro

2017-01-01

We examined the 68 Ga labeling of the α-helical peptide, DOTA-FAMP, and evaluated conformational changes during radiolabeling. 68 Ga-DOTA-FAMP is a positron emission tomography probe candidate for atherosclerotic plaques. The labeling yield achieved by Zhernosekov's method (using acetone for 68 Ga purification) was compared with that achieved by the original and 2 modified Mueller's methods (using NaCl solution). Modified method I involves desalting the 68 Ga prior to labeling, and modified method II involves the inclusion of ethanol in the labeling solution. The labeling yield using Zhernosekov's method was 62% ± 5.4%. In comparison, Mueller's original method gave 8.9% ± 1.7%. Modified method I gave a slight improvement of 32% ± 2.1%. Modified method II further increased the yield to 66% ± 3.4%. Conformational changes were determined by circular dichroism spectroscopy, revealing that these differences could be attributed to conformational changes. Heat treatment affects peptide conformation, which leads to aggregation and decreases the labeling yield. Mueller's method is simpler, but harsh conditions preclude its application to biomolecules. To suppress aggregation, we included a desalting process and added ethanol in the labeling solution. These changes significantly improved the labeling yield. Before use for imaging, conformational changes of biomolecules during radiolabeling should be evaluated by circular dichroism spectroscopy to ensure the homogeneity of the labeled product. Copyright © 2016 John Wiley & Sons, Ltd.

Leakage and slow allostery limit performance of single drug-sensing aptazyme molecules based on the hammerhead ribozyme

PubMed Central

de Silva, Chamaree; Walter, Nils G.

2009-01-01

Engineered “aptazymes” fuse in vitro selected aptamers with ribozymes to create allosteric enzymes as biosensing components and artificial gene regulatory switches through ligand-induced conformational rearrangement and activation. By contrast, activating ligand is employed as an enzymatic cofactor in the only known natural aptazyme, the glmS ribozyme, which is devoid of any detectable conformational rearrangements. To better understand this difference in biosensing strategy, we monitored by single molecule fluorescence resonance energy transfer (FRET) and 2-aminopurine (AP) fluorescence the global conformational dynamics and local base (un)stacking, respectively, of a prototypical drug-sensing aptazyme, built from a theophylline aptamer and the hammerhead ribozyme. Single molecule FRET reveals that a catalytically active state with distal Stems I and III of the hammerhead ribozyme is accessed both in the theophylline-bound and, if less frequently, in the ligand-free state. The resultant residual activity (leakage) in the absence of theophylline contributes to a limited dynamic range of the aptazyme. In addition, site-specific AP labeling shows that rapid local theophylline binding to the aptamer domain leads to only slow allosteric signal transduction into the ribozyme core. Our findings allow us to rationalize the suboptimal biosensing performance of the engineered compared to the natural aptazyme and to suggest improvement strategies. Our single molecule FRET approach also monitors in real time the previously elusive equilibrium docking dynamics of the hammerhead ribozyme between several inactive conformations and the active, long-lived, Y-shaped conformer. PMID:19029309

Effects of point mutations on the thermostability of B. subtilis lipase: investigating nonadditivity

NASA Astrophysics Data System (ADS)

Singh, Bipin; Bulusu, Gopalakrishnan; Mitra, Abhijit

2016-10-01

Molecular level understanding of mutational effects on stability and activity of enzymes is challenging particularly when several point mutations are incorporated during the directed evolution experiments. In our earlier study, we have suggested the lack of consistency in the effect of point mutations incorporated during the initial generations of directed evolution experiments, towards conformational stabilization of B. subtilis lipase mutants of later generations. Here, we report that the cumulative point mutations incorporated in mutants 2M (with two point mutations) to 6M (with six point mutations) possibly do not retain their original stabilizing nature in the most thermostable 12M mutant (with 12 point mutations). We have carried out MD simulations using structures incorporating reversal of different sets of point mutations to assess their effect on the conformational stability and activity of 12M. Our analysis has revealed that reversal of certain point mutations in 12M had little effect on its conformational stability, suggesting that these mutations were probably inconsequential towards the thermostability of the 12M mutant. Interestingly these mutations involved evolutionarily conserved residues. On the other hand, some of the other point mutations incorporated in nonconserved regions, appeared to contribute significantly towards the conformational stability and/or activity of 12M. Based on the analysis of dynamics of in silico mutants generated using the consensus sequence, we identified experimentally verifiable residue positions to further increase the conformational stability and activity of the 12M mutant.

Effects of point mutations on the thermostability of B. subtilis lipase: investigating nonadditivity.

PubMed

Singh, Bipin; Bulusu, Gopalakrishnan; Mitra, Abhijit

2016-10-01

Molecular level understanding of mutational effects on stability and activity of enzymes is challenging particularly when several point mutations are incorporated during the directed evolution experiments. In our earlier study, we have suggested the lack of consistency in the effect of point mutations incorporated during the initial generations of directed evolution experiments, towards conformational stabilization of B. subtilis lipase mutants of later generations. Here, we report that the cumulative point mutations incorporated in mutants 2M (with two point mutations) to 6M (with six point mutations) possibly do not retain their original stabilizing nature in the most thermostable 12M mutant (with 12 point mutations). We have carried out MD simulations using structures incorporating reversal of different sets of point mutations to assess their effect on the conformational stability and activity of 12M. Our analysis has revealed that reversal of certain point mutations in 12M had little effect on its conformational stability, suggesting that these mutations were probably inconsequential towards the thermostability of the 12M mutant. Interestingly these mutations involved evolutionarily conserved residues. On the other hand, some of the other point mutations incorporated in nonconserved regions, appeared to contribute significantly towards the conformational stability and/or activity of 12M. Based on the analysis of dynamics of in silico mutants generated using the consensus sequence, we identified experimentally verifiable residue positions to further increase the conformational stability and activity of the 12M mutant.

Structural insight into recruitment of translesion DNA polymerase Dpo4 to sliding clamp PCNA

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

Xing, G.; Kirouac, K.; Shin, Y.J.

2009-09-16

DNA polymerases are co-ordinated by sliding clamps (PCNA/{beta}-clamp) in translesion synthesis. It is unclear how these enzymes assemble on PCNA with geometric and functional compatibility. We report the crystal structure of a full-length Y-family polymerase, Dpo4, in complex with heterodimeric PCNA1-PCNA2 at 2.05 {angstrom} resolution. Dpo4 exhibits an extended conformation that differs from the Dpo4 structures in apo- or DNA-bound form. Two hinges have been identified in Dpo4, which render the multidomain polymerase flexible conformations and orientations relative to PCNA. Dpo4 binds specifically to PCNA1 on the conserved ligand binding site. The C-terminal peptide of Dpo4 becomes structured with amore » 3{sub 10} helix and dominates the specific binding. The Y-family polymerase also contacts PCNA1 with its finger, thumb and little finger domains, which are conformation-dependent protein-protein interactions that diversify the binding mode of Dpo4 on PCNA. The structure reveals a molecular model in which substrate/partner binding-coupled multiple conformations of a Y-family polymerase facilitate its recruitment and co-ordination on the sliding clamp. The conformational flexibility would turn the error-prone Y-family polymerase off when more efficient high-fidelity DNA polymerases work on undamaged DNA and turn it onto DNA templates to perform translesion synthesis when replication forks are stalled by DNA lesions.« less

Basic Theory of Fractional Conformal Invariance of Mei Symmetry and its Applications to Physics

NASA Astrophysics Data System (ADS)

Luo, Shao-Kai; Dai, Yun; Yang, Ming-Jing; Zhang, Xiao-Tian

2018-04-01

In this paper, we present a basic theory of fractional dynamics, i.e., the fractional conformal invariance of Mei symmetry, and find a new kind of conserved quantity led by fractional conformal invariance. For a dynamical system that can be transformed into fractional generalized Hamiltonian representation, we introduce a more general kind of single-parameter fractional infinitesimal transformation of Lie group, the definition and determining equation of fractional conformal invariance are given. And then, we reveal the fractional conformal invariance of Mei symmetry, and the necessary and sufficient condition whether the fractional conformal invariance would be the fractional Mei symmetry is found. In particular, we present the basic theory of fractional conformal invariance of Mei symmetry and it is found that, using the new approach, we can find a new kind of conserved quantity; as a special case, we find that an autonomous fractional generalized Hamiltonian system possesses more conserved quantities. Also, as the new method's applications, we, respectively, find the conserved quantities of a fractional general relativistic Buchduhl model and a fractional Duffing oscillator led by fractional conformal invariance of Mei symmetry.

Tuning Riboswitch Regulation through Conformational Selection

PubMed Central

Wilson, Ross C.; Smith, Angela M.; Fuchs, Ryan T.; Kleckner, Ian R.; Henkin, Tina M.; Foster, Mark P.

2010-01-01

SUMMARY The SMK box riboswitch, which represents one of three known classes of S-adenosylmethionine (SAM)-responsive riboswitches, regulates gene expression in bacteria at the level of translation initiation. In contrast to most riboswitches, which contain separate domains responsible for ligand recognition and gene regulation, the ligand-binding and regulatory domains of the SMK box riboswitch are coincident. This property was exploited to allow the first atomic-level characterization of a functionally intact riboswitch in both the ligand-bound and ligand-free states. NMR spectroscopy revealed distinct mutually exclusive RNA conformations that are differentially populated in the presence or absence of the effector metabolite. Isothermal titration calorimetry and in vivo reporter assay results revealed the thermodynamic and functional consequences of this conformational equilibrium. We present a comprehensive model of the structural, thermodynamic, and functional properties of this compact RNA regulatory element. PMID:21075119

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

PubMed

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

2016-06-01

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

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

NASA Astrophysics Data System (ADS)

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

2016-01-01

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

Stability and properties of quasi-stable conformational states in the LH2 light-harvesting complex of Rbl. acidophilus bacteria formed by hexacoordination of bacteriochlorophyll a magnesium atom

NASA Astrophysics Data System (ADS)

Belov, Aleksandr S.; Khokhlov, Daniil V.; Glebov, Ilya O.; Poddubnyy, Vladimir V.; Eremin, Vadim V.

2017-06-01

Single-molecule spectroscopic experiments on several light-harvesting complexes revealed the existence of a set of metastable conformational states with different spectroscopic properties and lifetimes spanning from milliseconds to tens of seconds. In the absence of explicit structural data, a number of probable structural changes underlying the observed spectroscopic shifts were proposed. We examine the donor-acceptor interaction between the magnesium atom and the acetyl group of the adjacent bacteriochlorophylls a as a possible origin of metastable conformational states in the LH2 light-harvesting complex of Rbl. acidophilus bacteria. The results of QM/MM and molecular dynamics simulations show that such ligation can occur at room temperature and leads to one metastable coordination bond per pair of bacteriochlorophylls in the B850 ring. According to the results of Poisson-TrESP modeling, such coordination lowers the energies of the excited states of the complex by up to 163 cm-1 which causes red spectral shift of the B850 band.

Accessing protein conformational ensembles using room-temperature X-ray crystallography

PubMed Central

Fraser, James S.; van den Bedem, Henry; Samelson, Avi J.; Lang, P. Therese; Holton, James M.; Echols, Nathaniel; Alber, Tom

2011-01-01

Modern protein crystal structures are based nearly exclusively on X-ray data collected at cryogenic temperatures (generally 100 K). The cooling process is thought to introduce little bias in the functional interpretation of structural results, because cryogenic temperatures minimally perturb the overall protein backbone fold. In contrast, here we show that flash cooling biases previously hidden structural ensembles in protein crystals. By analyzing available data for 30 different proteins using new computational tools for electron-density sampling, model refinement, and molecular packing analysis, we found that crystal cryocooling remodels the conformational distributions of more than 35% of side chains and eliminates packing defects necessary for functional motions. In the signaling switch protein, H-Ras, an allosteric network consistent with fluctuations detected in solution by NMR was uncovered in the room-temperature, but not the cryogenic, electron-density maps. These results expose a bias in structural databases toward smaller, overpacked, and unrealistically unique models. Monitoring room-temperature conformational ensembles by X-ray crystallography can reveal motions crucial for catalysis, ligand binding, and allosteric regulation. PMID:21918110

Analysis of the morphology, stability, and folding pathways of ring polymers with supramolecular topological constraints using molecular simulation and nonlinear manifold learning

NASA Astrophysics Data System (ADS)

Wang, Jiang; Ferguson, Andrew

Ring polymers offer a wide range of natural and engineered functions and applications, including as circular bacterial DNA, crown ethers for cation chelation, and ``molecular machines'' such as mechanical nanoswitches. The morphology and dynamics of ring polymers are governed by the chemistry and degree of polymerization of the ring, and intramolecular and supramolecular topological constraints such as knots or mechanically-interlocked rings. We perform molecular dynamics simulations of polyethylene ring polymers as a function of degree of polymerization and in different topological states, including a knotted state, catenane state (two interlocked rings), and borromean state (three interlocked rings). Applying nonlinear manifold learning to our all-atom simulation trajectories, we extract low-dimensional free energy surfaces governing the accessible conformational states and their relative thermodynamic stability. The free energy surfaces reveal how degree of polymerization and topological constraints affect the thermally accessible conformations, chiral symmetry breaking, and folding and collapse pathways of the rings, and present a means to rationally engineer ring size and topology to preferentially stabilize particular conformational states.

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

PubMed

Pal, Somedatta; Bandyopadhyay, Sanjoy

2013-05-16

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

Conformation Changes, N-terminal Involvement, and cGMP Signal Relay in the Phosphodiesterase-5 GAF Domain*

PubMed Central

Wang, Huanchen; Robinson, Howard; Ke, Hengming

2010-01-01

The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, which may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98–147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes. PMID:20861010

Folding of the 25 residue Abeta(12-36) peptide in TFE/water: temperature-dependent transition from a funneled free-energy landscape to a rugged one.

PubMed

Kamiya, Narutoshi; Mitomo, Daisuke; Shea, Joan-Emma; Higo, Junichi

2007-05-17

The free-energy landscape of the Alzheimer beta-amyloid peptide Abeta(12-36) in a 40% (v/v) 2,2,2-trifluoroethanol (TFE)/water solution was determined by using multicanonical molecular dynamics simulations. Simulations using this enhanced conformational sampling technique were initiated from a random unfolded polypeptide conformation. Our simulations reliably folded the peptide to the experimental NMR structure, which consists of two linked helices. The shape of the free energy landscape for folding was found to be strongly dependent on temperature: Above 325 K, the overall shape was funnel-like, with the bottom of the funnel coinciding exactly with the NMR structure. Below 325 K, on the other hand, the landscape became increasingly rugged, with the emergence of new conformational clusters connected by low free-energy pathways. Finally, our simulations reveal that water and TFE solvate the polypeptide in different ways: The hydrogen bond formation between TFE and Abeta was enhanced with decreasing temperature, while that between water and Abeta was depressed.

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.

Conformation changes, N-terminal involvement and cGMP signal relay in phosphodiesterase-5 GAF domain

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

Wang, H.; Robinson, H.; Ke, H.

2010-12-03

The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, whichmore » may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98-147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes.« less

Conformation Changes N-terminal Involvement and cGMP Signal Relay in the Phosphodiesterase-5 GAF Domain

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

H Wang; H Robinson; H Ke

2011-12-31

The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, whichmore » may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98-147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes.« less

77 FR 60887 - Airworthiness Directives; Alpha Aviation Concept Limited Airplanes

Federal Register 2010, 2011, 2012, 2013, 2014

2012-10-05

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2012-07-30

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Cryo-EM of the pathogenic VCP variant R155P reveals long-range conformational changes in the D2 ATPase ring

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

Mountassif, Driss; Fabre, Lucien; Zaid, Younes

Single amino acid mutations in valosin containing protein (VCP/p97), a highly conserved member of the ATPases associated with diverse cellular activities (AAA) family of ATPases has been linked to a severe degenerative disease affecting brain, muscle and bone tissue. Previous studies have demonstrated the role of VCP mutations in altering the ATPase activity of the D2 ring; however the structural consequences of these mutations remain unclear. In this study, we report the three-dimensional (3D) map of the pathogenic VCP variant, R155P, as revealed by single-particle Cryo-Electron Microscopy (EM) analysis at 14 Å resolution. We show that the N-terminal R155P mutation inducesmore » a large structural reorganisation of the D2 ATPase ring. Results from docking studies using crystal structure data of available wild-type VCP in the EM density maps indicate that the major difference is localized at the interface between two protomers within the D2 ring. Consistent with a conformational change, the VCP R155P variant shifted the isoelectric point of the protein and reduced its interaction with its well-characterized cofactor, nuclear protein localization-4 (Npl4). Together, our results demonstrate that a single amino acid substitution in the N-terminal domain can relay long-range conformational changes to the distal D2 ATPase ring. Our results provide the first structural clues of how VCP mutations may influence the activity and function of the D2 ATPase ring. - Highlights: • p97{sub R155P} and p97{sub A232E} decrease the ability of p97 to bind to its co-factor Npl4. • p97{sub R155P} has a different isoelectric point than that of p97{sub R95G}, p97{sub A232E} and p97{sub WT}. • Mutation R155P changes principally the conformation of the D2 ring. • Mutation R155P modifies the interface between two protomers within the D2 ring.« less

The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET.

PubMed

Yang, Mengyi; Peng, Sijia; Sun, Ruirui; Lin, Jingdi; Wang, Nan; Chen, Chunlai

2018-01-09

Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Biological oscillations: Fluorescence monitoring by confocal microscopy

NASA Astrophysics Data System (ADS)

Chattoraj, Shyamtanu; Bhattacharyya, Kankan

2016-09-01

Fluctuations play a vital role in biological systems. Single molecule spectroscopy has recently revealed many new kinds of fluctuations in biological molecules. In this account, we focus on structural fluctuations of an antigen-antibody complex, conformational dynamics of a DNA quadruplex, effects of taxol on dynamics of microtubules, intermittent red-ox oscillations at different organelles in a live cell (mitochondria, lipid droplets, endoplasmic reticulum and cell membrane) and stochastic resonance in gene silencing. We show that there are major differences in these dynamics between a cancer cell and the corresponding non-cancer cell.

