Dynamics of the His79-heme alkaline transition of yeast iso-1-cytochrome c probed by conformationally gated electron transfer with Co(II)bis(terpyridine).

PubMed

Cherney, Melisa M; Junior, Carolyn C; Bergquist, Bryan B; Bowler, Bruce E

2013-08-28

Alkaline conformers of cytochrome c may be involved in both its electron transport and apoptotic functions. We use cobalt(II)bis(terpyridine), Co(terpy)2(2+), as a reagent for conformationally gated electron-transfer (gated ET) experiments to study the alkaline conformational transition of K79H variants of yeast iso-1-cytochrome c expressed in Escherichia coli , WT*K79H, with alanine at position 72 and Saccharomyces cerevisiae , yK79H, with trimethyllysine (Tml) at position 72. Co(terpy)2(2+) is well-suited to the 100 ms to 1 s time scale of the His79-mediated alkaline conformational transition of these variants. Reduction of the His79-heme alkaline conformer by Co(terpy)2(2+) occurs primarily by gated ET, which involves conversion to the native state followed by reduction, with a small fraction of the His79-heme alkaline conformer directly reduced by Co(terpy)2(2+). The gated ET experiments show that the mechanism of formation of the His79-heme alkaline conformer involves only two ionizable groups. In previous work, we showed that the mechanism of the His73-mediated alkaline conformational transition requires three ionizable groups. Thus, the mechanism of heme crevice opening depends upon the position of the ligand mediating the process. The microscopic rate constants provided by gated ET studies show that mutation of Tml72 (yK79H variant) in the heme crevice loop to Ala72 (WT*K79H variant) affects the dynamics of heme crevice opening through a small destabilization of both the native conformer and the transition state relative to the His79-heme alkaline conformer. Previous pH jump data had indicated that the Tml72→Ala mutation primarily stabilized the transition state for the His79-mediated alkaline conformational transition.

Dynamics of the His79-heme Alkaline Transition of Yeast Iso-1-cytochrome c Probed by Conformationally-gated Electron Transfer with Co(II)bis(terpyridine)†

PubMed Central

Cherney, Melisa M.; Junior, Carolyn C.; Bergquist, Bryan B.; Bowler, Bruce E.

2013-01-01

Alkaline conformers of cytochrome c may be involved in both its electron transport and apoptotic functions. We use cobalt(II)bis(terpyridine), Co(terpy)22+, as a reagent for conformationally-gated electron transfer (gated ET) experiments to study the alkaline conformational transition of K79H variants of yeast iso-1-cytochrome c expressed in Escherichia coli, WT*K79H, with alanine at position 72, and Saccharomyces cerevisiae, yK79H, with trimethyllysine (Tml) at position 72. Co(terpy)22+ is well-suited to the 100 ms to 1 s time scale of the His79-mediated alkaline conformational transition of these variants. Reduction of the His79-heme alkaline conformer by Co(terpy)22+ occurs primarily by gated ET, which involves conversion to the native state followed by reduction, with a small fraction of the His79- heme alkaline conformer directly reduced by Co(terpy)22+. The gated ET experiments show that the mechanism of formation of the His79-heme alkaline conformer involves only two ionizable groups. In previous work, we showed that the mechanism of the His73-mediated alkaline conformational transition requires three ionizable groups. Thus, the mechanism of heme crevice opening depends upon the position of the ligand mediating the process. The microscopic rate constants provided by gated ET studies show that mutation of Tml72 (yK79H variant) in the heme crevice loop to Ala72 (WT*K79H variant) affects the dynamics of heme crevice opening through a small destabilization of both the native conformer and the transition state relative to the His79-heme alkaline conformer. Previous pH jump data had indicated that the Tml72→Ala mutation primarily stabilized the transition state for the His79-mediated alkaline conformational transition. PMID:23899348

Database of non-canonical base pairs found in known RNA structures

NASA Technical Reports Server (NTRS)

Nagaswamy, U.; Voss, N.; Zhang, Z.; Fox, G. E.

2000-01-01

Atomic resolution RNA structures are being published at an increasing rate. It is common to find a modest number of non-canonical base pairs in these structures in addition to the usual Watson-Crick pairs. This database summarizes the occurrence of these rare base pairs in accordance with standard nomenclature. The database, http://prion.bchs.uh.edu/, contains information such as sequence context, sugar pucker conformation, anti / syn base conformations, chemical shift, p K (a)values, melting temperature and free energy. Of the 29 anticipated pairs with two or more hydrogen bonds, 20 have been encountered to date. In addition, four unexpected pairs with two hydrogen bonds have been reported bringing the total to 24. Single hydrogen bond versions of five of the expected geometries have been encountered among the single hydrogen bond interactions. In addition, 18 different types of base triplets have been encountered, each of which involves three to six hydrogen bonds. The vast majority of the rare base pairs are antiparallel with the bases in the anti configuration relative to the ribose. The most common are the GU wobble, the Sheared GA pair, the Reverse Hoogsteen pair and the GA imino pair.

15 CFR 286.2 - Scope.

Code of Federal Regulations, 2014 CFR

2014-01-01

... accreditation body which satisfies the established requirements. (b) NIST operates the NVCASE program as follows: (1) Conformity level: Under this program NIST accepts requests for evaluations of U.S. bodies involved in activities related to conformity assessment. NIST does not perform conformity assessments as...