Structure of H/ACA RNP protein Nhp2p reveals cis/trans isomerization of a conserved proline at the RNA and Nop10 binding interface

PubMed Central

Koo, Bon-Kyung; Park, Chin-Ju; Fernandez, Cesar F.; Chim, Nicholas; Ding, Yi; Chanfreau, Guillaume; Feigon, Juli

2011-01-01

H/ACA small nucleolar and Cajal body ribonucleoproteins (RNPs) function in site-specific pseudouridylation of eukaryotic rRNA and snRNA, rRNA processing, and vertebrate telomerase biogenesis. Nhp2, one of four essential protein components of eukaryotic H/ACA RNPs, forms a core trimer with the pseudouridylase Cbf5 and Nop10 that specifically binds to H/ACA RNAs. Crystal structures of archaeal H/ACA RNPs have revealed how the protein components interact with each other and with the H/ACA RNA. However, in place of Nhp2p, archaeal H/ACA RNPs contain L7Ae, which binds specifically to an RNA K-loop motif absent in eukaryotic H/ACA RNPs, while Nhp2 binds a broader range of RNA structures. We report solution NMR studies of S. cerevisiae Nhp2 (Nhp2p), which reveal that Nhp2p exhibits two major conformations in solution due to cis/trans isomerization of the evolutionarily conserved Pro83. The equivalent proline is in the cis conformation in all reported structures of L7Ae and other homologous proteins. Nhp2p has the expected α-β-α fold, but the solution structures of the major conformation of Nhp2p with trans Pro83 and of Nhp2p-S82W with cis Pro83 reveal that Pro83 cis/trans isomerization affects the positions of numerous residues at the Nop10- and RNA-binding interface. An S82W substitution, which stabilizes the cis conformation, also stabilizes the association of Nhp2p with H/ACA snoRNPs in vivo. We propose that Pro83 plays a key role in the assembly of the eukaryotic H/ACA RNP, with the cis conformation locking in a stable Cbf5-Nop10-Nhp2 ternary complex and positioning the protein backbone to interact with the H/ACA RNA. PMID:21708174

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

Structural investigation of cellobiose dehydrogenase IIA: Insights from small angle scattering into intra- and intermolecular electron transfer mechanisms

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

Bodenheimer, Annette M.; O'Dell, William B.; Oliver, Ryan C.

Background: Cellobiose dehydrogenases have gained interest due to their potential applications in sectors from biofuel production to biomedical devices. The CDHIIA variant is comprised of a cytochrome domain (CYT), a dehydrogenase domain (DH), and a carbohydrate-binding module (CBM) that are connected by two flexible linkers. Upon cellobiose oxidation at the DH, intramolecular electron transfer (IaET) occurs from the DH to the CYT. In vivo, CDHIIA CYT subsequently performs intermolecular electron transfer (IeET) to a lytic polysaccharide monooxygenase (LPMO). The relevant solution-state CDH domain conformations for IaET and IeET have not been fully characterized.Methods: Small-angle X-ray and neutron scattering measurements ofmore » oxidized CDHIIA from Myriococcum thermophilum and Neurospora crassa were performed to investigate the structural landscape explored in solution by MtCDHIIA and NcCDHIIA in response to cations, pH, and the presence of an electron acceptor, LPMO9D from N. crassa.Results: The scattering data complemented by modeling show that, under oxidizing conditions, MtCDHIIA undergoes global conformational rearrangement in the presence of Ca2+. Oxidized NcCDHIIA exhibits conformational changes upon pH variation and, in the presence of NcLPMO9D, primarily adopts a compact conformation.Conclusions: These results demonstrate different conformational responses of oxidized MtCDHIIA and NcCDHIIA to changes in environment. The results also reveal a shift in the oxidized NcCDHIIA conformational landscape toward interdomain compaction upon co-incubation with NcLPMO9D.General significance: The present study is the first report on the structural landscapes explored in solution by oxidized cellobiose dehydrogenases under various cation concentrations, pH conditions and in the presence of an electron-accepting LPMO.« less

Structural investigation of cellobiose dehydrogenase IIA: Insights from small angle scattering into intra- and intermolecular electron transfer mechanisms

DOE PAGES

Bodenheimer, Annette M.; O'Dell, William B.; Oliver, Ryan C.; ...

2018-01-31

Background: Cellobiose dehydrogenases have gained interest due to their potential applications in sectors from biofuel production to biomedical devices. The CDHIIA variant is comprised of a cytochrome domain (CYT), a dehydrogenase domain (DH), and a carbohydrate-binding module (CBM) that are connected by two flexible linkers. Upon cellobiose oxidation at the DH, intramolecular electron transfer (IaET) occurs from the DH to the CYT. In vivo, CDHIIA CYT subsequently performs intermolecular electron transfer (IeET) to a lytic polysaccharide monooxygenase (LPMO). The relevant solution-state CDH domain conformations for IaET and IeET have not been fully characterized.Methods: Small-angle X-ray and neutron scattering measurements ofmore » oxidized CDHIIA from Myriococcum thermophilum and Neurospora crassa were performed to investigate the structural landscape explored in solution by MtCDHIIA and NcCDHIIA in response to cations, pH, and the presence of an electron acceptor, LPMO9D from N. crassa.Results: The scattering data complemented by modeling show that, under oxidizing conditions, MtCDHIIA undergoes global conformational rearrangement in the presence of Ca2+. Oxidized NcCDHIIA exhibits conformational changes upon pH variation and, in the presence of NcLPMO9D, primarily adopts a compact conformation.Conclusions: These results demonstrate different conformational responses of oxidized MtCDHIIA and NcCDHIIA to changes in environment. The results also reveal a shift in the oxidized NcCDHIIA conformational landscape toward interdomain compaction upon co-incubation with NcLPMO9D.General significance: The present study is the first report on the structural landscapes explored in solution by oxidized cellobiose dehydrogenases under various cation concentrations, pH conditions and in the presence of an electron-accepting LPMO.« less

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.

Raman characterization of Avocado Sunblotch viroid and its response to external perturbations and self-cleavage

PubMed Central

2014-01-01

Background Viroids are the smallest pathogens of plants. To date the structural and conformational details of the cleavage of Avocado sunblotch viroid (ASBVd) and the catalytic role of Mg2+ ions in efficient self-cleavage are of crucial interest. Results We report the first Raman characterization of the structure and activity of ASBVd, for plus and minus viroid strands. Both strands exhibit a typical A-type RNA conformation with an ordered double-helical content and a C3′-endo/anti sugar pucker configuration, although small but specific differences are found in the sugar puckering and base-stacking regions. The ASBVd(-) is shown to self-cleave 3.5 times more actively than ASBVd(+). Deuteration and temperature increase perturb differently the double-helical content and the phosphodiester conformation, as revealed by corresponding characteristic Raman spectral changes. Our data suggest that the structure rigidity and stability are higher and the D2O accessibility to H-bonding network is lower for ASBVd(+) than for ASBVd(-). Remarkably, the Mg2+-activated self-cleavage of the viroid does not induce any significant alterations of the secondary viroid structure, as evidenced from the absence of intensity changes of Raman marker bands that, however exhibit small but noticeable frequency downshifts suggesting several minor changes in phosphodioxy, internal loops and hairpins of the cleaved viroids. Conclusions Our results demonstrate the sensitivity of Raman spectroscopy in monitoring structural and conformational changes of the viroid and constitute the basis for further studies of its interactions with therapeutic agents and cell membranes. PMID:24655924

Principal component and clustering analysis on molecular dynamics data of the ribosomal L11·23S subdomain.

PubMed

Wolf, Antje; Kirschner, Karl N

2013-02-01

With improvements in computer speed and algorithm efficiency, MD simulations are sampling larger amounts of molecular and biomolecular conformations. Being able to qualitatively and quantitatively sift these conformations into meaningful groups is a difficult and important task, especially when considering the structure-activity paradigm. Here we present a study that combines two popular techniques, principal component (PC) analysis and clustering, for revealing major conformational changes that occur in molecular dynamics (MD) simulations. Specifically, we explored how clustering different PC subspaces effects the resulting clusters versus clustering the complete trajectory data. As a case example, we used the trajectory data from an explicitly solvated simulation of a bacteria's L11·23S ribosomal subdomain, which is a target of thiopeptide antibiotics. Clustering was performed, using K-means and average-linkage algorithms, on data involving the first two to the first five PC subspace dimensions. For the average-linkage algorithm we found that data-point membership, cluster shape, and cluster size depended on the selected PC subspace data. In contrast, K-means provided very consistent results regardless of the selected subspace. Since we present results on a single model system, generalization concerning the clustering of different PC subspaces of other molecular systems is currently premature. However, our hope is that this study illustrates a) the complexities in selecting the appropriate clustering algorithm, b) the complexities in interpreting and validating their results, and c) by combining PC analysis with subsequent clustering valuable dynamic and conformational information can be obtained.

Dynamic switching mechanisms of a CC chemokine, CCL5 (RANTES). A simulation study

NASA Astrophysics Data System (ADS)

Peter, Emanuel; Pivkin, Igor

CCL5 (RANTES) belongs to the class of pro-inflammatory chemokines which are part of the human immune-response. It is known to activate leukocytes through its associated chemokine receptor 5 (CCR5) and plays a key role in several malignancies, including HIV-1 infections and cancer. In this talk, we present our results from enhanced sampling simulations of the CCL5 (RANTES) monomer. We find that this protein can adopt 2 different conformations : a globular form, with an orthogonal alignment of the N-terminal part, and a 'cis' form, in which the N-terminus is aligned parallel to the β-strand interface. A detailed analysis of the structure reveals that each of these states is stabilized by salt-bridges along the sequence, and corresponds to a defined dihedral-geometry of the 2 disulfide bridges Cys10-34 and Cys11-50. We derive a uniform distribution of transitions from the globular form of CCL5 (RANTES), and find that each of the main conformers adopts different electrostatic patterns.

Functional rotation induced by alternating protonation states in the multidrug transporter AcrB: all-atom molecular dynamics simulations.

PubMed

Yamane, Tsutomu; Murakami, Satoshi; Ikeguchi, Mitsunori

2013-10-29

The multidrug transporter AcrB actively exports a wide variety of noxious compounds using proton-motive force as an energy source in Gram-negative bacteria. AcrB adopts an asymmetric structure comprising three protomers with different conformations that are sequentially converted during drug export; these cyclic conformational changes during drug export are referred to as functional rotation. To investigate functional rotation driven by proton-motive force, all-atom molecular dynamics simulations were performed. Using different protonation states for the titratable residues in the middle of the transmembrane domain, our simulations revealed the correlation between the specific protonation states and the side-chain configurations. Changing the protonation state for Asp408 induced a spontaneous structural transition, which suggests that the proton translocation stoichiometry may be one proton per functional rotation cycle. Furthermore, our simulations demonstrate that alternating the protonation states in the transmembrane domain induces functional rotation in the porter domain, which is primarily responsible for drug transport.

Conformational properties of chiral tobacco alkaloids by DFT calculations and vibrational circular dichroism: (-)-S-anabasine.

PubMed

Rodríguez Ortega, P G; Montejo, M; Márquez, F; López González, J J

2015-07-01

A thorough DFT and MM study of the conformational landscape, molecular and electronic structures of (-)-S-anabasine is reported aimed to reveal the mechanism controlling its conformational preference. Although the conformational flexibility and diversity of this system is quite extensive, only two structures are populated both in gas-phase and solution (CCl4 and DMSO). NBO-aided electronic structure analyses performed for the eight conformers representing minima in the potential energy surface of (-)-S-anabasine indicate that both steric and electrostatic factors are determinant in the conformational distribution of the sample in gas phase. Nonetheless, hyperconjugative effects are the key force tipping the balance in the conformational equilibrium between the two main rotamers. Increasing the polarity of the medium (using the IEF-PCM formalism) barely affect the conformational energy profile, although a slight increase in the theoretical population of those structures more affected by electrostatic interactions is predicted. The validity of the theoretical models and calculated conformers populations are endorsed by the accurate reproduction of the IR and VCD spectra (recorded in pure liquid and in CCl4 solution) of the sample (that have been firstly recorded and assigned in the present work) which are consistent with the occurrence of a 2:1 conformational ratio. Copyright © 2015 Elsevier Inc. All rights reserved.

Single-Molecule Spectroscopy and Imaging Studies of Protein Dynamics

NASA Astrophysics Data System (ADS)

Lu, H. Peter

2012-04-01

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

Application of MCR-ALS to reveal intermediate conformations in the thermally induced α-β transition of poly-L-lysine monitored by FT-IR spectroscopy

NASA Astrophysics Data System (ADS)

Alcaráz, Mirta R.; Schwaighofer, Andreas; Goicoechea, Héctor; Lendl, Bernhard

2017-10-01

Temperature-induced conformational transitions of poly-L-lysine were monitored with Fourier-transform infrared (FT-IR) spectroscopy between 10 °C and 70 °C. Chemometric analysis of dynamic IR spectra was performed by multivariate curve analysis-alternating least squares (MCR-ALS) of the amide I‧ and amide II‧ spectral region. With this approach, the pure spectral and concentration profiles of the conformational transition were obtained. Beside the initial α-helical, the intermediate random coil/extended helices and the final β-sheet structure, an additional intermediate PLL conformation was identified and attributed to a transient β-sheet structure.

Atomic structures of corkscrew-forming segments of SOD1 reveal varied oligomer conformations.

PubMed

Sangwan, Smriti; Sawaya, Michael R; Murray, Kevin A; Hughes, Michael P; Eisenberg, David S

2018-02-17

The aggregation cascade of disease-related amyloidogenic proteins, terminating in insoluble amyloid fibrils, involves intermediate oligomeric states. The structural and biochemical details of these oligomers have been largely unknown. Here we report crystal structures of variants of the cytotoxic oligomer-forming segment residues 28-38 of the ALS-linked protein, SOD1. The crystal structures reveal three different architectures: corkscrew oligomeric structure, nontwisting curved sheet structure and a steric zipper proto-filament structure. Our work highlights the polymorphism of the segment 28-38 of SOD1 and identifies the molecular features of amyloidogenic entities. © 2018 The Protein Society.

The multi-state energy landscape of the SAM-I riboswitch: A single-molecule Förster resonance energy transfer spectroscopy study

NASA Astrophysics Data System (ADS)

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

2018-03-01

RNA (ribonucleic acid) molecules are highly flexible biopolymers fluctuating at physiological temperatures among many different conformations that are represented by minima in a hierarchical conformational free energy landscape. Here we have employed single-molecule FRET (smFRET) to explore the energy landscape of the B. subtilis yitJ SAM-I riboswitch (RS). In this small RNA molecule, specific binding of an S-adenosyl-L-methionine (SAM) ligand in the aptamer domain regulates gene expression by inducing structural changes in another domain, the expression platform, causing transcription termination by the RNA polymerase. We have measured smFRET histograms over wide ranges of Mg2+ concentration for three RS variants that were specifically labeled with fluorescent dyes on different sites. In the analysis, different conformations are associated with discrete Gaussian model distributions, which are typically fairly broad on the FRET efficiency scale and thus can be extremely challenging to unravel due to their mutual overlap. Our earlier work on two SAM-I RS variants revealed four major conformations. By introducing a global fitting procedure which models both the Mg2+ concentration dependencies of the fractional populations and the average FRET efficiencies of the individual FRET distributions according to Mg2+ binding isotherms, we were able to consistently describe the histogram data of both variants at all studied Mg2+ concentrations. With the third FRET-labeled variant, however, we found significant deviations when applying the four-state model to the data. This can arise because the different FRET labeling of the new variant allows two states to be distinguished that were previously not separable due to overlap. Indeed, the resulting five-state model presented here consistently describes the smFRET histograms of all three variants as well as their variations with Mg2+ concentration. We also performed a triangulation of the donor position for two of the constructs to explore how the expression platform is oriented with respect to the aptamer.

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

Cross-protection in Neisseria meningitidis serogroups Y and W polysaccharides: A comparative conformational analysis.

PubMed

Kuttel, Michelle M; Timol, Zaheer; Ravenscroft, Neil

2017-06-29

The capsular polysaccharide is the main virulence factor in meningococcus. The capsular polysaccharides for meningococcal serogroups Y and W are almost identical polymers of hexose-sialic acid, suggesting the possibility of cross-protection between group Y and W vaccines. However, early studies indicated that they elicit different levels of cross-protection. Here we explore the conformations of the meningococcal Y and W polysaccharides with molecular dynamics simulations of three repeating unit oligosaccharide strands. We find differences in Y and W antigen conformation: the Y polysaccharide has a single dominant conformation, whereas W exhibits a family of conformations including the Y conformation. This result is supported by our NMR NOESY analysis, which indicates key close contacts for W that are not present in Y. These conformational differences provide an explanation for the different levels of cross-protection measured for the Y and W monovalent vaccines and the high group W responses observed in HibMenCY-TT vaccinees. Copyright © 2017 Elsevier Ltd. All rights reserved.

The calculations of small molecular conformation energy differences by density functional method

NASA Astrophysics Data System (ADS)

Topol, I. A.; Burt, S. K.

1993-03-01

The differences in the conformational energies for the gauche (G) and trans(T) conformers of 1,2-difluoroethane and for myo-and scyllo-conformer of inositol have been calculated by local density functional method (LDF approximation) with geometry optimization using different sets of calculation parameters. It is shown that in the contrast to Hartree—Fock methods, density functional calculations reproduce the correct sign and value of the gauche effect for 1,2-difluoroethane and energy difference for both conformers of inositol. The results of normal vibrational analysis for1,2-difluoroethane showed that harmonic frequencies calculated in LDF approximation agree with experimental data with the accuracy typical for scaled large basis set Hartree—Fock calculations.

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

Human immunoglobulin E flexes between acutely bent and extended conformations

PubMed Central

Keeble, Anthony H; Wright, Michael; Cain, Katharine; Hailu, Hanna; Oxbrow, Amanda; Delgado, Jean; Shuttleworth, Lindsay K; Kao, Michael W-P; McDonnell, James M; Beavil, Andrew J; Henry, Alistair J; Sutton, Brian J

2014-01-01

Crystallographic and solution studies have shown that IgE molecules are acutely bent in their Fc region. Crystal structures reveal the Cε2 domain pair folded back onto the Cε3-Cε4 domains, but is the molecule exclusively bent or can the Cε2 domains adopt extended conformations and even “flip” from one side of the molecule to the other? We report the crystal structure of IgE-Fc captured in a fully extended, symmetrical conformation and show by molecular dynamics, calorimetry, stopped-flow kinetic, SPR and FRET analyses, that the antibody can indeed adopt such extended conformations in solution. This diversity of conformational states available to IgE-Fc offers a new perspective on IgE function in allergen recognition, as part of the B cell receptor and as a therapeutic target in allergic disease. PMID:24632569

Structural basis for the metal-selective activation of the manganese transport regulator of Bacillus subtilis.

PubMed

Kliegman, Joseph I; Griner, Sarah L; Helmann, John D; Brennan, Richard G; Glasfeld, Arthur

2006-03-21

The manganese transport regulator (MntR) of Bacillus subtilis is activated by Mn(2+) to repress transcription of genes encoding transporters involved in the uptake of manganese. MntR is also strongly activated by cadmium, both in vivo and in vitro, but it is poorly activated by other metal cations, including calcium and zinc. The previously published MntR.Mn(2+) structure revealed a binuclear complex of manganese ions with a metal-metal separation of 3.3 A (herein designated the AB conformer). Analysis of four additional crystal forms of MntR.Mn(2+) reveals that the AB conformer is only observed in monoclinic crystals at 100 K, suggesting that this conformation may be stabilized by crystal packing forces. In contrast, monoclinic crystals analyzed at room temperature (at either pH 6.5 or pH 8.5), and a second hexagonal crystal form (analyzed at 100 K), all reveal the shift of one manganese ion by 2.5 A, thereby leading to a newly identified conformation (the AC conformer) with an internuclear distance of 4.4 A. Significantly, the cadmium and calcium complexes of MntR also contain binuclear complexes with a 4.4 A internuclear separation. In contrast, the zinc complex of MntR contains only one metal ion per subunit, in the A site. Isothermal titration calorimetry confirms the stoichiometry of Mn(2+), Cd(2+), and Zn(2+) binding to MntR. We propose that the specificity of MntR activation is tied to productive binding of metal ions at two sites; the A site appears to act as a selectivity filter, determining whether the B or C site will be occupied and thereby fully activate MntR.

Conformational flexibility of two RNA trimers explored by computational tools and database search.

PubMed

Fadrná, Eva; Koca, Jaroslav

2003-04-01

Two RNA sequences, AAA and AUG, were studied by the conformational search program CICADA and by molecular dynamics (MD) in the framework of the AMBER force field, and also via thorough PDB database search. CICADA was used to provide detailed information about conformers and conformational interconversions on the energy surfaces of the above molecules. Several conformational families were found for both sequences. Analysis of the results shows differences, especially between the energy of the single families, and also in flexibility and concerted conformational movement. Therefore, several MD trajectories (altogether 16 ns) were run to obtain more details about both the stability of conformers belonging to different conformational families and about the dynamics of the two systems. Results show that the trajectories strongly depend on the starting structure. When the MD start from the global minimum found by CICADA, they provide a stable run, while MD starting from another conformational family generates a trajectory where several different conformational families are visited. The results obtained by theoretical methods are compared with the thorough database search data. It is concluded that all except for the highest energy conformational families found in theoretical result also appear in experimental data. Registry numbers: adenylyl-(3' --> 5')-adenylyl-(3' --> 5')-adenosine [917-44-2] adenylyl-(3' --> 5')-uridylyl-(3' --> 5')-guanosine [3494-35-7].

Structural Polymorphism in a Self-Assembled Tri-Aromatic Peptide System.

PubMed

Brown, Noam; Lei, Jiangtao; Zhan, Chendi; Shimon, Linda J W; Adler-Abramovich, Lihi; Wei, Guanghong; Gazit, Ehud

2018-04-24

Self-assembly is a process of key importance in natural systems and in nanotechnology. Peptides are attractive building blocks due to their relative facile synthesis, biocompatibility, and other unique properties. Diphenylalanine (FF) and its derivatives are known to form nanostructures of various architectures and interesting and varied characteristics. The larger triphenylalanine peptide (FFF) was found to self-assemble as efficiently as FF, forming related but distinct architectures of plate-like and spherical nanostructures. Here, to understand the effect of triaromatic systems on the self-assembly process, we examined carboxybenzyl-protected diphenylalanine (z-FF) as a minimal model for such an arrangement. We explored different self-assembly conditions by changing solvent compositions and peptide concentrations, generating a phase diagram for the assemblies. We discovered that z-FF can form a variety of structures, including nanowires, fibers, nanospheres, and nanotoroids, the latter were previously observed only in considerably larger or co-assembly systems. Secondary structure analysis revealed that all assemblies possessed a β-sheet conformation. Additionally, in solvent combinations with high water ratios, z-FF formed rigid and self-healing hydrogels. X-ray crystallography revealed a "wishbone" structure, in which z-FF dimers are linked by hydrogen bonds mediated by methanol molecules, with a 2-fold screw symmetry along the c-axis. All-atom molecular dynamics (MD) simulations revealed conformations similar to the crystal structure. Coarse-grained MD simulated the assembly of the peptide into either fibers or spheres in different solvent systems, consistent with the experimental results. This work thus expands the building block library for the fabrication of nanostructures by peptide self-assembly.

Revealing Abrupt and Spontaneous Ruptures of Protein Native Structure under picoNewton Compressive Force Manipulation.

PubMed

Chowdhury, S Roy; Cao, Jin; He, Yufan; Lu, H Peter

2018-03-27

Manipulating protein conformations for exploring protein structure-function relationship has shown great promise. Although protein conformational changes under pulling force manipulation have been extensively studied, protein conformation changes under a compressive force have not been explored quantitatively. The latter is even more biologically significant and relevant in revealing protein functions in living cells associated with protein crowdedness, distribution fluctuations, and cell osmotic stress. Here we report our experimental observations on abrupt ruptures of protein native structures under compressive force, demonstrated and studied by single-molecule AFM-FRET spectroscopic nanoscopy. Our results show that the protein ruptures are abrupt and spontaneous events occurred when the compressive force reaches a threshold of 12-75 pN, a force amplitude accessible from thermal fluctuations in a living cell. The abrupt ruptures are sensitive to local environment, likely a general and important pathway of protein unfolding in living cells.

Functional dynamics of cell surface membrane proteins

NASA Astrophysics Data System (ADS)

Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

2014-04-01

Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

Functional dynamics of cell surface membrane proteins.

PubMed

Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

2014-04-01

Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules. Copyright © 2013 Elsevier Inc. All rights reserved.

Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms

PubMed Central

Vergara-Jaque, Ariela; Fenollar-Ferrer, Cristina; Kaufmann, Desirée; Forrest, Lucy R.

2015-01-01

Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to one or other side of the membrane, but not both simultaneously. Structures of a bacterial homolog of neuronal glutamate transporters, GltPh, in several different conformational states have revealed that the protein structure is asymmetric in the outward- and inward-open states, and that the conformational change connecting them involves a elevator-like movement of a substrate binding domain across the membrane. The structural asymmetry is created by inverted-topology repeats, i.e., structural repeats with similar overall folds whose transmembrane topologies are related to each other by two-fold pseudo-symmetry around an axis parallel to the membrane plane. Inverted repeats have been found in around three-quarters of secondary transporter folds. Moreover, the (a)symmetry of these systems has been successfully used as a bioinformatic tool, called “repeat-swap modeling” to predict structural models of a transporter in one conformation using the known structure of the transporter in the complementary conformation as a template. Here, we describe an updated repeat-swap homology modeling protocol, and calibrate the accuracy of the method using GltPh, for which both inward- and outward-facing conformations are known. We then apply this repeat-swap homology modeling procedure to a concentrative nucleoside transporter, VcCNT, which has a three-dimensional arrangement related to that of GltPh. The repeat-swapped model of VcCNT predicts that nucleoside transport also occurs via an elevator-like mechanism. PMID:26388773

Dosimetric differences between intraoperative and postoperative plans using Cs-131 in transrectal ultrasound–guided brachytherapy for prostatic carcinoma

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

Jones, Andrew, E-mail: aojones@geisinger.edu; Treas, Jared; Yavoich, Brian

2014-01-01

The aim of the study was to investigate the differences between intraoperative and postoperative dosimetry for transrectal ultrasound–guided transperineal prostate implants using cesium-131 ({sup 131}Cs). Between 2006 and 2010, 166 patients implanted with {sup 131}Cs had both intraoperative and postoperative dosimetry studies. All cases were monotherapy and doses of 115 were prescribed to the prostate. The dosimetric properties (D{sub 90}, V{sub 150}, and V{sub 100} for the prostate) of the studies were compared. Two conformity indices were also calculated and compared. Finally, the prostate was automatically sectioned into 6 sectors (anterior and posterior sectors at the base, midgland, and apex)more » and the intraoperative and postoperative dosimetry was compared in each individual sector. Postoperative dosimetry showed statistically significant changes (p < 0.01) in every dosimetric value except V{sub 150}. In each significant case, the postoperative plans showed lower dose coverage. The conformity indexes also showed a bimodal frequency distribution with the index indicating poorer dose conformity in the postoperative plans. Sector analysis revealed less dose coverage postoperatively in the base and apex sectors with an increase in dose to the posterior midgland sector. Postoperative dosimetry overall and in specific sectors of the prostate differs significantly from intraoperative planning. Care must be taken during the intraoperative planning stage to ensure complete dose coverage of the prostate with the understanding that the final postoperative dosimetry will show less dose coverage.« less

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

PubMed Central

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

2012-01-01

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

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

PubMed

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

2012-11-07

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

Structural distributions from single-molecule measurements as a tool for molecular mechanics

PubMed Central

Hanson, Jeffrey A.; Brokaw, Jason; Hayden, Carl C.; Chu, Jhih-Wei; Yang, Haw

2011-01-01

A mechanical view provides an attractive alternative for predicting the behavior of complex systems since it circumvents the resource-intensive requirements of atomistic models; however, it remains extremely challenging to characterize the mechanical responses of a system at the molecular level. Here, the structural distribution is proposed to be an effective means to extracting the molecular mechanical properties. End-to-end distance distributions for a series of short poly-L-proline peptides with the sequence PnCG3K-biotin (n = 8, 12, 15 and 24) were used to experimentally illustrate this new approach. High-resolution single-molecule Förster-type resonance energy transfer (FRET) experiments were carried out and the conformation-resolving power was characterized and discussed in the context of the conventional constant-time binning procedure for FRET data analysis. It was shown that the commonly adopted theoretical polymer models—including the worm-like chain, the freely jointed chain, and the self-avoiding chain—could not be distinguished by the averaged end-to-end distances, but could be ruled out using the molecular details gained by conformational distribution analysis because similar polymers of different sizes could respond to external forces differently. Specifically, by fitting the molecular conformational distribution to a semi-flexible polymer model, the effective persistence lengths for the series of short poly-L-proline peptides were found to be size-dependent with values of ~190 Å, ~67 Å, ~51 Å, and ~76 Å for n = 8, 12, 15, and 24, respectively. A comprehensive computational modeling was carried out to gain further insights for this surprising discovery. It was found that P8 exists as the extended all-trans isomaer whereas P12 and P15 predominantly contained one proline residue in the cis conformation. P24 exists as a mixture of one-cis (75%) and two-cis (25%) isomers where each isomer contributes to an experimentally resolvable conformational mode. This work demonstrates the resolving power of the distribution-based approach, and the capacity of integrating high-resolution single-molecule FRET experiments with molecular modeling to reveal detailed structural information about the conformation of molecules on the length scales relevant to the study of biological molecules. PMID:22661822

Near-Edge X-ray Absorption Fine Structure Studies of Electrospun Poly(dimethylsiloxane)/Poly (methyl methacrylate)/Multiwall Carbon Nanotube Composites

PubMed Central

Winter, A. Douglas; Larios, Eduardo; Alamgir, Faisal M.; Jaye, Cherno; Fischer, Daniel; Campo, Eva M.

2014-01-01

This work describes the near conduction band edge structure of electrospun mats of MWCNT-PDMS-PMMA by near edge X-Ray absorption fine structure (NEXAFS) spectroscopy. Effects of adding nanofillers of different sizes were addressed. Despite observed morphological variations and inhomogeneous carbon nanotube distribution, spun mats appeared homogeneous under NEXAFS analysis. Spectra revealed differences in emissions from glancing and normal spectra; which may evidence phase separation within the bulk of the micron-size fibers. Further, dichroic ratios show polymer chains did not align, even in the presence of nanofillers. Addition of nanofillers affected emissions in the C-H, C=O and C-C regimes, suggesting their involvement in interfacial matrix-carbon nanotube bonding. Spectral differences at glancing angles between pristine and composite mats suggest that geometric conformational configurations are taking place between polymeric chains and carbon nanotubes. These differences appear to be carbon nanotube-dimension dependent, and are promoted upon room temperature mixing and shear flow during electrospinning. CH-π bonding between polymer chains and graphitic walls, as well as H-bonds between impurities in the as-grown CNTs and polymer pendant groups are proposed bonding mechanisms promoting matrix conformation. PMID:24308286

Is the Conformational Ensemble of Alzheimer’s Aβ10-40 Peptide Force Field Dependent?

PubMed Central

Siwy, Christopher M.

2017-01-01

By applying REMD simulations we have performed comparative analysis of the conformational ensembles of amino-truncated Aβ10-40 peptide produced with five force fields, which combine four protein parameterizations (CHARMM36, CHARMM22*, CHARMM22/cmap, and OPLS-AA) and two water models (standard and modified TIP3P). Aβ10-40 conformations were analyzed by computing secondary structure, backbone fluctuations, tertiary interactions, and radius of gyration. We have also calculated Aβ10-40 3JHNHα-coupling and RDC constants and compared them with their experimental counterparts obtained for the full-length Aβ1-40 peptide. Our study led us to several conclusions. First, all force fields predict that Aβ adopts unfolded structure dominated by turn and random coil conformations. Second, specific TIP3P water model does not dramatically affect secondary or tertiary Aβ10-40 structure, albeit standard TIP3P model favors slightly more compact states. Third, although the secondary structures observed in CHARMM36 and CHARMM22/cmap simulations are qualitatively similar, their tertiary interactions show little consistency. Fourth, two force fields, OPLS-AA and CHARMM22* have unique features setting them apart from CHARMM36 or CHARMM22/cmap. OPLS-AA reveals moderate β-structure propensity coupled with extensive, but weak long-range tertiary interactions leading to Aβ collapsed conformations. CHARMM22* exhibits moderate helix propensity and generates multiple exceptionally stable long- and short-range interactions. Our investigation suggests that among all force fields CHARMM22* differs the most from CHARMM36. Fifth, the analysis of 3JHNHα-coupling and RDC constants based on CHARMM36 force field with standard TIP3P model led us to an unexpected finding that in silico Aβ10-40 and experimental Aβ1-40 constants are generally in better agreement than these quantities computed and measured for identical peptides, such as Aβ1-40 or Aβ1-42. This observation suggests that the differences in the conformational ensembles of Aβ10-40 and Aβ1-40 are small and the former can be used as proxy of the full-length peptide. Based on this argument, we concluded that CHARMM36 force field with standard TIP3P model produces the most accurate representation of Aβ10-40 conformational ensemble. PMID:28085875

Elucidating the ensemble of functionally-relevant transitions in protein systems with a robotics-inspired method

PubMed Central

2013-01-01

Background Many proteins tune their biological function by transitioning between different functional states, effectively acting as dynamic molecular machines. Detailed structural characterization of transition trajectories is central to understanding the relationship between protein dynamics and function. Computational approaches that build on the Molecular Dynamics framework are in principle able to model transition trajectories at great detail but also at considerable computational cost. Methods that delay consideration of dynamics and focus instead on elucidating energetically-credible conformational paths connecting two functionally-relevant structures provide a complementary approach. Effective sampling-based path planning methods originating in robotics have been recently proposed to produce conformational paths. These methods largely model short peptides or address large proteins by simplifying conformational space. Methods We propose a robotics-inspired method that connects two given structures of a protein by sampling conformational paths. The method focuses on small- to medium-size proteins, efficiently modeling structural deformations through the use of the molecular fragment replacement technique. In particular, the method grows a tree in conformational space rooted at the start structure, steering the tree to a goal region defined around the goal structure. We investigate various bias schemes over a progress coordinate for balance between coverage of conformational space and progress towards the goal. A geometric projection layer promotes path diversity. A reactive temperature scheme allows sampling of rare paths that cross energy barriers. Results and conclusions Experiments are conducted on small- to medium-size proteins of length up to 214 amino acids and with multiple known functionally-relevant states, some of which are more than 13Å apart of each-other. Analysis reveals that the method effectively obtains conformational paths connecting structural states that are significantly different. A detailed analysis on the depth and breadth of the tree suggests that a soft global bias over the progress coordinate enhances sampling and results in higher path diversity. The explicit geometric projection layer that biases the exploration away from over-sampled regions further increases coverage, often improving proximity to the goal by forcing the exploration to find new paths. The reactive temperature scheme is shown effective in increasing path diversity, particularly in difficult structural transitions with known high-energy barriers. PMID:24565158

Revealing the importance of linkers in K-series oxime reactivators for tabun-inhibited AChE using quantum chemical, docking and SMD studies

NASA Astrophysics Data System (ADS)

Ghosh, Shibaji; Chandar, Nellore Bhanu; Jana, Kalyanashis; Ganguly, Bishwajit

2017-08-01

Inhibition of acetylcholinesterase (AChE) with organophosphorus compounds has a detrimental effect on human life. Oxime K203 seems to be one of the promising reactivators for tabun-inhibited AChE than (K027, K127, and K628). These reactivators differ only in the linker units between the two pyridinium rings. The conformational analyses performed with quantum chemical RHF/6-31G* level for K027, K127, K203 and K628 showed that the minimum energy conformers have different orientations of the active and peripheral pyridinium rings for these reactivator molecules. K203 with (-CH2-CH=CH-CH2-) linker unit possesses more open conformation compared to the other reactivators. Such orientation of K203 experiences favorable interaction with the surrounding residues of catalytic anionic site (CAS) and peripheral anionic site (PAS) of tabun-inhibited AChE. From the steered molecular dynamics simulations, it has been observed that the oxygen atom of the oxime group of K203 reactivator approaches nearest to the P-atom of the SUN203 (3.75 Å) at lower time scales (less than 1000 ps) as compared to the other reactivators. K203 experiences less number of hydrophobic interaction with the PAS residues which is suggested to be an important factor for the efficient reactivation process. In addition, K203 crates large number of H-bonding with CAS residues SUN203, Phe295, Tyr337, Phe338 and His447. K203 barely changes its conformation during the SMD simulation process and hence the energy penalty to adopt any other conformation is minimal in this case as compared to the other reactivators. The molecular mechanics and Poisson-Boltzmann surface area binding energies obtained for the interaction of K203 inside the gorge of tabun inhibited AChE is substantially higher (-290.2 kcal/mol) than the corresponding K628 reactivator (-260.4 kcal/mol), which also possess unsaturated aromatic linker unit.

Variations in periplasmic loop interactions determine the pH-dependent activity of the hexameric urea transporter UreI from Helicobacter pylori: a molecular dynamics study.

PubMed

Cáceres-Delpiano, Javier; Teneb, Jaime; Mansilla, Rodrigo; García, Apolinaria; Salas-Burgos, Alexis

2015-06-26

Helicobacter pylori is an important factor in the development of diseases such as ulcer and gastric cancer. This bacterium uses a periplasmic transporter, UreI, to deliver urea to the intracelullar space, where later it is transformed into ammonia by the cytoplasmic enzyme urease to survive the acidic condition of the human stomach. The UreI transporter presents a pH-dependent activity, where this pH-dependence remains unknown at a structural level. Althought the existance of several protonable residues in the periplasmic loops are related to the pH-dependent activity, we find interesting to have a clear view of the conformational changes involved in this phenomena through a molecular dynamic study. Molecular dynamic simulations of the UreI transporter at three different pH conditions were performed, revealing two main pH-dependent conformations, which we present as the open and close states. We find that salt bridges between the periplasmic loops are crucial interactions that stabilize these conformations. Besides, a cooperative behaviour exists between the six subunits of the system that is necessary to fulfill the activity of this transporter. We found different pH-dependent conformations of the urea transporter UreI from Helicobacter pylori, which are related to salt-bridge interactions in the periplasmic regions. The behaviour of every channel in the system is not independent, given the existance of a cooperative behaviour through the formation of salt-bridges between the subunits of the hexameric system. We believe that our results will be related to the generation of new eradication therapies using this transporter as an attractive target, denoting that the knowledge of the possible pH-dependent conformations adopted for this transporter are important for the development of rational drug design approximations.

Multiple Conformational States Contribute to the 3D Structure of a Glucan Decasaccharide: A Combined SAXS and MD Simulation Study.