15 CFR 286.2 - Scope.

Code of Federal Regulations, 2012 CFR

2012-01-01

... accreditation body which satisfies the established requirements. (b) NIST operates the NVCASE program as follows: (1) Conformity level: Under this program NIST accepts requests for evaluations of U.S. bodies involved in activities related to conformity assessment. NIST does not perform conformity assessments as...

15 CFR 286.2 - Scope.

Code of Federal Regulations, 2011 CFR

2011-01-01

... accreditation body which satisfies the established requirements. (b) NIST operates the NVCASE program as follows: (1) Conformity level: Under this program NIST accepts requests for evaluations of U.S. bodies involved in activities related to conformity assessment. NIST does not perform conformity assessments as...

15 CFR 286.2 - Scope.

Code of Federal Regulations, 2010 CFR

2010-01-01

... accreditation body which satisfies the established requirements. (b) NIST operates the NVCASE program as follows: (1) Conformity level: Under this program NIST accepts requests for evaluations of U.S. bodies involved in activities related to conformity assessment. NIST does not perform conformity assessments as...

15 CFR 286.2 - Scope.

Code of Federal Regulations, 2013 CFR

2013-01-01

... accreditation body which satisfies the established requirements. (b) NIST operates the NVCASE program as follows: (1) Conformity level: Under this program NIST accepts requests for evaluations of U.S. bodies involved in activities related to conformity assessment. NIST does not perform conformity assessments as...

Human Blue Cone Opsin Regeneration Involves Secondary Retinal Binding with Analog Specificity.

PubMed

Srinivasan, Sundaramoorthy; Fernández-Sampedro, Miguel A; Morillo, Margarita; Ramon, Eva; Jiménez-Rosés, Mireia; Cordomí, Arnau; Garriga, Pere

2018-03-27

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigments. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Probing Conformational Changes and Interfacial Recognition Site of Lipases With Surfactants and Inhibitors.

PubMed

Mateos-Diaz, E; Amara, S; Roussel, A; Longhi, S; Cambillau, C; Carrière, F

2017-01-01

Structural studies on lipases by X-ray crystallography have revealed conformational changes occurring in the presence of surfactants/inhibitors and the pivotal role played by a molecular "lid" of variable size and structure depending on the enzyme. Besides controlling the access to the enzyme active site, the lid is involved in lipase activation, formation of the interfacial recognition site (IRS), and substrate docking within the active site. The combined use of surfactants and inhibitors has been critical for a better understanding of lipase structure-function relationships. An overview of crystal structures of lipases in complex with surfactants and inhibitors reveals common structural features and shows how surfactants monomers interact with the lid in its open conformation. The location of surfactants, inhibitors, and hydrophobic residues exposed upon lid opening provides insights into the IRS of lipases. The mechanism by which surfactants promote the lid opening can be further investigated in solution by site-directed spin labeling of lipase coupled to electron paramagnetic resonance spectroscopy. These experimental approaches are illustrated here by results obtained with mammalian digestive lipases, fungal lipases, and cutinases. © 2017 Elsevier Inc. All rights reserved.

Peer pressure and alcohol use in young men: a mediation analysis of drinking motives.

PubMed

Studer, Joseph; Baggio, Stéphanie; Deline, Stéphane; N'Goran, Alexandra A; Henchoz, Yves; Mohler-Kuo, Meichun; Daeppen, Jean-Bernard; Gmel, Gerhard

2014-07-01

Peer pressure (PP) has been shown to play a major role in the development and continuation of alcohol use and misuse. To date, almost all the studies investigating the association of PP with alcohol use only considered the PP for misconduct but largely ignored other aspects of PP, such as pressure for peer involvement and peer conformity. Moreover, it is not clear whether the association of PP with alcohol use is direct or mediated by other factors. The aim of the present study was to investigate the association of different aspects of peer pressure (PP) with drinking volume (DV) and risky single-occasion drinking (RSOD), and to explore whether these associations were mediated by drinking motives (DM). A representative sample of 5521 young Swiss men, aged around 20 years old, completed a questionnaire assessing their usual weekly DV, the frequency of RSOD, DM (i.e. enhancement, social, coping, and conformity motives), and 3 aspects of PP (i.e. misconduct, peer involvement, and peer conformity). Associations between PP and alcohol outcomes (DV and RSOD) as well as the mediation of DM were tested using structural equation models. Peer pressure to misconduct was associated with more alcohol use, whereas peer involvement and peer conformity were associated with less alcohol use. Associations of drinking outcomes with PP to misconduct and peer involvement were partially mediated by enhancement and coping motives, while the association with peer conformity was partially mediated by enhancement and conformity motives. Results suggest that PP to misconduct constitutes a risk factor, while peer conformity and peer involvement reflect protective factors with regard to alcohol use. Moreover, results from the mediation analyses suggest that part of the association of PP with alcohol use came indirectly through DM: PP was associated with DM, which in turn were associated with alcohol use. Copyright © 2014 Elsevier B.V. All rights reserved.

Multiple Replica Repulsion Technique for Efficient Conformational Sampling of Biological Systems

PubMed Central

Malevanets, Anatoly; Wodak, Shoshana J.

2011-01-01

Here, we propose a technique for sampling complex molecular systems with many degrees of freedom. The technique, termed “multiple replica repulsion” (MRR), does not suffer from poor scaling with the number of degrees of freedom associated with common replica exchange procedures and does not require sampling at high temperatures. The algorithm involves creation of multiple copies (replicas) of the system, which interact with one another through a repulsive potential that can be applied to the system as a whole or to portions of it. The proposed scheme prevents oversampling of the most populated states and provides accurate descriptions of conformational perturbations typically associated with sampling ground-state energy wells. The performance of MRR is illustrated for three systems of increasing complexity. A two-dimensional toy potential surface is used to probe the sampling efficiency as a function of key parameters of the procedure. MRR simulations of the Met-enkephalin pentapeptide, and the 76-residue protein ubiquitin, performed in presence of explicit water molecules and totaling 32 ns each, investigate the ability of MRR to characterize the conformational landscape of the peptide, and the protein native basin, respectively. Results obtained for the enkephalin peptide reflect more closely the extensive conformational flexibility of this peptide than previously reported simulations. Those obtained for ubiquitin show that conformational ensembles sampled by MRR largely encompass structural fluctuations relevant to biological recognition, which occur on the microsecond timescale, or are observed in crystal structures of ubiquitin complexes with other proteins. MRR thus emerges as a very promising simple and versatile technique for modeling the structural plasticity of complex biological systems. PMID:21843487

A UV-complete Composite Higgs model for Electroweak Symmetry Breaking: Minimal Conformal Technicolor

NASA Astrophysics Data System (ADS)

Tacchi, Ruggero Altair

The Large Hadron Collider is currently collecting data. One of the main goals of the experiment is to find evidence of the mechanism responsible for the breaking of the electroweak symmetry. There are many different models attempting to explain this breaking and traditionally most of them involve the use of supersymmetry near the scale of the breaking. This work is focused on exploring a viable model that is not based on a weakly coupled low scale supersymmetry sector to explain the electroweak symmetry breaking. We build a model based on a new strong interaction, in the fashion of theories commonly called "technicolor", name that is reminiscent of one of the first attempts of explaining the electroweak symmetry breaking using a strong interaction similar to the one whose charges are called colors. We explicitly study the minimal model of conformal technicolor, an SU(2) gauge theory near a strongly coupled conformal fixed point, with conformal symmetry softly broken by technifermion mass terms. Conformal symmetry breaking triggers chiral symmetry breaking in the pattern SU(4) → Sp (4), which gives rise to a pseudo-Nambu-Goldstone boson that can act as a composite Higgs boson. There is an additional composite pseudoscalar A with mass larger than mh and suppressed direct production at LHC. We discuss the electroweak fit in this model in detail. A good fit requires fine tuning at the 10% level. We construct a complete, realistic, and natural UV completion of the model, that explains the origin of quark and lepton masses and mixing angles. We embed conformal technicolor in a supersymmetric theory, with supersymmetry broken at a high scale. The effective theory below the supersymmetry breaking scale is minimal conformal technicolor with an additional light technicolor gaugino that might give rise to an additional pseudo Nambu-Goldstone boson that is observable at the LHC.

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…

Cyclo-biphenalenyl biradicaloid molecular materials: conformation, tautomerization, magnetism, and thermochromism

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

Huang, Jingsong; Sumpter, Bobby G; Meunier, Vincent

2011-01-01

Phenalenyl and its derivatives have recently attracted a great deal of interest as a result of a 2-electron multicenter (2e/mc) covalent pi-pi bonding between two pi-stacked phenalenyl units. The 2e/mc bonded pi-dimers are close in energy to the sigma-dimers of phenalenyl and therefore fickle properties may emerge from bond fluctuation, yielding smart pi-functional materials. Here we examine the valence tautomerization of two cyclo-biphenalenyl biradicaloid molecular materials with chair- and boat-conformations by spin-restricted (R) and unrestricted (U) DFT using the M06 and B3LYP functionals. We found that the chair-conformation involves a 2e/4c pi-pi bonded structure while the boat-conformation involves a 2e/12cmore » pi-pi bonded structure on their potential energy surfaces. The global minimum for the chair-conformation is the sigma-bonded structure while it is the pi-pi bonded structure for the boat-conformation. The chair-conformation exhibits a stepwise [3,3]-sigmatropic rearrangement, and calculations predict a negligible paramagnetic susceptibility near room temperature. In comparison, the paramagnetism of the boat-conformation should be observable by SQUID and/or ESR. According to the difference of the global minima of the two conformations and the parameterized UV-Vis calculations, the color of the chair-conformation is expected to become darker while that of the boat-conformation become lighter with increasing temperature.« less

77 FR 9625 - Presentation of Final Conventional Conformance Test Criteria and Common Air Interface (CAI...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-17

... Tests for Inclusion in the Program AGENCY: National Institute of Standards and Technology (NIST... meeting is to present the final requirements for CAI conventional conformance tests for inclusion in the... suitability for inclusion in the P25 CAP is below: Conformance tests should limit devices in the test...

Hierarchical sampling for metastable conformers determines biomolecular recognition: the case of malectin and diglucosylated N-glycan interactions.