PubMed

Jo, Sunhwan; Myatt, Daniel; Qi, Yifei; Doutch, James; Clifton, Luke A; Im, Wonpil; Widmalm, Göran

2018-01-25

The inherent flexibility of carbohydrates is dependent on stereochemical arrangements, and characterization of their influence and importance will give insight into the three-dimensional structure and dynamics. In this study, a β-(1→4)/β-(1→3)-linked glucosyl decasaccharide is experimentally investigated by synchrotron small-angle X-ray scattering from which its radius of gyration (R g ) is obtained. Molecular dynamics (MD) simulations of the decasaccharide show four populated states at each glycosidic linkage, namely, syn- and anti-conformations. The calculated R g values from the MD simulation reveal that in addition to syn-conformers the presence of anti-ψ conformational states is required to reproduce experimental scattering data, unveiling inherent glycosidic linkage flexibility. The CHARMM36 force field for carbohydrates thus describes the conformational flexibility of the decasaccharide very well and captures the conceptual importance that anti-conformers are to be anticipated at glycosidic linkages of carbohydrates.

Synthesis and stereochemical analysis of β-nitromethane substituted γ-amino acids and peptides.

PubMed

Ganesh Kumar, Mothukuri; Mali, Sachitanand M; Gopi, Hosahudya N

2013-02-07

The high diastereoselectivity in the Michael addition of nitromethane to α,β-unsaturated γ-amino esters, crystal conformations of β-nitromethane substituted γ-amino acids and peptides are studied. Results suggest that the N-Boc protected amide NH, conformations of α,β-unsaturated γ-amino esters and alkyl side chains play a crucial role in dictating the high diastereoselectivity of nitromethane addition to E-vinylogous amino esters. Investigation of the crystal conformations of both α,β-unsaturated γ-amino esters and the Michael addition products suggests that an H-C(γ)-C(β)=C(α) eclipsed conformer of the unsaturated amino ester leads to the major (anti) product compared to that of an N-C(γ)-C(β)=C(α) eclipsed conformer. The major diastereomers were separated and subjected to the peptide synthesis. The single crystal analysis of the dipeptide containing β-nitromethane substituted γ-amino acids reveals a helical type of folded conformation with an isolated H-bond involving a nine-atom pseudocycle.

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

Movie of the structural changes during a catalytic cycle of nucleoside monophosphate kinases.

PubMed

Vonrhein, C; Schlauderer, G J; Schulz, G E

1995-05-15

There are 17 crystal structures of nucleoside monophosphate kinases known. As expected for kinases, they show large conformational changes upon binding of substrates. These are concentrated in two chain segments, or domains, of 30 and 38 residues that are involved in binding of the substrates N1TP and N2MP (nucleoside tri- and monophosphates with bases N1 and N2), respectively. After aligning the 17 structures on the main parts of their polypeptide chains, two domains in various conformational states were revealed. These states were caused by bound substrate (or analogues) and by crystal-packing forces, and ranged between a 'closed' conformation and a less well defined 'open' conformation. The structures were visually sorted yielding an approximately evenly spaced series of domain states that outlines the closing motions when the substrates bind. The packing forces in the crystals are weak, leaving the natural domain trajectories essentially intact. Packing is necessary, however, to produce stable intermediates. The ordered experimental structures were then recorded as still pictures of a movie and animated to represent the motions of the molecule during a catalytic cycle. The motions were smoothed out by adding interpolated structures to the observed ones. The resulting movies are available through the World Wide Web (http:@bio5.chemie.uni-freiburg.de/ak movie.html). Given the proliferating number of homologous proteins known to exist in different conformational states, it is becoming possible to outline the motions of chain segments and combine them into a movie, which can then represent protein action much more effectively than static pictures alone are able to do.

A Schiff base connectivity switch in sensory rhodopsin signaling

PubMed Central

Sineshchekov, Oleg A.; Sasaki, Jun; Phillips, Brian J.; Spudich, John L.

2008-01-01

Sensory rhodopsin I (SRI) in Halobacterium salinarum acts as a receptor for single-quantum attractant and two-quantum repellent phototaxis, transmitting light stimuli via its bound transducer HtrI. Signal-inverting mutations in the SRI–HtrI complex reverse the single-quantum response from attractant to repellent. Fast intramolecular charge movements reported here reveal that the unphotolyzed SRI–HtrI complex exists in two conformational states, which differ by their connection of the retinylidene Schiff base in the SRI photoactive site to inner or outer half-channels. In single-quantum photochemical reactions, the conformer with the Schiff base connected to the cytoplasmic (CP) half-channel generates an attractant signal, whereas the conformer with the Schiff base connected to the extracellular (EC) half-channel generates a repellent signal. In the wild-type complex the conformer equilibrium is poised strongly in favor of that with CP-accessible Schiff base. Signal-inverting mutations shift the equilibrium in favor of the EC-accessible Schiff base form, and suppressor mutations shift the equilibrium back toward the CP-accessible Schiff base form, restoring the wild-type phenotype. Our data show that the sign of the behavioral response directly correlates with the state of the connectivity switch, not with the direction of proton movements or changes in acceptor pKa. These findings identify a shared fundamental process in the mechanisms of transport and signaling by the rhodopsin family. Furthermore, the effects of mutations in the HtrI subunit of the complex on SRI Schiff base connectivity indicate that the two proteins are tightly coupled to form a single unit that undergoes a concerted conformational transition. PMID:18852467

A Schiff base connectivity switch in sensory rhodopsin signaling.

PubMed

Sineshchekov, Oleg A; Sasaki, Jun; Phillips, Brian J; Spudich, John L

2008-10-21

Sensory rhodopsin I (SRI) in Halobacterium salinarum acts as a receptor for single-quantum attractant and two-quantum repellent phototaxis, transmitting light stimuli via its bound transducer HtrI. Signal-inverting mutations in the SRI-HtrI complex reverse the single-quantum response from attractant to repellent. Fast intramolecular charge movements reported here reveal that the unphotolyzed SRI-HtrI complex exists in two conformational states, which differ by their connection of the retinylidene Schiff base in the SRI photoactive site to inner or outer half-channels. In single-quantum photochemical reactions, the conformer with the Schiff base connected to the cytoplasmic (CP) half-channel generates an attractant signal, whereas the conformer with the Schiff base connected to the extracellular (EC) half-channel generates a repellent signal. In the wild-type complex the conformer equilibrium is poised strongly in favor of that with CP-accessible Schiff base. Signal-inverting mutations shift the equilibrium in favor of the EC-accessible Schiff base form, and suppressor mutations shift the equilibrium back toward the CP-accessible Schiff base form, restoring the wild-type phenotype. Our data show that the sign of the behavioral response directly correlates with the state of the connectivity switch, not with the direction of proton movements or changes in acceptor pK(a). These findings identify a shared fundamental process in the mechanisms of transport and signaling by the rhodopsin family. Furthermore, the effects of mutations in the HtrI subunit of the complex on SRI Schiff base connectivity indicate that the two proteins are tightly coupled to form a single unit that undergoes a concerted conformational transition.

Towards Solvation of a Chiral Alpha-Hydroxy Ester: Broadband Chirp and Narrow Band Cavity Fouirier Transform Microwave Spectroscopy of Methyl Lactate-Water Clusters

NASA Astrophysics Data System (ADS)

Thomas, Javix; Sukhorukov, Oleksandr; Jaeger, Wolfgang; Xu, Yunjie

2013-06-01

Methyl lactate (ML), a chiral alpha-hydroxy ester, has attracted much attention as a prototype system in studies of chirality transfer,[1] solvation effects on chiroptical signatures,[2] and chirality recognition.[3] It has multiple functional groups which can serve both as a hydrogen donor and acceptor. By applying rotational spectroscopy and high level ab initio calculations, we examine the delicate competition between inter- and intramolecular hydrogen-bonding in the ML-water clusters. Broadband rotational spectra obtained with a chirp Fourier transform microwave (FTMW) spectrometer, reveal that the insertion conformations are the most favourable ones in the binary and ternary solvated complexes. In the insertion conformations, the water molecule(s) inserts itself (themselves) into the existing intramolecular hydrogen-bonded ring formed between the alcoholic hydroxyl group and the oxygen of the carbonyl group of ML. The final frequency measurements have been carried out using a cavity based FTMW instrument where internal rotation splittings due to the ester methyl group have also been detected. A number of insertion conformers with subtle structural differences for both the binary and ternary complexes have been identified theoretically. The interconversion dynamics of these conformers and the identification of the most favorable conformers will be discussed. 1. C. Merten, Y. Xu, Angew. Chem. Int. Ed., 2013, 52, 2073 -2076. 2. M. Losada, Y. Xu, Phys. Chem. Chem. Phys., 2007, 9, 3127-3135; Y. Liu, G. Yang, M. Losada, Y. Xu, J. Chem. Phys., 2010, 132, 234513/1-11. 3. A. Zehnacker, M. Suhm, Angew. Chem. Int. Ed. 2008, 47, 6970 - 6992.

Allosteric response and substrate sensitivity in peptide binding of the signal recognition particle.

PubMed

Wang, Connie Y; Miller, Thomas F

2014-10-31

We characterize the conformational dynamics and substrate selectivity of the signal recognition particle (SRP) using a thermodynamic free energy cycle approach and microsecond timescale molecular dynamics simulations. The SRP is a central component of the co-translational protein targeting machinery that binds to the N-terminal signal peptide (SP) of nascent proteins. We determined the shift in relative conformational stability of the SRP upon substrate binding to quantify allosteric coupling between SRP domains. In particular, for dipeptidyl aminopeptidase, an SP that is recognized by the SRP for co-translational targeting, it is found that substrate binding induces substantial changes in the SRP toward configurations associated with targeting of the nascent protein, and it is found that the changes are modestly enhanced by a mutation that increases the hydrophobicity of the SP. However, for alkaline phosphatase, an SP that is recognized for post-translational targeting, substrate binding induces the reverse change in the SRP conformational distribution away from targeting configurations. Microsecond timescale trajectories reveal the intrinsic flexibility of the SRP conformational landscape and provide insight into recent single molecule studies by illustrating that 10-nm lengthscale changes between FRET pairs occur via the rigid-body movement of SRP domains connected by the flexible linker region. In combination, these results provide direct evidence for the hypothesis that substrate-controlled conformational switching in the SRP provides a mechanism for discriminating between different SPs and for connecting substrate binding to downstream steps in the protein targeting pathway. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed Central

McCammon, J. Andrew

2011-01-01

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

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

PubMed

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

2011-10-01

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

Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

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

Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the nativemore » ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.« less

Binding of ncd to microtubules induces a conformational change near the junction of the motor domain with the neck.

PubMed

Naber, N; Cooke, R; Pate, E

1997-08-12

We have covalently attached an electron paramagnetic resonance (EPR) spin probe to Cys-670 of the motor domain of ncd (nonclaret disjunctional protein) in order to investigate conformational changes associated with the chemomechanical cycle. Spin-labeling is highly specific and does not affect ncd function as monitored by either the binding affinity to microtubules or the rate of ATP hydrolysis. The EPR spectra can be deconvoluted into two components, one that is highly mobile with respect to the protein and one that is strongly immobilized. In the absence of microtubules, the relative proportions of these two components varied with temperature, showing that the transition between them involves a large change in enthalpy (DeltaH degrees = -75 kJ/mol). This result implies that the two populations represent very different protein conformations. Binding to microtubules results in virtually all probes shifting into the immobilized component, independent of the nucleotide bound. Superposition of the structures of ncd and myosin subfragment 1 reveals that the labeled cysteine is very close to the region which is homologous to the helix containing the two reactive sulfhydryls in myosin and is approximately 10 A from the junction of the motor domain with the remainder of the molecule. We conclude that the binding of ncd to microtubules results in a conformational change in this region which may be involved in the working power stroke.

Cleavage reaction of HDV ribozymes in the presence of Mg2+ is accompanied by a conformational change.

PubMed

Tanaka, Yoichiro; Tagaya, Mitsuhiro; Hori, Tamaki; Sakamoto, Taiichi; Kurihara, Yasuyuki; Katahira, Masato; Uesugi, Seiichi

2002-06-01

Hepatitis delta virus (HDV) ribozymes cleave RNA in the presence of divalent metal ions. We have previously elucidated the solution conformation of a minimized trans-acting HDV ribozyme and obtained evidence by NMR study that an Mg2+ ion binds to a site close to the cleavage site. We examined two ribozyme systems: a pre-cleavage complex with a non-cleavable substrate analogue (mS8) and a post-cleavage complex with a 3' cleavage product (P7). Upon titration with MgCl2, the complex with P7 showed a profound spectral change, while that with mS8 showed broadening of the signals. Analysis of the NOESY spectra of the P7 complex at high Mg2+ concentration revealed that a G:U pair is formed within the L3 loop, and the P1 and P4 stems are stabilized with respect to those of the pre-cleavage complex. The present analysis indicates that the cleavage reaction of the HDV ribozyme produces a big conformational change. Furthermore, presence of the 5'-terminal cytidine residue prevents this conformational change and its absence stabilizes the product-ribozyme complex in the presence of Mg2+. The structure of the Mg2+-bound P7 complex is similar to the crystal structure found for a product-ribozyme complex but is different from the pre-cleavage structure.

The Role of Magnesium for Geometry and Charge in GTP Hydrolysis, Revealed by Quantum Mechanics/Molecular Mechanics Simulations

PubMed Central

Rudack, Till; Xia, Fei; Schlitter, Jürgen; Kötting, Carsten; Gerwert, Klaus

2012-01-01

The coordination of the magnesium ion in proteins by triphosphates plays an important role in catalytic hydrolysis of GTP or ATP, either in signal transduction or energy conversion. For example, in Ras the magnesium ion contributes to the catalysis of GTP hydrolysis. The cleavage of GTP to GDP and Pi in Ras switches off cellular signaling. We analyzed GTP hydrolysis in water, Ras, and Ras·Ras-GTPase-activating protein using quantum mechanics/molecular mechanics simulations. By comparison of the theoretical IR-difference spectra for magnesium ion coordinated triphosphate to experimental ones, the simulations are validated. We elucidated thereby how the magnesium ion contributes to catalysis. It provides a temporary storage for the electrons taken from the triphosphate and it returns them after bond cleavage and Pi release back to the diphosphate. Furthermore, the Ras·Mg2+ complex forces the triphosphate into a stretched conformation in which the β- and γ-phosphates are coordinated in a bidentate manner. In this conformation, the triphosphate elongates the bond, which has to be cleaved during hydrolysis. Furthermore, the γ-phosphate adopts a more planar structure, driving the conformation of the molecule closer to the hydrolysis transition state. GTPase-activating protein enhances these changes in GTP conformation and charge distribution via the intruding arginine finger. PMID:22853907

Polarimetry as a tool for the study of solutions of chiral solutes.

PubMed

Orlova, Anna V; Andrade, Renato R; da Silva, Clarissa O; Zinin, Alexander I; Kononov, Leonid O

2014-01-13

Optical rotation of aqueous solutions of D-levoglucosan was studied experimentally in the 0.03-4.0 mol L(-1) concentration range and a nonlinear concentration dependence of specific optical rotation (SR) was revealed. Discontinuities observed in the concentration plot of SR (at 0.1, 0.3, 0.5, 1.0, and 2.0 mol L(-1)) are well correlated with those found by static and dynamic light scattering and identify concentration ranges in which different solution domains (supramers) may exist. The average SR experimental value for a D-levoglucosan aqueous solution ([α]D(28) -58.5±8.7 deg dm(-1) cm(-3) g(-1)) was found to be in good agreement with values obtained by theoretical calculation (TD-DFT/GIAO) of SR for 15 different conformers revealed by conformational sampling at the PCM/B3LYP/6-311++G(2d,2p)//B3LYP/6-31+G(d,p) level, which were shown to be strongly affected by the solvation microenvironment (0, 1, 2, and 3 explicit solvent molecules considered) due to local geometrical changes induced in the solute molecule. This exceptionally high sensitivity of SR makes polarimetry a unique method capable of sensing changes in the structure of supramers detected in this study. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular simulations reveal that the long range fluctuations of human DPP III change upon ligand binding.

PubMed

Tomić, A; Berynskyy, M; Wade, R C; Tomić, S

2015-11-01

The experimentally determined structures of human dipeptidyl peptidase III (DPP III) for the wild-type protein and for the complex of its E451A mutant with the peptide substrate, tynorphin, differ significantly in their overall shape. The two domains of the enzyme are separated by a wide cleft in the structure of the ligand-free enzyme, while in the ligand-bound mutant they are very close to each other, and the protein structure is extremely compact. Here, we applied a range of molecular dynamics simulation techniques to investigate the DPP III conformational landscape and the influence of ligand binding on the protein structure and dynamics. We used conventional, accelerated and steered methods to simulate DPP III and its complexes with tynorphin and with the preferred, synthetic, substrate Arg-Arg-2-naphthylamide. We found that DPP III can adopt a number of different forms in solution. The compact forms are more stable, but the open and partially closed states, spanning a wide range of conformations, can more effectively recognize the substrate which preferentially binds to the five-stranded β-core of the lower DPP III domain. The simulations indicated the existence of a dynamic equilibrium between open and semi-closed states and revealed two ways that the protein can close, leading to two distinct compact structures. The way in which the protein closes depends on the presence of the ligand.

Conformational Structure of Tyrosine, Tyrosyl-Glycine, and Tyrosyl-Glycyl-Glycine by Double Resonance Spectroscopy

NASA Technical Reports Server (NTRS)

Abo-Riziq, Ali; Grace, Louis; Crews, Bridgit; Callahan, Michael P,; van Mourik, Tanja; de Vries, Mattanjah S,

2011-01-01

We investigated the variation in conformation for the amino acid tyrosine (Y), alone and in the small peptides tyrosine-glycine (YC) and tyrosine-glycine-glycine (YGG), in the gas phase by using UV-UV and IR-UV double resonance spectroscopy and density functional theory calculations. For tyrosine we found seven different conformations, for YG we found four different conformations, and for YGG we found three different conformations. As the peptides get larger, we observe fewer stable conformers, despite the increasing complexity and number of degrees of freedom. We find structural trends similar to those in phenylalanine-glycine glycine (FGG) and tryptophan-glycine-glycine (WGG)j however) the effect of dispersive forces in FGG for stabilizing a folded structure is replaced by that of hydrogen bonding in YGG.

Discovering Conformational Sub-States Relevant to Protein Function

PubMed Central

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

2011-01-01

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

NMR and rotational angles in solution conformation of polypeptides

NASA Astrophysics Data System (ADS)

Bystrov, V. F.

1985-01-01

Professor San-Ichiro Mizushima and Professor Yonezo Morino's classical contributions provided unique means and firm basis for understanding of conformational states and internal rotation in polypeptide molecules. Now the NMR spectroscopy is the best choice to study molecular conformation, mechanism of action and structure-functional relationships of peptide and proteins in solution under conditions approaching those of their physiological environments. Crucial details of spatial structure and interactions of these molecules in solution are revealed by using proton-proton and carbon-proton vicinal coupling constants, proton nuclear Overhauser effect and spectral perturbation techniques. The results of NMR conformational analysis are presented for valinomycin "bracelet", gramicidin A double helices, honey-bee neurotoxin apamin, scorpion insectotoxins and snake neurotoxins of long and short types.