PubMed

Mamidi, Ashalatha Sreshty; Surolia, Avadhesha

2015-01-01

Structural information over the entire course of binding interactions based on the analyses of energy landscapes is described, which provides a framework to understand the events involved during biomolecular recognition. Conformational dynamics of malectin's exquisite selectivity for diglucosylated N-glycan (Dig-N-glycan), a highly flexible oligosaccharide comprising of numerous dihedral torsion angles, are described as an example. For this purpose, a novel approach based on hierarchical sampling for acquiring metastable molecular conformations constituting low-energy minima for understanding the structural features involved in a biologic recognition is proposed. For this purpose, four variants of principal component analysis were employed recursively in both Cartesian space and dihedral angles space that are characterized by free energy landscapes to select the most stable conformational substates. Subsequently, k-means clustering algorithm was implemented for geometric separation of the major native state to acquire a final ensemble of metastable conformers. A comparison of malectin complexes was then performed to characterize their conformational properties. Analyses of stereochemical metrics and other concerted binding events revealed surface complementarity, cooperative and bidentate hydrogen bonds, water-mediated hydrogen bonds, carbohydrate-aromatic interactions including CH-π and stacking interactions involved in this recognition. Additionally, a striking structural transition from loop to β-strands in malectin CRD upon specific binding to Dig-N-glycan is observed. The interplay of the above-mentioned binding events in malectin and Dig-N-glycan supports an extended conformational selection model as the underlying binding mechanism.

Investigating ion channel conformational changes using voltage clamp fluorometry.

PubMed

Talwar, Sahil; Lynch, Joseph W

2015-11-01

Ion channels are membrane proteins whose functions are governed by conformational changes. The widespread distribution of ion channels, coupled with their involvement in most physiological and pathological processes and their importance as therapeutic targets, renders the elucidation of these conformational mechanisms highly compelling from a drug discovery perspective. Thanks to recent advances in structural biology techniques, we now have high-resolution static molecular structures for members of the major ion channel families. However, major questions remain to be resolved about the conformational states that ion channels adopt during activation, drug modulation and desensitization. Patch-clamp electrophysiology has long been used to define ion channel conformational states based on functional criteria. It achieves this by monitoring conformational changes at the channel gate and cannot detect conformational changes occurring in regions distant from the gate. Voltage clamp fluorometry involves labelling cysteines introduced into domains of interest with environmentally sensitive fluorophores and inferring structural rearrangements from voltage or ligand-induced fluorescence changes. Ion channel currents are monitored simultaneously to verify the conformational status. By defining real time conformational changes in domains distant from the gate, this technique provides unexpected new insights into ion channel structure and function. This review aims to summarise the methodology and highlight recent innovative applications of this powerful technique. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'. Copyright © 2015 Elsevier Ltd. All rights reserved.

Infrared multiple photon dissociation action spectroscopy of deprotonated RNA mononucleotides: gas-phase conformations and energetics.

PubMed

Nei, Y-w; Crampton, K T; Berden, G; Oomens, J; Rodgers, M T

2013-10-17

The IRMPD action spectra of the deprotonated forms of the four common RNA mononucleotides, adenosine-5'-monophosphate (A5'p), guanosine-5'-monophosphate (G5'p), cytidine-5'-monophosphate (C5'p), and uridine-5'-monophosphate (U5'p), are measured to probe their gas-phase structures. The IRMPD action spectra of all four deprotonated RNA mononucleotides exhibit distinct IR signatures in the frequency region investigated, 570-1900 cm(-1), that allows these deprotonated mononucleotides to be easily differentiated from one other. Comparison of the measured IRMPD action spectra to the linear IR spectra calculated at the B3LYP/6-31+G(d,p) level of theory finds that the most stable conformations of the deprotonated forms of A5'p, C5'p, and U5'p are accessed in the experiments, and these conformers adopt the C3' endo conformation of the ribose moiety and the anti conformation of the nucleobase. In the case of deprotonated G5'p, the most stable conformer is also accessed in the experiments. However, the ground-state conformer differs from the other three deprotonated RNA mononucleotides in that it adopts the syn rather than anti conformation for the nucleobase. Present results are compared to results previously obtained for the deprotonated forms of the four common DNA mononucleotides to examine the fundamental conformational differences between these species, and thus elucidate the effects of the 2'-hydroxyl group on their structure, stability, and fragmentation behavior.

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.

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

PubMed

Weininger, Ulrich; Respondek, Michal; Akke, Mikael

2012-09-01

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

Molecular Mechanics

PubMed Central

Vanommeslaeghe, Kenno; Guvench, Olgun; MacKerell, Alexander D.

2014-01-01

Molecular Mechanics (MM) force fields are the methods of choice for protein simulations, which are essential in the study of conformational flexibility. Given the importance of protein flexibility in drug binding, MM is involved in most if not all Computational Structure-Based Drug Discovery (CSBDD) projects. This section introduces the reader to the fundamentals of MM, with a special emphasis on how the target data used in the parametrization of force fields determine their strengths and weaknesses. Variations and recent developments such as polarizable force fields are discussed. The section ends with a brief overview of common force fields in CSBDD. PMID:23947650

Pulse EPR distance measurements to study multimers and multimerisation

NASA Astrophysics Data System (ADS)

Ackermann, Katrin; Bode, Bela E.

2018-06-01

Pulse dipolar electron paramagnetic resonance (PD-EPR) has become a powerful tool for structural biology determining distances on the nanometre scale. Recent advances in hardware, methodology, and data analysis have widened the scope to complex biological systems. PD-EPR can be applied to systems containing lowly populated conformers or displaying large intrinsic flexibility, making them all but intractable for cryo-electron microscopy and crystallography. Membrane protein applications are of particular interest due to the intrinsic difficulties for obtaining high-resolution structures of all relevant conformations. Many drug targets involved in critical cell functions are multimeric channels or transporters. Here, common approaches for introducing spin labels for PD-EPR cause the presence of more than two electron spins per multimeric complex. This requires careful experimental design to overcome detrimental multi-spin effects and to secure sufficient distance resolution in presence of multiple distances. In addition to obtaining mere distances, PD-EPR can also provide information on multimerisation degrees allowing to study binding equilibria and to determine dissociation constants.