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

PubMed Central

Câmara, Amanda Souza

2018-01-01

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

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

PubMed

Câmara, Amanda Souza; Horjales, Eduardo

2018-01-01

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

Biosensors Paving the Way to Understanding the Interaction between Cadmium and the Estrogen Receptor Alpha

PubMed Central

Fechner, Peter; Damdimopoulou, Pauliina; Gauglitz, Günter

2011-01-01

Cadmium is a toxic heavy metal ubiquitously present in the environment and subsequently in the human diet. Cadmium has been proposed to disrupt the endocrine system, targeting in particular the estrogen signaling pathway already at environmentally relevant concentrations. Thus far, the reports on the binding affinity of cadmium towards human estrogen receptor alpha (hERα) have been contradicting, as have been the reports on the in vivo estrogenicity of cadmium. Hence, the mode of interaction between cadmium and the receptor remains unclear. Here, we investigated the interaction between cadmium and hERα on a molecular level by applying a novel, label-free biosensor technique based on reflectometric interference spectroscopy (RIfS). We studied the binding of cadmium to hERα, and the conformation of the receptor following cadmium treatment. Our data reveals that cadmium interacts with the ligand binding domain (LBD) of the ERα and affects the conformation of the receptor. However, the binding event, as well as the induced conformation change, greatly depends on the accessibility of the cysteine tails in the LBD. As the LBD cysteine residues have been reported as targets of post-translational modifications in vivo, we present a hypothesis according to which different cellular pools of ERα respond to cadmium differently. Our proposed theory could help to explain some of the previously contradicting results regarding estrogen-like activity of cadmium. PMID:21829690

Multiple Gas-Phase Conformations of a Synthetic Linear Poly(acrylamide) Polymer Observed Using Ion Mobility-Mass Spectrometry.

PubMed

Haler, Jean R N; Far, Johann; Aqil, Abdelhafid; Claereboudt, Jan; Tomczyk, Nick; Giles, Kevin; Jérôme, Christine; De Pauw, Edwin

2017-11-01

Ion mobility-mass spectrometry (IM-MS) has emerged as a powerful separation and identification tool to characterize synthetic polymer mixtures and topologies (linear, cyclic, star-shaped,…). Electrospray coupled to IM-MS already revealed the coexistence of several charge state-dependent conformations for a single charge state of biomolecules with strong intramolecular interactions, even when limited resolving power IM-MS instruments were used. For synthetic polymers, the sample's polydispersity allows the observation of several chain lengths. A unique collision cross-section (CCS) trend is usually observed when increasing the degree of polymerization (DP) at constant charge state, allowing the deciphering of different polymer topologies. In this paper, we report multiple coexisting CCS trends when increasing the DP at constant charge state for linear poly(acrylamide) PAAm in the gas phase. This is similar to observations on peptides and proteins. Biomolecules show in addition population changes when collisionally heating the ions. In the case of synthetic PAAm, fragmentation occurred before reaching the energy for conformation conversion. These observations, which were made on two different IM-MS instruments (SYNAPT G2 HDMS and high resolution multi-pass cyclic T-Wave prototype from Waters), limit the use of ion mobility for synthetic polymer topology interpretations to polymers where unique CCS values are observed for each DP at constant charge state. Graphical Abstract ᅟ.

Multiple Gas-Phase Conformations of a Synthetic Linear Poly(acrylamide) Polymer Observed Using Ion Mobility-Mass Spectrometry

NASA Astrophysics Data System (ADS)

Haler, Jean R. N.; Far, Johann; Aqil, Abdelhafid; Claereboudt, Jan; Tomczyk, Nick; Giles, Kevin; Jérôme, Christine; De Pauw, Edwin

2017-08-01

Ion mobility-mass spectrometry (IM-MS) has emerged as a powerful separation and identification tool to characterize synthetic polymer mixtures and topologies (linear, cyclic, star-shaped,…). Electrospray coupled to IM-MS already revealed the coexistence of several charge state-dependent conformations for a single charge state of biomolecules with strong intramolecular interactions, even when limited resolving power IM-MS instruments were used. For synthetic polymers, the sample's polydispersity allows the observation of several chain lengths. A unique collision cross-section (CCS) trend is usually observed when increasing the degree of polymerization (DP) at constant charge state, allowing the deciphering of different polymer topologies. In this paper, we report multiple coexisting CCS trends when increasing the DP at constant charge state for linear poly(acrylamide) PAAm in the gas phase. This is similar to observations on peptides and proteins. Biomolecules show in addition population changes when collisionally heating the ions. In the case of synthetic PAAm, fragmentation occurred before reaching the energy for conformation conversion. These observations, which were made on two different IM-MS instruments (SYNAPT G2 HDMS and high resolution multi-pass cyclic T-Wave prototype from Waters), limit the use of ion mobility for synthetic polymer topology interpretations to polymers where unique CCS values are observed for each DP at constant charge state. [Figure not available: see fulltext.

A structural portrait of the PDZ domain family.

PubMed

Ernst, Andreas; Appleton, Brent A; Ivarsson, Ylva; Zhang, Yingnan; Gfeller, David; Wiesmann, Christian; Sidhu, Sachdev S

2014-10-23

PDZ (PSD-95/Discs-large/ZO1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signaling complexes in multicellular organisms. We have analyzed the existing database of PDZ domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side-chain and main-chain interactions. Most PDZ domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended β-strand conformation by interacting in an antiparallel fashion with a PDZ β-strand. However, a subset of PDZ domains bind peptides with a bent main-chain conformation and the specificities of these non-canonical domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ domain family, which serves as a guide in understanding the structural basis for the diverse specificities across the family. Copyright © 2014 Elsevier Ltd. All rights reserved.

Crystal Structures of T Cell Receptor (Beta) Chains Related to Rheumatoid Arthritis

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

Li,H.; van Vranken, S.; Zhao, Y.

The crystal structures of the V{beta}17+ {beta} chains of two human T cell receptors (TCRs), originally derived from the synovial fluid (SF4) and tissue (C5-1) of a patient with rheumatoid arthritis (RA), have been determined in native (SF4) and mutant (C5-1{sub F104{yields}Y/C187{yields}S}) forms, respectively. These TCR {beta} chains form homo-dimers in solution and in crystals. Structural comparison reveals that the main-chain conformations in the CDR regions of the C5-1 and SF4 V{beta}17 closely resemble those of a V{beta}17 JM22 in a bound form; however, the CDR3 region shows different conformations among these three V{beta}17 structures. At the side-chain level, conformationalmore » differences were observed at the CDR2 regions between our two ligand-free forms and the bound JM22 form. Other significant differences were observed at the V{beta} regions 8-12, 40-44, and 82-88 between C5-1/SF4 and JM22 V{beta}17, implying that there is considerable variability in the structures of very similar {beta} chains. Structural alignments also reveal a considerable variation in the V{beta}-C{beta} associations, and this may affect ligand recognition. The crystal structures also provide insights into the structure basis of T cell recognition of Mycoplasma arthritidis mitogen (MAM), a superantigen that may be implicated in the development of human RA. Structural comparisons of the V{beta} domains of known TCR structures indicate that there are significant similarities among V{beta} regions that are MAM-reactive, whereas there appear to be significant structural differences among those V{beta} regions that lack MAM-reactivity. It further reveals that CDR2 and framework region (FR) 3 are likely to account for the binding of TCR to MAM.« less

Gas-Phase Folding of Small Glutamine Containing Peptides: Sidechain Hydrogen Bonding Stabilizes β-turns

NASA Astrophysics Data System (ADS)

Walsh, Patrick S.; Blodgett, Karl N.; McBurney, Carl; Gellman, Samuel H.; Zwier, Timothy S.

Glutamine is vitally important to a class of neurodegenerative diseases called poly-glutamine (poly-Q) repeat diseases such as Huntington's Disease (HD). Recent studies have revealed a pathogenic pathway that proceeds through misfolding of poly-Q regions into characteristic β-turn/ β-hairpin structures that are highly correlated with toxicity. The inherent conformational preferences of small glutamine containing peptides (Ac-Q-Q-NHBn and Ac-A-Q-NHBn) were studied using conformation-specific IR and UV spectroscopies, with the goal of probing the delicate interplay between three competitive hydrogen bonding motifs: backbone-backbone, sidechain-backbone, and sidechain-sidechain hydrogen bonds. Laser desorption, coupled with a supersonic expansion, was used to introduce the non-thermally labile sample into the gas-phase. Resonant ion-dip infrared (RIDIR) spectroscopy is a powerful tool for recording the vibrational spectra of single conformational isomers and was used here to reveal the innate structural preferences of the glutamine containing peptides. Experimental results are compared against density functional calculations to arrive at firm conformational assignments. Our results demonstrate a striking preference for β-turn formation in the non-polar environment of the gas-phase. Previous Affiliation: Purdue University, Department of Chemistry.

The α-Helical Structure of Prodomains Promotes Translocation of Intrinsically Disordered Neuropeptide Hormones into the Endoplasmic Reticulum*

PubMed Central

Dirndorfer, Daniela; Seidel, Ralf P.; Nimrod, Guy; Miesbauer, Margit; Ben-Tal, Nir; Engelhard, Martin; Zimmermann, Richard; Winklhofer, Konstanze F.; Tatzelt, Jörg

2013-01-01

Different neuropeptide hormones, which are either too small to adopt a stable conformation or are predicted to be intrinsically disordered, are synthesized as larger precursors containing a prodomain in addition to an N-terminal signal peptide. We analyzed the biogenesis of three unstructured neuropeptide hormones and observed that translocation of these precursors into the lumen of the endoplasmic reticulum (ER) is critically dependent on the presence of the prodomain. The hormone domains could be deleted from the precursors without interfering with ER import and secretion, whereas constructs lacking the prodomain remained in the cytosol. Domain-swapping experiments revealed that the activity of the prodomains to promote productive ER import resides in their ability to adopt an α-helical structure. Removal of the prodomain from the precursor did not interfere with co-translational targeting of the nascent chain to the Sec61 translocon but with its subsequent productive translocation into the ER lumen. Our study reveals a novel function of prodomains to enable import of small or intrinsically disordered secretory proteins into the ER based on their ability to adopt an α-helical conformation. PMID:23532840

The α-helical structure of prodomains promotes translocation of intrinsically disordered neuropeptide hormones into the endoplasmic reticulum.

PubMed

Dirndorfer, Daniela; Seidel, Ralf P; Nimrod, Guy; Miesbauer, Margit; Ben-Tal, Nir; Engelhard, Martin; Zimmermann, Richard; Winklhofer, Konstanze F; Tatzelt, Jörg

2013-05-17

Different neuropeptide hormones, which are either too small to adopt a stable conformation or are predicted to be intrinsically disordered, are synthesized as larger precursors containing a prodomain in addition to an N-terminal signal peptide. We analyzed the biogenesis of three unstructured neuropeptide hormones and observed that translocation of these precursors into the lumen of the endoplasmic reticulum (ER) is critically dependent on the presence of the prodomain. The hormone domains could be deleted from the precursors without interfering with ER import and secretion, whereas constructs lacking the prodomain remained in the cytosol. Domain-swapping experiments revealed that the activity of the prodomains to promote productive ER import resides in their ability to adopt an α-helical structure. Removal of the prodomain from the precursor did not interfere with co-translational targeting of the nascent chain to the Sec61 translocon but with its subsequent productive translocation into the ER lumen. Our study reveals a novel function of prodomains to enable import of small or intrinsically disordered secretory proteins into the ER based on their ability to adopt an α-helical conformation.

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

Akabayov, B.; Akabayov, S; Lee , S

Gene 5 of bacteriophage T7 encodes a DNA polymerase (gp5) responsible for the replication of the phage DNA. Gp5 polymerizes nucleotides with low processivity, dissociating after the incorporation of 1 to 50 nucleotides. Thioredoxin (trx) of Escherichia coli binds tightly (Kd = 5 nM) to a unique segment in the thumb subdomain of gp5 and increases processivity. We have probed the molecular basis for the increase in processivity. A single-molecule experiment reveals differences in rates of enzymatic activity and processivity between gp5 and gp5/trx. Small angle X-ray scattering studies combined with nuclease footprinting reveal two conformations of gp5, one inmore » the free state and one upon binding to trx. Comparative analysis of the DNA binding clefts of DNA polymerases and DNA binding proteins show that the binding surface contains more hydrophobic residues than other DNA binding proteins. The balanced composition between hydrophobic and charged residues of the binding site allows for efficient sliding of gp5/trx on the DNA. We propose a model for trx-induced conformational changes in gp5 that enhance the processivity by increasing the interaction of gp5 with DNA.« less

Ligand-Binding Properties and Conformational Dynamics of Autolysin Repeat Domains in Staphylococcal Cell Wall Recognition

PubMed Central

Zoll, Sebastian; Schlag, Martin; Shkumatov, Alexander V.; Rautenberg, Maren; Svergun, Dmitri I.; Götz, Friedrich

2012-01-01

The bifunctional major autolysin Atl plays a key role in staphylococcal cell separation. Processing of Atl yields catalytically active amidase (AM) and glucosaminidase (GL) domains that are each fused to repeating units. The two repeats of AM (R1 and R2) target the enzyme to the septum, where it cleaves murein between dividing cells. We have determined the crystal structure of R2, which reveals that each repeat folds into two half-open β-barrel subunits. We further demonstrate that lipoteichoic acid serves as a receptor for the repeats and that this interaction depends on conserved surfaces in each subunit. Small-angle X-ray scattering of the mature amidase reveals the presence of flexible linkers separating the AM, R1, and R2 units. Different levels of flexibility for each linker provide mechanistic insights into the conformational dynamics of the full-length protein and the roles of its components in cell wall association and catalysis. Our analysis supports a model in which the repeats direct the catalytic AM domain to the septum, where it can optimally perform the final step of cell division. PMID:22609916

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

PubMed Central

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

2018-01-01

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

Exploration of the folding dynamics of human telomeric G-quadruplex with a hybrid atomistic structure-based model

NASA Astrophysics Data System (ADS)

Bian, Yunqiang; Ren, Weitong; Song, Feng; Yu, Jiafeng; Wang, Jihua

2018-05-01

Structure-based models or Gō-like models, which are built from one or multiple particular experimental structures, have been successfully applied to the folding of proteins and RNAs. Recently, a variant termed the hybrid atomistic model advances the description of backbone and side chain interactions of traditional structure-based models, by borrowing the description of local interactions from classical force fields. In this study, we assessed the validity of this model in the folding problem of human telomeric DNA G-quadruplex, where local dihedral terms play important roles. A two-state model was developed and a set of molecular dynamics simulations was conducted to study the folding dynamics of sequence Htel24, which was experimentally validated to adopt two different (3 + 1) hybrid G-quadruplex topologies in K+ solution. Consistent with the experimental observations, the hybrid-1 conformation was found to be more stable and the hybrid-2 conformation was kinetically more favored. The simulations revealed that the hybrid-2 conformation folded in a higher cooperative manner, which may be the reason why it was kinetically more accessible. Moreover, by building a Markov state model, a two-quartet G-quadruplex state and a misfolded state were identified as competing states to complicate the folding process of Htel24. Besides, the simulations also showed that the transition between hybrid-1 and hybrid-2 conformations may proceed an ensemble of hairpin structures. The hybrid atomistic structure-based model reproduced the kinetic partitioning folding dynamics of Htel24 between two different folds, and thus can be used to study the complex folding processes of other G-quadruplex structures.

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.

Dynamic properties of the native free antithrombin from molecular dynamics simulations: computational evidence for solvent- exposed Arg393 side chain.

PubMed

Tóth, László; Fekete, Attila; Balogh, Gábor; Bereczky, Zsuzsanna; Komáromi, István

2015-09-01

While antithrombin (AT) has small basal inhibitory activity, it reaches its full inhibitory potential against activated blood coagulation factors, FXa, FIXa, and FIIa (thrombin), via an allosteric and/or template (bridging) mechanism by the action of heparin, heparan sulfate, or heparin-mimetic pentasaccharides (PS). From the numerous X-ray structures available for different conformational states of AT, only indirect and incomplete conclusions can be drawn on the inherently dynamic properties of AT. As a typical example, the basal inhibitory activity of AT cannot be interpreted on the basis of "non-activated" free antithrombin X-ray structures since the Arg393 side chain, playing crucial role in antithrombin-proteinase interaction, is not exposed. In order to reveal the intrinsic dynamic properties and the reason of basal inhibitory activity of antithrombin, 2 μs molecular dynamics simulations were carried out on its native free-forms. It was shown from the simulation trajectories that the reactive center loop which is functioning as "bait" for proteases, even without any biasing potential can populate conformational state in which the Arg393 side chain is solvent exposed. It is revealed from the trajectory analysis that the peptide sequences correspond to the helix D extension, and new helix P formation can be featured with especially large root-mean-square fluctuations. Mutual information analyses of the trajectory showed remarkable (generalized) correlation between those regions of antithrombin which changed their conformations as the consequence of AT-PS complex formation. This suggests that allosteric information propagation pathways are present even in the non-activated native form of AT.

Effect of temperature on the conformation of natively unfolded protein 4E-BP1 in aqueous and mixed solutions containing trifluoroethanol and hexafluoroisopropanol.

PubMed

Hackl, Ellen V

2015-02-01

Natively unfolded (intrinsically disordered) proteins have attracted growing attention due to their high abundance in nature, involvement in various signalling and regulatory pathways and direct association with many diseases. In the present work the combined effect of temperature and alcohols, trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP), on the natively unfolded 4E-BP1 protein was studied to elucidate the balance between temperature-induced folding and unfolding in intrinsically disordered proteins. It was shown that elevated temperatures induce reversible partial folding of 4E-BP1 both in buffer and in the mixed solutions containing denaturants. In the mixed solutions containing TFE (HFIP) 4E-BP1 adopts a partially folded helical conformation. As the temperature increases, the initial temperature-induced protein folding is replaced by irreversible unfolding/melting only after a certain level of the protein helicity has been reached. Onset unfolding temperature decreases with TFE (HFIP) concentration in solution. It was shown that an increase in the temperature induces two divergent processes in a natively unfolded protein--hydrophobicity-driven folding and unfolding. Balance between these two processes determines thermal behaviour of a protein. The correlation between heat-induced protein unfolding and the amount of helical content in a protein is revealed. Heat-induced secondary structure formation can be a valuable test to characterise minor changes in the conformations of natively unfolded proteins as a result of site-directed mutagenesis. Mutants with an increased propensity to fold into a structured form reveal different temperature behaviour.

Unveiling the Sweet Conformations of Ketohexoses

NASA Astrophysics Data System (ADS)

Bermudez, C.; Pena, I.; Cabezas, C.; Daly, A. M.; Mata, S.; Alonso, J. L.