Influence of ethanol on the metabolism of alprazolam.

PubMed

Huang, Zhibin; Xu, Zhiru; Wang, Hao; Zhao, Z Q; Rao, Yulan

2018-06-01

Alprazolam is a commonly used benzodiazepine in clinical practice, and when coingested with ethanol, alprazolam can increase behavioral irritability and aggression. However, the mechanism of its interaction with ethanol remains unknown. The pharmacokinetics of alprazolam was studied in vivo in rat experiments involving the simultaneous administration of alprazolam and ethanol, and the interactions between ethanol and alprazolam were investigated in vitro in human liver microsomes. In silico molecular docking was applied to analyze the change in the CYP3A4-alprazolam-binding conformation when ethanol was coadministered with alprazolam. Compared with alprazolam administered alone (2 mg/kg), the C max of alprazolam increased when ethanol was simultaneously administered at 3 g/kg. The concentrations of alprazolam significantly increased by 39%, 17%, 105%, and 642% at 5, 10, 30, and 120 min intervals in the brain when coadministered with ethanol, respectively. Molecular docking results suggested that the conformation of CYP3A4 with alprazolam changed when ethanol was bound to the SER119 residue, which seems critical in the process of CYP3A4-alprazolam binding. Ethanol might increase the toxicity of alprazolam by inhibiting the activity of CYP3A4, although other pharmacokinetic processes may be affected. Ethanol could change the conformation of CYP3A4 and affect alprazolam binding.

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

Insights into the conformational switching mechanism of the human vascular endothelial growth factor receptor type 2 kinase domain.

PubMed

Chioccioli, Matteo; Marsili, Simone; Bonaccini, Claudia; Procacci, Piero; Gratteri, Paola

2012-02-27

Human vascular endothelial growth factor receptor type 2 (h-VEFGR2) is a receptor tyrosine kinase involved in the angiogenesis process and regarded as an interesting target for the design of anticancer drugs. Its activation/inactivation mechanism is related to conformational changes in its cytoplasmatic kinase domain, involving first among all the αC-helix in N-lobe and the A-loop in C-lobe. Affinity of inhibitors for the active or inactive kinase form could dictate the open or closed conformation of the A-loop, thus making the different conformations of the kinase domain receptor (KDR) domain different drug targets in drug discovery. In this view, a detailed knowledge of the conformational landscape of KDR domain is of central relevance to rationalize the efficiency and selectivity of kinase inhibitors. Here, molecular dynamics simulations were used to gain insight into the conformational switching activity of the KDR domain and to identify intermediate conformations between the two limiting active and inactive conformations. Specific energy barriers have been selectively removed to induce, and hence highlight at the atomistic level, the regulation mechanism of the A-loop opening. The proposed strategy allowed to repeatedly observe the escape of the KDR domain from the DFG-out free energy basin and to identify rare intermediate conformations between the DFG-out and the DFG-in structures to be employed in a structure-based drug discovery process.

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.

A surprising role for conformational entropy in protein function

PubMed Central

Wand, A. Joshua; Moorman, Veronica R.; Harpole, Kyle W.

2014-01-01

Formation of high-affinity complexes is critical for the majority of enzymatic reactions involving proteins. The creation of the family of Michaelis and other intermediate complexes during catalysis clearly involves a complicated manifold of interactions that are diverse and complex. Indeed, computing the energetics of interactions between proteins and small molecule ligands using molecular structure alone remains a grand challenge. One of the most difficult contributions to the free energy of protein-ligand complexes to experimentally access is that due to changes in protein conformational entropy. Fortunately, recent advances in solution nuclear magnetic resonance (NMR) relaxation methods have enabled the use of measures-of-motion between conformational states of a protein as a proxy for conformational entropy. This review briefly summarizes the experimental approaches currently employed to characterize fast internal motion in proteins, how this information is used to gain insight into conformational entropy, what has been learned and what the future may hold for this emerging view of protein function. PMID:23478875

Histone H3 Tails Containing Dimethylated Lysine and Adjacent Phosphorylated Serine Modifications Adopt a Specific Conformation during Mitosis and Meiosis▿ †

PubMed Central

Eberlin, Adrien; Grauffel, Cédric; Oulad-Abdelghani, Mustapha; Robert, Flavie; Torres-Padilla, Maria-Elena; Lambrot, Romain; Spehner, Danièle; Ponce-Perez, Lourdes; Würtz, Jean-Marie; Stote, Roland H.; Kimmins, Sarah; Schultz, Patrick; Dejaegere, Annick; Tora, Laszlo

2008-01-01

Condensation of chromatin, mediated in part by posttranslational modifications of histones, is essential for cell division during mitosis. Histone H3 tails are dimethylated on lysine (Kme2) and become phosphorylated on serine (Sp) residues during mitosis. We have explored the possibility that these double modifications are involved in the establishment of H3 tail conformations during the cell cycle. Here we describe a specific chromatin conformation occurring at Kme2 and adjacently phosphorylated S of H3 tails upon formation of a hydrogen bond. This conformation appears exclusively between early prophase and early anaphase of the mitosis, when chromatin condensation is highest. Moreover, we observed that the conformed H3Kme2Sp tail is present at the diplotene and metaphase stages in spermatocytes and oocytes. Our data together with results obtained by cryoelectron microscopy suggest that the conformation of Kme2Sp-modified H3 tails changes during mitosis and meiosis. This is supported by biostructural modeling of a modified histone H3 tail bound by an antibody, indicating that Kme2Sp-modified H3 tails can adopt at least two different conformations. Thus, the H3K9me2S10p and the H3K27me2S28p sites are involved in the acquisition of specific chromatin conformations during chromatin condensation for cell division. PMID:18180282

Fabrication challenges associated with conformal optics

NASA Astrophysics Data System (ADS)

Schaefer, John; Eichholtz, Richard A.; Sulzbach, Frank C.

2001-09-01

A conformal optic is typically an optical window that conforms smoothly to the external shape of a system platform to improve aerodynamics. Conformal optics can be on-axis, such as an ogive missile dome, or off-axis, such as in a free form airplane wing. A common example of conformal optics is the automotive head light window that conforms to the body of the car aerodynamics and aesthetics. The unusual shape of conformal optics creates tremendous challenges for design, manufacturing, and testing. This paper will discuss fabrication methods that have been successfully demonstrated to produce conformal missile domes and associated wavefront corrector elements. It will identify challenges foreseen with more complex free-form configurations. Work presented in this paper was directed by the Precision Conformal Optics Consortium (PCOT). PCOT is comprised of both industrial and academic members who teamed to develop and demonstrate conformal optical systems suitable for insertion into future military programs. The consortium was funded under DARPA agreement number MDA972-96-9-08000.

Pilot Non-Conformance to Alerting System Commands

NASA Technical Reports Server (NTRS)

Pritchett, Amy R.; Hansman, R. John

1997-01-01

Instances of pilot non-conformance to alerting system commands have been identified in previous studies. Pilot non-conformance changes the final behavior of the system, and therefore may reduce actual performance from that anticipated. A simulator study has examined pilot non-conformance, using the task of collision avoidance during closely spaced parallel approaches as a case study. Consonance between the display and the alerting system was found to significantly improve subject agreement with automatic alerts. Based on these results, a more general discussion of the factors involved in pilot conformance is given, and design guidelines for alerting systems are given.

The arrestin-1 finger loop interacts with two distinct conformations of active rhodopsin.

PubMed

Elgeti, Matthias; Kazmin, Roman; Rose, Alexander S; Szczepek, Michal; Hildebrand, Peter W; Bartl, Franz J; Scheerer, Patrick; Hofmann, Klaus Peter

2018-03-23

Signaling of the prototypical G protein-coupled receptor (GPCR) rhodopsin through its cognate G protein transducin (G t ) is quenched when arrestin binds to the activated receptor. Although the overall architecture of the rhodopsin/arrestin complex is known, many questions regarding its specificity remain unresolved. Here, using FTIR difference spectroscopy and a dual pH/peptide titration assay, we show that rhodopsin maintains certain flexibility upon binding the "finger loop" of visual arrestin (prepared as synthetic peptide ArrFL-1). We found that two distinct complexes can be stabilized depending on the protonation state of E3.49 in the conserved (D)ERY motif. Both complexes exhibit different interaction modes and affinities of ArrFL-1 binding. The plasticity of the receptor within the rhodopsin/ArrFL-1 complex stands in contrast to the complex with the C terminus of the G t α-subunit (GαCT), which stabilizes only one specific substate out of the conformational ensemble. However, G t α-subunit binding and both ArrFL-1-binding modes involve a direct interaction to conserved R3.50, as determined by site-directed mutagenesis. Our findings highlight the importance of receptor conformational flexibility and cytoplasmic proton uptake for modulation of rhodopsin signaling and thereby extend the picture provided by crystal structures of the rhodopsin/arrestin and rhodopsin/ArrFL-1 complexes. Furthermore, the two binding modes of ArrFL-1 identified here involve motifs of conserved amino acids, which indicates that our results may have elucidated a common modulation mechanism of class A GPCR-G protein/-arrestin signaling. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Asymmetric processing of a substrate protein in sequential allosteric cycles of AAA+ nanomachines

NASA Astrophysics Data System (ADS)

Kravats, Andrea N.; Tonddast-Navaei, Sam; Bucher, Ryan J.; Stan, George

2013-09-01

Essential protein quality control includes mechanisms of substrate protein (SP) unfolding and translocation performed by powerful ring-shaped AAA+ (ATPases associated with various cellular activities) nanomachines. These SP remodeling actions are effected by mechanical forces imparted by AAA+ loops that protrude into the central channel. Sequential intra-ring allosteric motions, which underlie repetitive SP-loop interactions, have been proposed to comprise clockwise (CW), counterclockwise (CCW), or random (R) conformational transitions of individual AAA+ subunits. To probe the effect of these allosteric mechanisms on unfoldase and translocase functions, we perform Langevin dynamics simulations of a coarse-grained model of an all-alpha SP processed by the single-ring ClpY ATPase or by the double-ring p97 ATPase. We find that, in all three allosteric mechanisms, the SP undergoes conformational transitions along a common set of pathways, which reveals that the active work provided by the ClpY machine involves single loop-SP interactions. Nevertheless, the rates and yields of SP unfolding and translocation are controlled by mechanism-dependent loop-SP binding events, as illustrated by faster timescales of SP processing in CW allostery compared with CCW and R allostery. The distinct efficacy of allosteric mechanisms is due to the asymmetric collaboration of adjacent subunits, which involves CW-biased structural motions of AAA+ loops and results in CW-compatible torque applied onto the SP. Additional simulations of mutant ClpY rings, which render a subset of subunits catalytically-defective or reduce their SP binding affinity, reveal that subunit-based conformational transitions play the major role in SP remodeling. Based on these results we predict that the minimally functional AAA+ ring includes three active subunits, only two of which are adjacent.