2013-06-01

The conformational behavior of ketohexoses D-Fructose, L-Sorbose, D-Tagatose and D-Psicose has been revealed from their rotational spectra. A broadband microwave spectrometer (CP-FTMW) has been used to rapidly acquire the rotational spectra in the 6 to 12 GHz frequency range. All observed species are stabilized by complicated intramolecular hydrogen-bonding networks. Structural motifs related to the sweetness of ketohexoses are revealed. G. G. Brown, B. C. Dian, K. O. Douglass, S. M. Geyer, S. T. Shipman, B. H. Pate, Rev. Sci. Instrum. 2008, 79, 053103. S. Mata, I. Peña, C. Cabezas, J. C. López, J. L. Alonso, J. Mol. Spectrosc. 2012, 280, 91.

Theoretical studies on absorption, emission, and resonance Raman spectra of Coumarin 343 isomers

NASA Astrophysics Data System (ADS)

Wu, Wenpeng; Cao, Zexing; Zhao, Yi

2012-03-01

The vibrationally resolved spectral method and quantum chemical calculations are employed to reveal the structural and spectral properties of Coumarin 343 (C343), an ideal candidate for organic dye photosensitizers, in vacuum and solution. The results manifest that the ground-state energies are dominantly determined by different placements of hydrogen atom in carboxylic group of C343 conformations. Compared to those in vacuum, the electronic absorption spectra in methanol solvent show a hyperchromic property together with the redshift and blueshift for the neutral C343 isomers and their deprotonated anions, respectively. From the absorption, emission, and resonance Raman spectra, it is found that the maximal absorption and emission come from low-frequency modes whereas the high-frequency modes have high Raman activities. The detailed spectra are further analyzed for the identification of the conformers and understanding the potential charge transfer mechanism in their photovoltaic applications.

Competing Intramolecular vs. Intermolecular Hydrogen Bonds in Solution

PubMed Central

Nagy, Peter I.

2014-01-01

A hydrogen bond for a local-minimum-energy structure can be identified according to the definition of the International Union of Pure and Applied Chemistry (IUPAC recommendation 2011) or by finding a special bond critical point on the density map of the structure in the framework of the atoms-in-molecules theory. Nonetheless, a given structural conformation may be simply favored by electrostatic interactions. The present review surveys the in-solution competition of the conformations with intramolecular vs. intermolecular hydrogen bonds for different types of small organic molecules. In their most stable gas-phase structure, an intramolecular hydrogen bond is possible. In a protic solution, the intramolecular hydrogen bond may disrupt in favor of two solute-solvent intermolecular hydrogen bonds. The balance of the increased internal energy and the stabilizing effect of the solute-solvent interactions regulates the new conformer composition in the liquid phase. The review additionally considers the solvent effects on the stability of simple dimeric systems as revealed from molecular dynamics simulations or on the basis of the calculated potential of mean force curves. Finally, studies of the solvent effects on the type of the intermolecular hydrogen bond (neutral or ionic) in acid-base complexes have been surveyed. PMID:25353178

Role of conformational sampling in computing mutation-induced changes in protein structure and stability.

PubMed

Kellogg, Elizabeth H; Leaver-Fay, Andrew; Baker, David

2011-03-01

The prediction of changes in protein stability and structure resulting from single amino acid substitutions is both a fundamental test of macromolecular modeling methodology and an important current problem as high throughput sequencing reveals sequence polymorphisms at an increasing rate. In principle, given the structure of a wild-type protein and a point mutation whose effects are to be predicted, an accurate method should recapitulate both the structural changes and the change in the folding-free energy. Here, we explore the performance of protocols which sample an increasing diversity of conformations. We find that surprisingly similar performances in predicting changes in stability are achieved using protocols that involve very different amounts of conformational sampling, provided that the resolution of the force field is matched to the resolution of the sampling method. Methods involving backbone sampling can in some cases closely recapitulate the structural changes accompanying mutations but not surprisingly tend to do more harm than good in cases where structural changes are negligible. Analysis of the outliers in the stability change calculations suggests areas needing particular improvement; these include the balance between desolvation and the formation of favorable buried polar interactions, and unfolded state modeling. Copyright © 2010 Wiley-Liss, Inc.

Sorbitol counteracts temperature- and chemical-induced denaturation of a recombinant α-amylase from alkaliphilic Bacillus sp. TS-23.

PubMed

Chi, Meng-Chun; Wu, Tai-Jung; Chen, Hsing-Ling; Lo, Huei-Fen; Lin, Long-Liu

2012-12-01

Enzymes are highly complex systems with a substantial degree of structural variability in their folded state. In the presence of cosolvents, fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways; alternatively, certain conformations can be stabilized or destabilized. To understand the contribution of osmolytes to the stabilization of structural changes and enzymatic activity of a truncated Bacillus sp. TS-23 α-amylase (BACΔNC), we monitored amylolytic activity, circular dichroism, and fluorescence as a function of osmolytes. In the presence of trimethylamine N-oxide (TMAO) and sorbitol, BACΔNC activity was retained significantly at elevated temperatures. As compared to the control, the secondary structures of this enzyme were essentially conserved upon the addition of these two kinds of osmolytes. Fluorescence results revealed that the temperature-induced conformational change of BACΔNC was prevented by TMAO and sorbitol. However, glycerol did not provide profound protection against thermal denaturation of the enzyme. Sorbitol was further found to counteract guanidine hydrochloride- and SDS-induced denaturation of BACΔNC. Thus, some well-known naturally occurring osmolytes make a dominant contribution to the stabilization of BACΔNC.

Intrinsically Disordered Energy Landscapes

PubMed Central

Chebaro, Yassmine; Ballard, Andrew J.; Chakraborty, Debayan; Wales, David J.

2015-01-01

Analysis of an intrinsically disordered protein (IDP) reveals an underlying multifunnel structure for the energy landscape. We suggest that such ‘intrinsically disordered’ landscapes, with a number of very different competing low-energy structures, are likely to characterise IDPs, and provide a useful way to address their properties. In particular, IDPs are present in many cellular protein interaction networks, and several questions arise regarding how they bind to partners. Are conformations resembling the bound structure selected for binding, or does further folding occur on binding the partner in a induced-fit fashion? We focus on the p53 upregulated modulator of apoptosis (PUMA) protein, which adopts an -helical conformation when bound to its partner, and is involved in the activation of apoptosis. Recent experimental evidence shows that folding is not necessary for binding, and supports an induced-fit mechanism. Using a variety of computational approaches we deduce the molecular mechanism behind the instability of the PUMA peptide as a helix in isolation. We find significant barriers between partially folded states and the helix. Our results show that the favoured conformations are molten-globule like, stabilised by charged and hydrophobic contacts, with structures resembling the bound state relatively unpopulated in equilibrium. PMID:25999294

A combined spectroscopic and theoretical study of propofol.(H2O)3

NASA Astrophysics Data System (ADS)

León, Iker; Cocinero, Emilio J.; Millán, Judith; Rijs, Anouk M.; Usabiaga, Imanol; Lesarri, Alberto; Castaño, Fernando; Fernández, José A.

2012-08-01

Propofol (2,6-di-isopropylphenol) is probably the most widely used general anesthetic. Previous studies focused on its complexes containing 1 and 2 water molecules. In this work, propofol clusters containing three water molecules were formed using supersonic expansions and probed by means of a number of mass-resolved laser spectroscopic techniques. The 2-color REMPI spectrum of propofol.(H2O)3 contains contributions from at least two conformational isomers, as demonstrated by UV/UV hole burning. Using the infrared IR/UV double resonance technique, the IR spectrum of each isomer was obtained both in ground and first excited electronic states and interpreted in the light of density functional theory (DFT) calculations at M06-2X/6-311++G(d,p) and B3LYP/6-311++G(d,p) levels. The spectral analysis reveals that in both isomers the water molecules are forming cyclic hydrogen bond networks around propofol's OH moiety. Furthermore, some evidences point to the existence of isomerization processes, due to a complicated conformational landscape and the existence of multiple paths with low energy barriers connecting the different conformers. Such processes are discussed with the aid of DFT calculations.

Sex Differences in Influenceability

ERIC Educational Resources Information Center

Eagly, Alice H.

1978-01-01

Examines the hypothesis that women are more easily influenced than men by reviewing the literature on persuasion and conformity research. Persuasion research and conformity studies not involving group pressure show scant empirical support for sex differences. For group pressure conformity research, a substantial minority of studies support the…

Neuronal Calcium Sensor-1 Binds the D2 Dopamine Receptor and G-protein-coupled Receptor Kinase 1 (GRK1) Peptides Using Different Modes of Interactions.

PubMed

Pandalaneni, Sravan; Karuppiah, Vijaykumar; Saleem, Muhammad; Haynes, Lee P; Burgoyne, Robert D; Mayans, Olga; Derrick, Jeremy P; Lian, Lu-Yun

2015-07-24

Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca(2+)-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca(2+)/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca(2+)/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178-Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca(2+)/NCS-1·D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178-Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Neuronal Calcium Sensor-1 Binds the D2 Dopamine Receptor and G-protein-coupled Receptor Kinase 1 (GRK1) Peptides Using Different Modes of Interactions*

PubMed Central

Pandalaneni, Sravan; Karuppiah, Vijaykumar; Saleem, Muhammad; Haynes, Lee P.; Burgoyne, Robert D.; Mayans, Olga; Derrick, Jeremy P.; Lian, Lu-Yun

2015-01-01

Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca2+-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca2+/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca2+/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178–Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca2+/NCS-1·D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178–Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1. PMID:25979333

First passage analysis of the folding of a β-sheet miniprotein: is it more realistic than the standard equilibrium approach?

PubMed

Kalgin, Igor V; Chekmarev, Sergei F; Karplus, Martin

2014-04-24

Simulations of first-passage folding of the antiparallel β-sheet miniprotein beta3s, which has been intensively studied under equilibrium conditions by A. Caflisch and co-workers, show that the kinetics and dynamics are significantly different from those for equilibrium folding. Because the folding of a protein in a living system generally corresponds to the former (i.e., the folded protein is stable and unfolding is a rare event), the difference is of interest. In contrast to equilibrium folding, the Ch-curl conformations become very rare because they contain unfavorable parallel β-strand arrangements, which are difficult to form dynamically due to the distant N- and C-terminal strands. At the same time, the formation of helical conformations becomes much easier (particularly in the early stage of folding) due to short-range contacts. The hydrodynamic descriptions of the folding reaction have also revealed that while the equilibrium flow field presented a collection of local vortices with closed "streamlines", the first-passage folding is characterized by a pronounced overall flow from the unfolded states to the native state. The flows through the locally stable structures Cs-or and Ns-or, which are conformationally close to the native state, are negligible due to detailed balance established between these structures and the native state. Although there are significant differences in the general picture of the folding process from the equilibrium and first-passage folding simulations, some aspects of the two are in agreement. The rate of transitions between the clusters of characteristic protein conformations in both cases decreases approximately exponentially with the distance between the clusters in the hydrogen bond distance space of collective variables, and the folding time distribution in the first-passage segments of the equilibrium trajectory is in good agreement with that for the first-passage folding simulations.

First Passage Analysis of the Folding of a β-Sheet Miniprotein: Is it More Realistic Than the Standard Equilibrium Approach?

PubMed Central

2015-01-01

Simulations of first-passage folding of the antiparallel β-sheet miniprotein beta3s, which has been intensively studied under equilibrium conditions by A. Caflisch and co-workers, show that the kinetics and dynamics are significantly different from those for equilibrium folding. Because the folding of a protein in a living system generally corresponds to the former (i.e., the folded protein is stable and unfolding is a rare event), the difference is of interest. In contrast to equilibrium folding, the Ch-curl conformations become very rare because they contain unfavorable parallel β-strand arrangements, which are difficult to form dynamically due to the distant N- and C-terminal strands. At the same time, the formation of helical conformations becomes much easier (particularly in the early stage of folding) due to short-range contacts. The hydrodynamic descriptions of the folding reaction have also revealed that while the equilibrium flow field presented a collection of local vortices with closed ”streamlines”, the first-passage folding is characterized by a pronounced overall flow from the unfolded states to the native state. The flows through the locally stable structures Cs-or and Ns-or, which are conformationally close to the native state, are negligible due to detailed balance established between these structures and the native state. Although there are significant differences in the general picture of the folding process from the equilibrium and first-passage folding simulations, some aspects of the two are in agreement. The rate of transitions between the clusters of characteristic protein conformations in both cases decreases approximately exponentially with the distance between the clusters in the hydrogen bond distance space of collective variables, and the folding time distribution in the first-passage segments of the equilibrium trajectory is in good agreement with that for the first-passage folding simulations. PMID:24669953

Conformational equilibrium and hydrogen bonding in liquid 2-phenylethylamine explored by Raman spectroscopy and theoretical calculations.

PubMed

Xie, Min; Qi, Yajing; Hu, Yongjun

2011-04-14

2-Phenylethylamine (PEA) is the simplest aromatic amine neurotransmitter, as well as one of the most important. In this work, the conformational equilibrium and hydrogen bonding in liquid PEA were studied by means of Raman spectroscopy and theoretical calculations (DFT/MP2). By changing the orientation of the ethyl and the NH(2) group, nine possible conformers of PEA were found, including four degenerate conformers. Comparison of the experimental Raman spectra of liquid PEA and the calculated Raman spectra of the five typical conformers in selected regions (550-800 and 1250-1500 cm(-1)) revealed that the five conformers can coexist in conformational equilibrium in the liquid. The NH(2) stretching mode of the liquid is red-shifted by ca. 30 cm(-1) relative to that of an isolated PEA molecule (measured previously), implying that intermolecular N-H···N hydrogen bonds play an important role in liquid PEA. The relative intensity of the Raman band at 762 cm(-1) was found to increase with increasing temperature, indicating that the anti conformer might be favorable in liquid PEA at room temperature. The blue shift of the band for the bonded N-H stretch with increasing temperature also provides evidence of the existence of intermolecular N-H···N hydrogen bonds.

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

PubMed

Rajeshwar T, Rajitha; Krishnan, Marimuthu

2017-05-25

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

Conformational Sampling and Nucleotide-Dependent Transitions of the GroEL Subunit Probed by Unbiased Molecular Dynamics Simulations

PubMed Central

Skjaerven, Lars; Grant, Barry; Muga, Arturo; Teigen, Knut; McCammon, J. Andrew; Reuter, Nathalie; Martinez, Aurora

2011-01-01

GroEL is an ATP dependent molecular chaperone that promotes the folding of a large number of substrate proteins in E. coli. Large-scale conformational transitions occurring during the reaction cycle have been characterized from extensive crystallographic studies. However, the link between the observed conformations and the mechanisms involved in the allosteric response to ATP and the nucleotide-driven reaction cycle are not completely established. Here we describe extensive (in total long) unbiased molecular dynamics (MD) simulations that probe the response of GroEL subunits to ATP binding. We observe nucleotide dependent conformational transitions, and show with multiple 100 ns long simulations that the ligand-induced shift in the conformational populations are intrinsically coded in the structure-dynamics relationship of the protein subunit. Thus, these simulations reveal a stabilization of the equatorial domain upon nucleotide binding and a concomitant “opening” of the subunit, which reaches a conformation close to that observed in the crystal structure of the subunits within the ADP-bound oligomer. Moreover, we identify changes in a set of unique intrasubunit interactions potentially important for the conformational transition. PMID:21423709

Populations of the Minor α-Conformation in AcGXGNH2 and the α-Helical Nucleation Propensities

NASA Astrophysics Data System (ADS)

Zhou, Yanjun; He, Liu; Zhang, Wenwen; Hu, Jingjing; Shi, Zhengshuang

2016-06-01

Intrinsic backbone conformational preferences of different amino acids are important for understanding the local structure of unfolded protein chains. Recent evidence suggests α-structure is relatively minor among three major backbone conformations for unfolded proteins. The α-helices are the dominant structures in many proteins. For these proteins, how could the α-structures occur from the least in unfolded to the most in folded states? Populations of the minor α-conformation in model peptides provide vital information. Reliable determination of populations of the α-conformers in these peptides that exist in multiple equilibriums of different conformations remains a challenge. Combined analyses on data from AcGXPNH2 and AcGXGNH2 peptides allow us to derive the populations of PII, β and α in AcGXGNH2. Our results show that on average residue X in AcGXGNH2 adopt PII, β, and α 44.7%, 44.5% and 10.8% of time, respectively. The contents of α-conformations for different amino acids define an α-helix nucleation propensity scale. With derived PII, β and α-contents, we can construct a free energy-conformation diagram on each AcGXGNH2 in aqueous solution for the three major backbone conformations. Our results would have broad implications on early-stage events of protein folding.

FRET detection of lymphocyte function–associated antigen-1 conformational extension

PubMed Central

Chigaev, Alexandre; Smagley, Yelena; Haynes, Mark K.; Ursu, Oleg; Bologa, Cristian G.; Halip, Liliana; Oprea, Tudor; Waller, Anna; Carter, Mark B.; Zhang, Yinan; Wang, Wei; Buranda, Tione; Sklar, Larry A.

2015-01-01

Lymphocyte function–associated antigen 1 (LFA-1, CD11a/CD18, αLβ2-integrin) and its ligands are essential for adhesion between T-cells and antigen-presenting cells, formation of the immunological synapse, and other immune cell interactions. LFA-1 function is regulated through conformational changes that include the modulation of ligand binding affinity and molecular extension. However, the relationship between molecular conformation and function is unclear. Here fluorescence resonance energy transfer (FRET) with new LFA-1–specific fluorescent probes showed that triggering of the pathway used for T-cell activation induced rapid unquenching of the FRET signal consistent with extension of the molecule. Analysis of the FRET quenching at rest revealed an unexpected result that can be interpreted as a previously unknown LFA-1 conformation. PMID:25378583

Conformational fluctuation of Synaptotagmin-1 observed with single molecule fluorescence resonance energy transfer (smFRET)

NASA Astrophysics Data System (ADS)

Choi, Ucheor; Weninger, Keith

2008-10-01

Calcium dependent neurotransmitter release at the synapses involves a synaptic vesicle protein synaptotagmin-1, a calcium sensor, to regulate exocytosis. It has been known that Synaptotagmin-1 interacts with assembled SNARE complexes, but it is unclear how their molecular mechanisms are coupled. X-ray studies in the absence of calcium revealed a closed conformation of synaptotagmin-1 and with calcium bound to the C2 domains of synaptotagmin-3 found extensive interactions holding the domains open. Suggesting the two conformations can be the key to the two functions of synaptotagmin in regulating neurotransmission. Here we use single molecule fluorescence resonance energy transfer (smFRET) to study synaptotagmin interactions with SNARE complexes and the spontaneous conformational changes of synaptotagmin-1 when calcium is induced.

On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein.

PubMed

Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo; Roitberg, Adrian E; Fernandez-Alberti, Sebastian

2015-06-28

The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data.

On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein

NASA Astrophysics Data System (ADS)

Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo; Roitberg, Adrian E.; Fernandez-Alberti, Sebastian

2015-06-01

The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data.

Molecular docking and molecular dynamics studies on the interactions of hydroxylated polybrominated diphenyl ethers to estrogen receptor alpha.

PubMed

Lu, Qun; Cai, Zhengqing; Fu, Jie; Luo, Siyi; Liu, Chunsheng; Li, Xiaolin; Zhao, Dongye

2014-03-01

Environmental estrogens have attracted great concerns. Recent studies have indicated that some hydroxylated polybrominated diphenyl ethers (HO-PBDEs) can interact with estrogen receptor (ER), and exhibit estrogenic activity. However, interactions between HO-PBDEs and ER are not well understood. In this work, molecular docking and molecular dynamics (MD) simulations were performed to characterize interactions of two HO-PBDEs (4'-HO-BDE30 and 4'-HO-BDE121) with ERα. Surflex-Dock was employed to reveal the probable binding conformations of the compounds at the active site of ERα; MD simulation was used to determine the detailed binding process. The driving forces of the binding between HO-PBDEs and ERα were van der Waals and electrostatic interactions. The decomposition of the binding free energy indicated that the hydrogen bonds between the residues Glu353, Gly521 and ligands were crucial for anchoring the ligands into the active site of ERα and stabilizing their conformations. The results showed that different interaction modes and different specific interactions with some residues were responsible for the different estrogenic activities of the two HO-PBDEs. Copyright © 2013 Elsevier Inc. All rights reserved.

Covalent dye attachment influences the dynamics and conformational properties of flexible peptides

PubMed Central

Crevenna, Alvaro H.; Bomblies, Rainer; Lamb, Don C.

2017-01-01

Fluorescence spectroscopy techniques like Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) have become important tools for the in vitro and in vivo investigation of conformational dynamics in biomolecules. These methods rely on the distance-dependent quenching of the fluorescence signal of a donor fluorophore either by a fluorescent acceptor fluorophore (FRET) or a non-fluorescent quencher, as used in FCS with photoinduced electron transfer (PET). The attachment of fluorophores to the molecule of interest can potentially alter the molecular properties and may affect the relevant conformational states and dynamics especially of flexible biomolecules like intrinsically disordered proteins (IDP). Using the intrinsically disordered S-peptide as a model system, we investigate the impact of terminal fluorescence labeling on the molecular properties. We perform extensive molecular dynamics simulations on the labeled and unlabeled peptide and compare the results with in vitro PET-FCS measurements. Experimental and simulated timescales of end-to-end fluctuations were found in excellent agreement. Comparison between simulations with and without labels reveal that the π-stacking interaction between the fluorophore labels traps the conformation of S-peptide in a single dominant state, while the unlabeled peptide undergoes continuous conformational rearrangements. Furthermore, we find that the open to closed transition rate of S-peptide is decreased by at least one order of magnitude by the fluorophore attachment. Our approach combining experimental and in silico methods provides a benchmark for the simulations and reveals the significant effect that fluorescence labeling can have on the conformational dynamics of small biomolecules, at least for inherently flexible short peptides. The presented protocol is not only useful for comparing PET-FCS experiments with simulation results but provides a strategy to minimize the influence on molecular properties when chosing labeling positions for fluorescence experiments. PMID:28542243

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

PubMed

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

2017-05-23

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

How and How Much Molecular Conformation Affects Electronic Circular Dichroism: The Case of 1,1-Diarylcarbinols.

PubMed

Padula, Daniele; Pescitelli, Gennaro

2018-01-09

Chiroptical spectra such as electronic circular dichroism (ECD) are said to be much more sensitive to conformation than their non-chiroptical counterparts, however, it is difficult to demonstrate such a common notion in a clear-cut way. We run DFT and TDDFT calculations on two closely related 1,1-diarylmethanols which show mirror-image ECD spectra for the same absolute configuration. We demonstrate that the main reason for the different chiroptical response of the two compounds lies in different conformational ensembles, caused by a single hydrogen-to-methyl substitution. We conclude that two compounds, having the same configuration but different conformation, may exhibit mirror-image ECD signals, stressing the importance and impact of conformational factors on ECD spectra.

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.

Probing Conformational Changes in Human DNA Topoisomerase IIα by Pulsed Alkylation Mass Spectrometry*

PubMed Central

Chen, Yu-tsung; Collins, Tammy R. L.; Guan, Ziqiang; Chen, Vincent B.; Hsieh, Tao-Shih

2012-01-01

Type II topoisomerases are essential enzymes for solving DNA topological problems by passing one segment of DNA duplex through a transient double-strand break in a second segment. The reaction requires the enzyme to precisely control DNA cleavage and gate opening coupled with ATP hydrolysis. Using pulsed alkylation mass spectrometry, we were able to monitor the solvent accessibilities around 13 cysteines distributed throughout human topoisomerase IIα by measuring the thiol reactivities with monobromobimane. Most of the measured reactivities are in accordance with the predicted ones based on a homology structural model generated from available crystal structures. However, these results reveal new information for both the residues not covered in the structural model and potential differences between the modeled and solution holoenzyme structures. Furthermore, on the basis of the reactivity changes of several cysteines located at the N-gate and DNA gate, we could monitor the movement of topoisomerase II in the presence of cofactors and detect differences in the DNA gate between two closed clamp enzyme conformations locked by either 5′-adenylyl β,γ-imidodiphosphate or the anticancer drug ICRF-193. PMID:22679013

Domain motions of Argonaute, the catalytic engine of RNA interference

PubMed Central

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

2007-01-01

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

Conformational properties of two exopolysaccharides produced by Inquilinus limosus, a cystic fibrosis lung pathogen.

PubMed

Kuttel, Michelle; Ravenscroft, Neil; Foschiatti, Michela; Cescutti, Paola; Rizzo, Roberto

2012-03-01

Inquilinus limosus is a multi-resistant bacterium found in the respiratory tract of patients with cystic fibrosis. This bacterium produces two unique fully pyruvylated exopolysaccharides in similar quantities: an α-(1→2)-linked mannan and a β-(1→3)-linked glucan. We employed molecular modelling methods to probe the characteristic conformations and dynamics of these polysaccharides, with corroboration from potentiometric titrations and circular dichroism experiments. Our calculations reveal different structural motifs for the mannan and glucan polysaccharides: the glucan forms primarily right-handed helices with a wide range of extensions, while the mannan forms only left-handed helices. This finding is supported by our circular dichroism experiments. Our calculations also show that the (1→3)-β-d-Glcp linkage is more dynamically flexible than the (1→2)-α-d-Manp: the glucan characteristically forms a range of wide helices with large central cavities. In contrast, the mannan forms rigid regular 'bottlebrush' helices with a minimal central cavity. The widely different character of these two polymers suggests a possible differentiation of biological roles. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

PubMed

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

2007-11-30

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

Conformational interconversions in peptide beta-turns: analysis of turns in proteins and computational estimates of barriers.

PubMed

Gunasekaran, K; Gomathi, L; Ramakrishnan, C; Chandrasekhar, J; Balaram, P

1998-12-18

The two most important beta-turn features in peptides and proteins are the type I and type II turns, which differ mainly in the orientation of the central peptide unit. Facile conformational interconversion is possible, in principle, by a flip of the central peptide unit. Homologous crystal structures afford an opportunity to structurally characterize both possible conformational states, thus allowing identification of sites that are potentially stereochemically mobile. A representative data set of 250 high-resolution (

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

PubMed

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

2015-01-01

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

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

PubMed Central

McGeagh, John D.; Mulholland, Adrian J.

2015-01-01

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

Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process

NASA Astrophysics Data System (ADS)

Iacovache, Ioan; de Carlo, Sacha; Cirauqui, Nuria; Dal Peraro, Matteo; van der Goot, F. Gisou; Zuber, Benoît

2016-07-01

Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry.

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

PubMed Central

Shrivastava, Indira; LaLonde, Judith M.

2012-01-01

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

The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs.

PubMed

Groves, M R; Hanlon, N; Turowski, P; Hemmings, B A; Barford, D

1999-01-08

The PR65/A subunit of protein phosphatase 2A serves as a scaffolding molecule to coordinate the assembly of the catalytic subunit and a variable regulatory B subunit, generating functionally diverse heterotrimers. Mutations of the beta isoform of PR65 are associated with lung and colon tumors. The crystal structure of the PR65/Aalpha subunit, at 2.3 A resolution, reveals the conformation of its 15 tandemly repeated HEAT sequences, degenerate motifs of approximately 39 amino acids present in a variety of proteins, including huntingtin and importin beta. Individual motifs are composed of a pair of antiparallel alpha helices that assemble in a mainly linear, repetitive fashion to form an elongated molecule characterized by a double layer of alpha helices. Left-handed rotations at three interrepeat interfaces generate a novel left-hand superhelical conformation. The protein interaction interface is formed from the intrarepeat turns that are aligned to form a continuous ridge.

Isolation of Ion-Driven Conformations in Diphenylacetylene Molecular Switches Using Cryogenic Infrared Spectroscopy

NASA Astrophysics Data System (ADS)

Wolk, Arron B.; Garand, Etienne; Jones, Ian M.; Kamrath, Michael Z.; Hamilton, Rew; Johnson, Mark A.

2012-06-01

We report the infrared predissociation spectra of a family of ionic diphenylacetylene molecular switch complexes. The electrosprayed complexes were trapped and cooled in a cryogenic (10K) quadrupole ion trap and tagged with molecular deuterium. The infrared spectra of the vibrationally cold species reveal sharp transitions over a wide energy range (800 - 3800 cm-1), facilitating comparison to harmonic spectra. The evolution of the band pattern upon derivatization of the complexes exposes the signatures of the amide, urea, and carbonyl functionalities, enabling unambiguous identification of the non-covalent interactions that control the secondary structure of the molecule. Complexation with the tetramethylammonium cation reveals a conformation analogous to that of the neutral molecule, while halide ion attachment induces a conformational change similar to that observed earlier in solution. In several cases, both the donor and acceptor groups involved in the multidentate H-bonds are observed, providing a microscopic mechanical picture of the interactions at play. I. Jones, and A. Hamilton, Angew. Chem. Intl. Edit. 50, 4597 (2011).

Concentrated Solutions of Single-Chain Nanoparticles: A Simple Model for Intrinsically Disordered Proteins under Crowding Conditions.

PubMed

Moreno, Angel J; Lo Verso, Federica; Arbe, Arantxa; Pomposo, José A; Colmenero, Juan

2016-03-03

By means of large-scale computer simulations and small-angle neutron scattering (SANS), we investigate solutions of single-chain nanoparticles (SCNPs), covering the whole concentration range from infinite dilution to melt density. The analysis of the conformational properties of the SCNPs reveals that these synthetic nano-objects share basic ingredients with intrinsically disordered proteins (IDPs), as topological polydispersity, generally sparse conformations, and locally compact domains. We investigate the role of the architecture of the SCNPs in their collapse behavior under macromolecular crowding. Unlike in the case of linear macromolecules, which experience the usual transition from self-avoiding to Gaussian random-walk conformations, crowding leads to collapsed conformations of SCNPs resembling those of crumpled globules. This behavior is already found at volume fractions (about 30%) that are characteristic of crowding in cellular environments. The simulation results are confirmed by the SANS experiments. Our results for SCNPs--a model system free of specific interactions--propose a general scenario for the effect of steric crowding on IDPs: collapse from sparse conformations at high dilution to crumpled globular conformations in cell environments.

Dielectric Properties of Poly(ethylene oxide) from Molecular Dynamics Simulations

NASA Technical Reports Server (NTRS)

Smith, Grant D.

1994-01-01

The order, conformations and dynamics of poly(oxyethylene) (POE) melts have been investigated through molecular dynamics simulations. The potential energy functions were determined from detailed ab initio electronic structure calculations of the conformational energies of the model molecules 1,2-dimethoxyethane (DME) and diethylether. The x-ray structure factor for POE from simulation will be compared to experiment. In terms of conformation, simulations reveal that chains are extended in the melt relative to isolated chains due to the presence of strong intermolecular O...H interactions, which occur at the expense of intramolecular O...H interactions. Conformational dynamics about the C-C bond were found to be significantly faster than in polymethylene, while conformational dynamics about the C-O bond even faster than the C-C dynamics. The faster local dynamics in POE relative to polymethylene is consistent with C-13 NMR spin-lattice relaxation experiments. Conformational transitions showed significant second-neighbor correlation, as was found for polymethylene. This correlation of transitions with C-C neighbors was found to be reduced relative to C-O neighbors. Dielectric relaxation from simulation will also be compared with experiment.

The major autoantibody epitope on factor H in atypical hemolytic uremic syndrome is structurally different from its homologous site in factor H-related protein 1, supporting a novel model for induction of autoimmunity in this disease.

PubMed

Bhattacharjee, Arnab; Reuter, Stefanie; Trojnár, Eszter; Kolodziejczyk, Robert; Seeberger, Harald; Hyvärinen, Satu; Uzonyi, Barbara; Szilágyi, Ágnes; Prohászka, Zoltán; Goldman, Adrian; Józsi, Mihály; Jokiranta, T Sakari

2015-04-10

Atypical hemolytic uremic syndrome (aHUS) is characterized by complement attack against host cells due to mutations in complement proteins or autoantibodies against complement factor H (CFH). It is unknown why nearly all patients with autoimmune aHUS lack CFHR1 (CFH-related protein-1). These patients have autoantibodies against CFH domains 19 and 20 (CFH19-20), which are nearly identical to CFHR1 domains 4 and 5 (CFHR14-5). Here, binding site mapping of autoantibodies from 17 patients using mutant CFH19-20 constructs revealed an autoantibody epitope cluster within a loop on domain 20, next to the two buried residues that are different in CFH19-20 and CFHR14-5. The crystal structure of CFHR14-5 revealed a difference in conformation of the autoantigenic loop in the C-terminal domains of CFH and CFHR1, explaining the variation in binding of autoantibodies from some aHUS patients to CFH19-20 and CFHR14-5. The autoantigenic loop on CFH seems to be generally flexible, as its conformation in previously published structures of CFH19-20 bound to the microbial protein OspE and a sialic acid glycan is somewhat altered. Cumulatively, our data suggest that association of CFHR1 deficiency with autoimmune aHUS could be due to the structural difference between CFHR1 and the autoantigenic CFH epitope, suggesting a novel explanation for CFHR1 deficiency in the pathogenesis of autoimmune aHUS. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Concurrent Increases and Decreases in Local Stability and Conformational Heterogeneity in Cu, Zn Superoxide Dismutase Variants Revealed by Temperature-Dependence of Amide Chemical Shifts.

PubMed

Doyle, Colleen M; Rumfeldt, Jessica A; Broom, Helen R; Sekhar, Ashok; Kay, Lewis E; Meiering, Elizabeth M

2016-03-08

The chemical shifts of backbone amide protons in proteins are sensitive reporters of local structural stability and conformational heterogeneity, which can be determined from their readily measured linear and nonlinear temperature-dependences, respectively. Here we report analyses of amide proton temperature-dependences for native dimeric Cu, Zn superoxide dismutase (holo pWT SOD1) and structurally diverse mutant SOD1s associated with amyotrophic lateral sclerosis (ALS). Holo pWT SOD1 loses structure with temperature first at its periphery and, while having extremely high global stability, nevertheless exhibits extensive conformational heterogeneity, with ∼1 in 5 residues showing evidence for population of low energy alternative states. The holo G93A and E100G ALS mutants have moderately decreased global stability, whereas V148I is slightly stabilized. Comparison of the holo mutants as well as the marginally stable immature monomeric unmetalated and disulfide-reduced (apo(2SH)) pWT with holo pWT shows that changes in the local structural stability of individual amides vary greatly, with average changes corresponding to differences in global protein stability measured by differential scanning calorimetry. Mutants also exhibit altered conformational heterogeneity compared to pWT. Strikingly, substantial increases as well as decreases in local stability and conformational heterogeneity occur, in particular upon maturation and for G93A. Thus, the temperature-dependence of amide shifts for SOD1 variants is a rich source of information on the location and extent of perturbation of structure upon covalent changes and ligand binding. The implications for potential mechanisms of toxic misfolding of SOD1 in disease and for general aspects of protein energetics, including entropy-enthalpy compensation, are discussed.

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.

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

PubMed Central

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

2009-01-01

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

Mechanism of allosteric propagation across a β-sheet structure investigated by molecular dynamics simulations.

PubMed

Interlandi, Gianluca; Thomas, Wendy E

2016-07-01

The bacterial adhesin FimH consists of an allosterically regulated mannose-binding lectin domain and a covalently linked inhibitory pilin domain. Under normal conditions, the two domains are bound to each other, and FimH interacts weakly with mannose. However, under tensile force, the domains separate and the lectin domain undergoes conformational changes that strengthen its bond with mannose. Comparison of the crystallographic structures of the low and the high affinity state of the lectin domain reveals conformational changes mainly in the regulatory inter-domain region, the mannose binding site and a large β sheet that connects the two distally located regions. Here, molecular dynamics simulations investigated how conformational changes are propagated within and between different regions of the lectin domain. It was found that the inter-domain region moves towards the high affinity conformation as it becomes more compact and buries exposed hydrophobic surface after separation of the pilin domain. The mannose binding site was more rigid in the high affinity state, which prevented water penetration into the pocket. The large central β sheet demonstrated a soft spring-like twisting. Its twisting motion was moderately correlated to fluctuations in both the regulatory and the binding region, whereas a weak correlation was seen in a direct comparison of these two distal sites. The results suggest a so called "population shift" model whereby binding of the lectin domain to either the pilin domain or mannose locks the β sheet in a rather twisted or flat conformation, stabilizing the low or the high affinity state, respectively. Proteins 2016; 84:990-1008. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Resonance Raman spectra of an O2-binding H-NOX domain reveal heme relaxation upon mutation.

PubMed

Tran, Rosalie; Boon, Elizabeth M; Marletta, Michael A; Mathies, Richard A

2009-09-15

Resonance Raman spectra were measured for the wild type Heme-Nitric oxide/OXygen binding domain from Thermoanaerobacter tengcongensis (Tt H-NOX WT) and three other Tt H-NOX proteins containing mutations at key conserved residues to determine the heme conformation in solution. The most dramatic changes in heme conformation occurred in the O2-bound forms, and the single Tt H-NOX P115A mutation was sufficient to generate a significant relaxation of the chromophore. Clear evidence of heme relaxation in the Tt H-NOX I5L, P115A, and I5L/P115A mutants in solution is demonstrated by the observation of reduced resonance Raman intensities for several out-of-plane low frequency modes (e.g., gamma11, gamma12, gamma13, and gamma15) in the 400-750 cm(-1) region known to be sensitive to ruffling and saddling deformations, as well as increased vibrational frequencies for the core heme skeletal stretching modes, nu3, nu2, and nu10. In addition, all three mutants exhibited some degree of heme conformational heterogeneity based on several broad skeletal markers (e.g., nu10) in the high frequency region. These results are comparable to those observed by Olea et al. for Tt H-NOX P115A in crystal form, where four different heme structures were determined from a single unit cell. On the basis of the resonance Raman spectra, it is clear that the actual heme conformation for Tt H-NOX P115A in solution is considerably more relaxed than that of the WT protein, with increased flexibility within the protein pocket, allowing for rapid sampling of alternate conformations.