Direct patterning of highly-conductive graphene@copper composites using copper naphthenate as a resist for graphene device applications.

PubMed

Bi, Kaixi; Xiang, Quan; Chen, Yiqin; Shi, Huimin; Li, Zhiqin; Lin, Jun; Zhang, Yongzhe; Wan, Qiang; Zhang, Guanhua; Qin, Shiqiao; Zhang, Xueao; Duan, Huigao

2017-11-09

We report an electron-beam lithography process to directly fabricate graphene@copper composite patterns without involving metal deposition, lift-off and etching processes using copper naphthenate as a high-resolution negative-tone resist. As a commonly used industrial painting product, copper naphthenate is extremely cheap with a long shelf time but demonstrates an unexpected patterning resolution better than 10 nm. With appropriate annealing under a hydrogen atmosphere, the produced graphene@copper composite patterns show high conductivity of ∼400 S cm -1 . X-ray diffraction, conformal Raman spectroscopy and X-ray photoelectron spectroscopy were used to analyze the chemical composition of the final patterns. With the properties of high resolution and high conductivity, the patterned graphene@copper composites could be used as conductive pads and interconnects for graphene electronic devices with ohmic contacts. Compared to common fabrication processes involving metal evaporation and lift-off steps, this pattern-transfer-free fabrication process using copper naphthenate resist is direct and simple but allows comparable device performance in practical device applications.

Weight shifting operators and conformal blocks

NASA Astrophysics Data System (ADS)

Karateev, Denis; Kravchuk, Petr; Simmons-Duffin, David

2018-02-01

We introduce a large class of conformally-covariant differential operators and a crossing equation that they obey. Together, these tools dramatically simplify calculations involving operators with spin in conformal field theories. As an application, we derive a formula for a general conformal block (with arbitrary internal and external representations) in terms of derivatives of blocks for external scalars. In particular, our formula gives new expressions for "seed conformal blocks" in 3d and 4d CFTs. We also find simple derivations of identities between external-scalar blocks with different dimensions and internal spins. We comment on additional applications, including deriving recursion relations for general conformal blocks, reducing inversion formulae for spinning operators to inversion formulae for scalars, and deriving identities between general 6 j symbols (Racah-Wigner coefficients/"crossing kernels") of the conformal group.

Structural studies of Saccharomyces cerevesiae mitochondrial NADP-dependent isocitrate dehydrogenase in different enzymatic states reveal substantial conformational changes during the catalytic reaction.

PubMed

Peng, Yingjie; Zhong, Chen; Huang, Wei; Ding, Jianping

2008-09-01

Isocitrate dehydrogenases (IDHs) catalyze oxidative decarboxylation of isocitrate (ICT) into alpha-ketoglutarate (AKG). We report here the crystal structures of Saccharomyces cerevesiae mitochondrial NADP-IDH Idp1p in binary complexes with coenzyme NADP, or substrate ICT, or product AKG, and in a quaternary complex with NADPH, AKG, and Ca(2+), which represent different enzymatic states during the catalytic reaction. Analyses of these structures identify key residues involved in the binding of these ligands. Comparisons among these structures and with the previously reported structures of other NADP-IDHs reveal that eukaryotic NADP-IDHs undergo substantial conformational changes during the catalytic reaction. Binding or release of the ligands can cause significant conformational changes of the structural elements composing the active site, leading to rotation of the large domain relative to the small and clasp domains along two hinge regions (residues 118-124 and residues 284-287) while maintaining the integrity of its secondary structural elements, and thus, formation of at least three distinct overall conformations. Specifically, the enzyme adopts an open conformation when bound to NADP, a quasi-closed conformation when bound to ICT or AKG, and a fully closed conformation when bound to NADP, ICT, and Ca(2+) in the pseudo-Michaelis complex or with NADPH, AKG, and Ca(2+) in the product state. The conformational changes of eukaryotic NADP-IDHs are quite different from those of Escherichia coli NADP-IDH, for which significant conformational changes are observed only between two forms of the apo enzyme, suggesting that the catalytic mechanism of eukaryotic NADP-IDHs is more complex than that of EcIDH, and involves more fine-tuned conformational changes.

Structural studies of Saccharomyces cerevesiae mitochondrial NADP-dependent isocitrate dehydrogenase in different enzymatic states reveal substantial conformational changes during the catalytic reaction