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

Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo

The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use inmore » combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data.« less

Transferability of different classical force fields for right and left handed α-helices constructed from enantiomeric amino acids.

PubMed

Biswas, Santu; Sarkar, Sujit; Pandey, Prithvi Raj; Roy, Sudip

2016-02-21

Amino acids can form d and l enantiomers, of which the l enantiomer is abundant in nature. The naturally occurring l enantiomer has a greater preference for a right handed helical conformation, and the d enantiomer for a left handed helical conformation. The other conformations, that is, left handed helical conformations of the l enantiomers and right handed helical conformations of the d enantiomers, are not common. The energetic differences between left and right handed alpha helical peptide chains constructed from enantiomeric amino acids are investigated using quantum chemical calculations (using the M06/6-311g(d,p) level of theory). Further, the performances of commonly used biomolecular force fields (OPLS/AA, CHARMM27/CMAP and AMBER) to represent the different helical conformations (left and right handed) constructed from enantiomeric (D and L) amino acids are evaluated. 5- and 10-mer chains from d and l enantiomers of alanine, leucine, lysine, and glutamic acid, in right and left handed helical conformations, are considered in the study. Thus, in total, 32 α-helical polypeptides (4 amino acids × 4 conformations of 5-mer and 10-mer) are studied. Conclusions, with regards to the performance of the force fields, are derived keeping the quantum optimized geometry as the benchmark, and on the basis of phi and psi angle calculations, hydrogen bond analysis, and different long range helical order parameters.

Ionic tethering contributes to the conformational stability and function of complement C3b.

PubMed

López-Perrote, Andrés; Harrison, Reed E S; Subías, Marta; Alcorlo, Martín; Rodríguez de Córdoba, Santiago; Morikis, Dimitrios; Llorca, Oscar

2017-05-01

C3b, the central component of the alternative pathway (AP) of the complement system, coexists as a mixture of conformations in solution. These conformational changes can affect interactions with other proteins and complement regulators. Here we combine a computational model for electrostatic interactions within C3b with molecular imaging to study the conformation of C3b. The computational analysis shows that the TED domain in C3b is tethered ionically to the macroglobulin (MG) ring. Monovalent counterion concentration affects the magnitude of electrostatic forces anchoring the TED domain to the rest of the C3b molecule in a thermodynamic model. This is confirmed by observing NaCl concentration dependent conformational changes using single molecule electron microscopy (EM). We show that the displacement of the TED domain is compatible with C3b binding to Factor B (FB), suggesting that the regulation of the C3bBb convertase could be affected by conditions that promote movement in the TED domain. Our molecular model also predicts mutations that could alter the positioning of the TED domain, including the common R102G polymorphism, a risk variant for developing age-related macular degeneration. The common C3b isoform, C3bS, and the risk isoform, C3bF, show distinct energetic barriers to displacement in the TED that are related to a network of electrostatic interactions at the interface of the TED and MG-ring domains of C3b. These computational predictions agree with experimental evidence that shows differences in conformation observed in C3b isoforms purified from homozygous donors. Altogether, we reveal an ionic, reversible attachment of the TED domain to the MG ring that may influence complement regulation in some mutations and polymorphisms of C3b. Copyright © 2016 Elsevier Ltd. All rights reserved.

Conformation of a Group 2 Late Embryogenesis Abundant Protein from Soybean. Evidence of Poly (l-Proline)-type II Structure1

PubMed Central

Soulages, Jose L.; Kim, Kangmin; Arrese, Estela L.; Walters, Christina; Cushman, John C.

2003-01-01

Late embryogenesis abundant (LEA) proteins are members of a large group of hydrophilic, glycine-rich proteins found in plants, algae, fungi, and bacteria known collectively as hydrophilins that are preferentially expressed in response to dehydration or hyperosmotic stress. Group 2 LEA (dehydrins or responsive to abscisic acid) proteins are postulated to stabilize macromolecules against damage by freezing, dehydration, ionic, or osmotic stress. However, the structural and physicochemical properties of group 2 LEA proteins that account for such functions remain unknown. We have analyzed the structural properties of a recombinant form of a soybean (Glycine max) group 2 LEA (rGmDHN1). Differential scanning calorimetry of purified rGmDHN1 demonstrated that the protein does not display a cooperative unfolding transition upon heating. Ultraviolet absorption and circular dichroism spectroscopy revealed that the protein is in a largely hydrated and unstructured conformation in solution. However, ultraviolet absorption and circular dichroism measurements collected at different temperatures showed that the protein exists in equilibrium between two extended conformational states: unordered and left-handed extended helical or poly (l-proline)-type II structures. It is estimated that 27% of the residues of rGmDHN1 adopt or poly (l-proline)-type II-like helical conformation at 12°C. The content of extended helix gradually decreases to 15% as the temperature is increased to 80°C. Studies of the conformation of the protein in solution in the presence of liposomes, trifluoroethanol, and sodium dodecyl sulfate indicated that rGmDHN1 has a very low intrinsic ability to adopt α-helical structure and to interact with phospholipid bilayers through amphipathic α-helices. The ability of the protein to remain in a highly extended conformation at low temperatures could constitute the basis of the functional role of GmDHN1 in the prevention of freezing, desiccation, ionic, or osmotic stress-related damage to macromolecular structures. PMID:12644649

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

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

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

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

Dynamic Conformational Changes in MUNC18 Prevent Syntaxin Binding

PubMed Central

Bar-On, Dana; Nachliel, Esther; Gutman, Menachem; Ashery, Uri

2011-01-01

The Sec1/munc18 protein family is essential for vesicle fusion in eukaryotic cells via binding to SNARE proteins. Protein kinase C modulates these interactions by phosphorylating munc18a thereby reducing its affinity to one of the central SNARE members, syntaxin-1a. The established hypothesis is that the reduced affinity of the phosphorylated munc18a to syntaxin-1a is a result of local electrostatic repulsion between the two proteins, which interferes with their compatibility. The current study challenges this paradigm and offers a novel mechanistic explanation by revealing a syntaxin-non-binding conformation of munc18a that is induced by the phosphomimetic mutations. In the present study, using molecular dynamics simulations, we explored the dynamics of the wild-type munc18a versus phosphomimetic mutant munc18a. We focused on the structural changes that occur in the cavity between domains 3a and 1, which serves as the main syntaxin-binding site. The results of the simulations suggest that the free wild-type munc18a exhibits a dynamic equilibrium between several conformations differing in the size of its cavity (the main syntaxin-binding site). The flexibility of the cavity's size might facilitate the binding or unbinding of syntaxin. In silico insertion of phosphomimetic mutations into the munc18a structure induces the formation of a conformation where the syntaxin-binding area is rigid and blocked as a result of interactions between residues located on both sides of the cavity. Therefore, we suggest that the reduced affinity of the phosphomimetic mutant/phosphorylated munc18a is a result of the closed-cavity conformation, which makes syntaxin binding energetically and sterically unfavorable. The current study demonstrates the potential of phosphoryalation, an essential biological process, to serve as a driving force for dramatic conformational changes of proteins modulating their affinity to target proteins. PMID:21390273

Precursory signatures of protein folding/unfolding: From time series correlation analysis to atomistic mechanisms

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

Hsu, P. J.; Lai, S. K., E-mail: sklai@coll.phy.ncu.edu.tw; Molecular Science and Technology Program, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan

Folded conformations of proteins in thermodynamically stable states have long lifetimes. Before it folds into a stable conformation, or after unfolding from a stable conformation, the protein will generally stray from one random conformation to another leading thus to rapid fluctuations. Brief structural changes therefore occur before folding and unfolding events. These short-lived movements are easily overlooked in studies of folding/unfolding for they represent momentary excursions of the protein to explore conformations in the neighborhood of the stable conformation. The present study looks for precursory signatures of protein folding/unfolding within these rapid fluctuations through a combination of three techniques: (1)more » ultrafast shape recognition, (2) time series segmentation, and (3) time series correlation analysis. The first procedure measures the differences between statistical distance distributions of atoms in different conformations by calculating shape similarity indices from molecular dynamics simulation trajectories. The second procedure is used to discover the times at which the protein makes transitions from one conformation to another. Finally, we employ the third technique to exploit spatial fingerprints of the stable conformations; this procedure is to map out the sequences of changes preceding the actual folding and unfolding events, since strongly correlated atoms in different conformations are different due to bond and steric constraints. The aforementioned high-frequency fluctuations are therefore characterized by distinct correlational and structural changes that are associated with rate-limiting precursors that translate into brief segments. Guided by these technical procedures, we choose a model system, a fragment of the protein transthyretin, for identifying in this system not only the precursory signatures of transitions associated with α helix and β hairpin, but also the important role played by weaker correlations in such protein folding dynamics.« less

Precursory signatures of protein folding/unfolding: From time series correlation analysis to atomistic mechanisms

NASA Astrophysics Data System (ADS)

Hsu, P. J.; Cheong, S. A.; Lai, S. K.

2014-05-01

Folded conformations of proteins in thermodynamically stable states have long lifetimes. Before it folds into a stable conformation, or after unfolding from a stable conformation, the protein will generally stray from one random conformation to another leading thus to rapid fluctuations. Brief structural changes therefore occur before folding and unfolding events. These short-lived movements are easily overlooked in studies of folding/unfolding for they represent momentary excursions of the protein to explore conformations in the neighborhood of the stable conformation. The present study looks for precursory signatures of protein folding/unfolding within these rapid fluctuations through a combination of three techniques: (1) ultrafast shape recognition, (2) time series segmentation, and (3) time series correlation analysis. The first procedure measures the differences between statistical distance distributions of atoms in different conformations by calculating shape similarity indices from molecular dynamics simulation trajectories. The second procedure is used to discover the times at which the protein makes transitions from one conformation to another. Finally, we employ the third technique to exploit spatial fingerprints of the stable conformations; this procedure is to map out the sequences of changes preceding the actual folding and unfolding events, since strongly correlated atoms in different conformations are different due to bond and steric constraints. The aforementioned high-frequency fluctuations are therefore characterized by distinct correlational and structural changes that are associated with rate-limiting precursors that translate into brief segments. Guided by these technical procedures, we choose a model system, a fragment of the protein transthyretin, for identifying in this system not only the precursory signatures of transitions associated with α helix and β hairpin, but also the important role played by weaker correlations in such protein folding dynamics.

Statistical thermodynamics of protein folding: Comparison of a mean-field theory with Monte Carlo simulations

NASA Astrophysics Data System (ADS)

Hao, Ming-Hong; Scheraga, Harold A.

1995-01-01

A comparative study of protein folding with an analytical theory and computer simulations, respectively, is reported. The theory is based on an improved mean-field formalism which, in addition to the usual mean-field approximations, takes into account the distributions of energies in the subsets of conformational states. Sequence-specific properties of proteins are parametrized in the theory by two sets of variables, one for the energetics of mean-field interactions and one for the distribution of energies. Simulations are carried out on model polypeptides with different sequences, with different chain lengths, and with different interaction potentials, ranging from strong biases towards certain local chain states (bond angles and torsional angles) to complete absence of local conformational preferences. Theoretical analysis of the simulation results for the model polypeptides reveals three different types of behavior in the folding transition from the statistical coiled state to the compact globular state; these include a cooperative two-state transition, a continuous folding, and a glasslike transition. It is found that, with the fitted theoretical parameters which are specific for each polypeptide under a different potential, the mean-field theory can describe the thermodynamic properties and folding behavior of the different polypeptides accurately. By comparing the theoretical descriptions with simulation results, we verify the basic assumptions of the theory and, thereby, obtain new insights about the folding transitions of proteins. It is found that the cooperativity of the first-order folding transition of the model polypeptides is determined mainly by long-range interactions, in particular the dipolar orientation; the local interactions (e.g., bond-angle and torsion-angle potentials) have only marginal effect on the cooperative characteristic of the folding, but have a large impact on the difference in energy between the folded lowest-energy structure and the unfolded conformations of a protein.

Conformation-Specific Spectroscopy of a Prototypical γ-PEPTIDE-WATER Complex: Ac-γ2-hPhe-NHMe-(H2O)1

NASA Astrophysics Data System (ADS)

Buchanan, Evan G.; James, William H., III; Zwier, Timothy S.; Guo, Li; Gellman, Samuel H.

2010-06-01

The prototypical γ-peptide, Ac-γ2-hPhe-NHMe, has been previously studied in a supersonic jet expansion, with three different conformers observed. Two of the monomers form nine atom, intramolecular hydrogen bonded rings, which differ by the position of the aromatic chromophore relative to the backbone. The third monomer conformer has no intramolecular H-bonds, but forms instead an intramolecular, amide-amide stacked structure unique to the γ-peptide backbone. This talk focuses attention on the conformation-specific IR spectra of the Ac-γ2-hPhe-NHMe-(H2O)1 complex, which is observed to form six unique conformational isomers, all of which preserve the two distinct monomer structural motifs. Three conformers are assigned to the nine atom intramolecular hydrogen bond family with the water hydrogen bonded to it as donor in different locations. The other three belong to the amide-amide stacking family with the water forming a bridge between the two amide planes. Infrared photodissocation of the water molecule from the complex to form γ-peptide monomer conformations will also be discussed.

Knowledge-based versus experimentally acquired distance and angle constraints for NMR structure refinement.

PubMed

Cui, Feng; Jernigan, Robert; Wu, Zhijun

2008-04-01

Nuclear Overhauser effects (NOE) distance constraints and torsion angle constraints are major conformational constraints for nuclear magnetic resonance (NMR) structure refinement. In particular, the number of NOE constraints has been considered as an important determinant for the quality of NMR structures. Of course, the availability of torsion angle constraints is also critical for the formation of correct local conformations. In our recent work, we have shown how a set of knowledge-based short-range distance constraints can also be utilized for NMR structure refinement, as a complementary set of conformational constraints to the NOE and torsion angle constraints. In this paper, we show the results from a series of structure refinement experiments by using different types of conformational constraints--NOE, torsion angle, or knowledge-based constraints--or their combinations, and make a quantitative assessment on how the experimentally acquired constraints contribute to the quality of structural models and whether or not they can be combined with or substituted by the knowledge-based constraints. We have carried out the experiments on a small set of NMR structures. Our preliminary calculations have revealed that the torsion angle constraints contribute substantially to the quality of the structures, but require to be combined with the NOE constraints to be fully effective. The knowledge-based constraints can be functionally as crucial as the torsion angle constraints, although they are statistical constraints after all and are not meant to be able to replace the latter.

Atomic structure of recombinant thaumatin II reveals flexible conformations in two residues critical for sweetness and three consecutive glycine residues.

PubMed

Masuda, Tetsuya; Mikami, Bunzo; Tani, Fumito

2014-11-01

Thaumatin, an intensely sweet-tasting protein used as a sweetener, elicits a sweet taste at 50 nM. Although two major variants designated thaumatin I and thaumatin II exist in plants, there have been few dedicated thaumatin II structural studies and, to date, data beyond atomic resolution had not been obtained. To identify the detailed structural properties explaining why thaumatin elicits a sweet taste, the structure of recombinant thaumatin II was determined at the resolution of 0.99 Å. Atomic resolution structural analysis with riding hydrogen atoms illustrated the differences in the direction of the side-chains more precisely and the electron density maps of the C-terminal regions were markedly improved. Though it had been suggested that the three consecutive glycine residues (G142-G143-G144) have highly flexible conformations, G143, the central glycine residue was successfully modelled in two conformations for the first time. Furthermore, the side chain r.m.s.d. values for two residues (R67 and R82) critical for sweetness exhibited substantially higher values, suggesting that these residues are highly disordered. These results demonstrated that the flexible conformations in two critical residues favoring their interaction with sweet taste receptors are prominent features of the intensely sweet taste of thaumatin. Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

A pH-induced conformational switch in a tyrosine kinase inhibitor identified by electronic spectroscopy and quantum chemical calculations.

PubMed

Khattab, Muhammad; Wang, Feng; Clayton, Andrew H A

2017-11-24

Tyrosine kinase inhibitors (TKIs) are a major class of drug utilised in the clinic. During transit to their cognate kinases, TKIs will encounter different pH environments that could have a major influence on TKI structure. To address this, we report UV-Vis spectroscopic and computational studies of the TKI, AG1478, as a function of pH. The electronic absorption spectrum of AG1478 shifted by 10 nm (from 342 nm to 332 nm) from acid to neutral pH and split into two peaks (at 334 nm and 345 nm) in highly alkaline conditions. From these transitions, the pKa value was calculated as 5.58 ± 0.01. To compute structures and spectra, time-dependent density functional theory (TD-DFT) calculations were performed along with conductor-like polarizable continuum model (CPCM) to account for implicit solvent effect. On the basis of the theoretical spectra, we could assign the AG1478 experimental spectrum at acidic pH to a mixture of two twisted conformers (71% AG1478 protonated at quinazolyl nitrogen N(1) and 29% AG1478 protonated at quinazolyl nitrogen N(3)) and at neutral pH to the neutral planar conformer. The AG1478 absorption spectrum (pH 13.3) was fitted to a mixture of neutral (70%) and NH-deprotonated species (30%). These studies reveal a pH-induced conformational transition in a TKI.

Enzymatic Detoxication, Conformational Selection, and the Role of Molten Globule Active Sites*

PubMed Central

Honaker, Matthew T.; Acchione, Mauro; Zhang, Wei; Mannervik, Bengt; Atkins, William M.

2013-01-01

The role of conformational ensembles in enzymatic reactions remains unclear. Discussion concerning “induced fit” versus “conformational selection” has, however, ignored detoxication enzymes, which exhibit catalytic promiscuity. These enzymes dominate drug metabolism and determine drug-drug interactions. The detoxication enzyme glutathione transferase A1–1 (GSTA1–1), exploits a molten globule-like active site to achieve remarkable catalytic promiscuity wherein the substrate-free conformational ensemble is broad with barrierless transitions between states. A quantitative index of catalytic promiscuity is used to compare engineered variants of GSTA1–1 and the catalytic promiscuity correlates strongly with characteristics of the thermodynamic partition function, for the substrate-free enzymes. Access to chemically disparate transition states is encoded by the substrate-free conformational ensemble. Pre-steady state catalytic data confirm an extension of the conformational selection model, wherein different substrates select different starting conformations. The kinetic liability of the conformational breadth is minimized by a smooth landscape. We propose that “local” molten globule behavior optimizes detoxication enzymes. PMID:23649628

Using simulation to interpret experimental data in terms of protein conformational ensembles.

PubMed

Allison, Jane R

2017-04-01

In their biological environment, proteins are dynamic molecules, necessitating an ensemble structural description. Molecular dynamics simulations and solution-state experiments provide complimentary information in the form of atomically detailed coordinates and averaged or distributions of structural properties or related quantities. Recently, increases in the temporal and spatial scale of conformational sampling and comparison of the more diverse conformational ensembles thus generated have revealed the importance of sampling rare events. Excitingly, new methods based on maximum entropy and Bayesian inference are promising to provide a statistically sound mechanism for combining experimental data with molecular dynamics simulations. Copyright © 2016 Elsevier Ltd. All rights reserved.