PubMed Central

Peng, Yingjie; Zhong, Chen; Huang, Wei; Ding, Jianping

2008-01-01

Isocitrate dehydrogenases (IDHs) catalyze oxidative decarboxylation of isocitrate (ICT) into α-ketoglutarate (AKG). We report here the crystal structures of Saccharomyces cerevesiae mitochondrial NADP-IDH Idp1p in binary complexes with coenzyme NADP, or substrate ICT, or product AKG, and in a quaternary complex with NADPH, AKG, and Ca2+, which represent different enzymatic states during the catalytic reaction. Analyses of these structures identify key residues involved in the binding of these ligands. Comparisons among these structures and with the previously reported structures of other NADP-IDHs reveal that eukaryotic NADP-IDHs undergo substantial conformational changes during the catalytic reaction. Binding or release of the ligands can cause significant conformational changes of the structural elements composing the active site, leading to rotation of the large domain relative to the small and clasp domains along two hinge regions (residues 118–124 and residues 284–287) while maintaining the integrity of its secondary structural elements, and thus, formation of at least three distinct overall conformations. Specifically, the enzyme adopts an open conformation when bound to NADP, a quasi-closed conformation when bound to ICT or AKG, and a fully closed conformation when bound to NADP, ICT, and Ca2+ in the pseudo-Michaelis complex or with NADPH, AKG, and Ca2+ in the product state. The conformational changes of eukaryotic NADP-IDHs are quite different from those of Escherichia coli NADP-IDH, for which significant conformational changes are observed only between two forms of the apo enzyme, suggesting that the catalytic mechanism of eukaryotic NADP-IDHs is more complex than that of EcIDH, and involves more fine-tuned conformational changes. PMID:18552125

Multiple Drug Transport Pathways through human P-Glycoprotein(†)

PubMed Central

McCormick, James W.; Vogel, Pia D.; Wise, John G.

2015-01-01

P-glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11 to 12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methyl-pyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar is presented that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp. PMID:26125482

Multiple Drug Transport Pathways through Human P-Glycoprotein.

PubMed

McCormick, James W; Vogel, Pia D; Wise, John G

2015-07-21

P-Glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11-12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methylpyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp is presented.

Hydrogen Bond Networks and Hydrophobic Effects in the Amyloid β30-35 Chain in Water: A Molecular Dynamics Study.

PubMed

Jong, KwangHyok; Grisanti, Luca; Hassanali, Ali

2017-07-24

We have studied the conformational landscape of the C-terminal fragment of the amyloid protein Aβ 30-35 in water using well-tempered metadynamics simulations and found that it resembles an intrinsically disordered protein. The conformational fluctuations of the protein are facilitated by a collective reorganization of both protein and water hydrogen bond networks, combined with electrostatic interactions between termini as well as hydrophobic interactions of the side chains. The stabilization of hydrophobic interactions in one of the conformers involves a collective collapse of the side chains along with a squeeze-out of water sandwiched between them. The charged N- and C-termini play a critical role in stabilizing different types of protein conformations, including those involving contact-ion salt bridges as well as solvent-mediated interactions of the termini and the amide backbone. We have examined this by probing the distribution of directed water wires forming the hydrogen bond network enveloping the polypeptide. Water wires and their fluctuations form an integral part of structural signature of the protein conformation.

Worming our way to novel drug discovery with the Caenorhabditis elegans proteostasis network, stress response and insulin-signaling pathways.

PubMed

O'Reilly, Linda P; Benson, Joshua A; Cummings, Erin E; Perlmutter, David H; Silverman, Gary A; Pak, Stephen C

2014-09-01

Many human diseases result from a failure of a single protein to achieve the correct folding and tertiary conformation. These so-called 'conformational diseases' involve diverse proteins and distinctive cellular pathologies. They all engage the proteostasis network (PN), to varying degrees in an attempt to mange cellular stress and restore protein homeostasis. The insulin/insulin-like growth factor signaling (IIS) pathway is a master regulator of cellular stress response, which is implicated in regulating components of the PN. This review focuses on novel approaches to target conformational diseases. The authors discuss the evidence supporting the involvement of the IIS pathway in modulating the PN and regulating proteostasis in Caenorhabditis elegans. Furthermore, they review previous PN and IIS drug screens and explore the possibility of using C. elegans for whole organism-based drug discovery for modulators of IIS-proteostasis pathways. An alternative approach to develop individualized therapy for each conformational disease is to modulate the global PN. The involvement of the IIS pathway in regulating longevity and response to a variety of stresses is well documented. Increasing data now provide evidence for the close association between the IIS and the PN pathways. The authors believe that high-throughput screening campaigns, which target the C. elegans IIS pathway, may identify drugs that are efficacious in treating numerous conformational diseases.

Dissecting Antibodies with Regards to Linear and Conformational Epitopes

PubMed Central

Forsström, Björn; Bisławska Axnäs, Barbara; Rockberg, Johan; Danielsson, Hanna; Bohlin, Anna; Uhlen, Mathias

2015-01-01

An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets. PMID:25816293

Structure based aggregation studies reveal the presence of helix-rich intermediate during α-Synuclein aggregation

PubMed Central

Ghosh, Dhiman; Singh, Pradeep K.; Sahay, Shruti; Jha, Narendra Nath; Jacob, Reeba S.; Sen, Shamik; Kumar, Ashutosh; Riek, Roland; Maji, Samir K.

2015-01-01

Mechanistic understanding of nucleation dependent polymerization by α-synuclein (α-Syn) into toxic oligomers and amyloids is important for the drug development against Parkinson's disease. However the structural and morphological characterization during nucleation and subsequent fibrillation process of α-Syn is not clearly understood. Using a variety of complementary biophysical techniques monitoring entire pathway of nine different synucleins, we found that transition of unstructured conformation into β-sheet rich fibril formation involves helix-rich intermediates. These intermediates are common for all aggregating synucleins, contain high solvent-exposed hydrophobic surfaces, are cytotoxic to SHSY-5Y cells and accelerate α-Syn aggregation efficiently. A multidimensional NMR study characterizing the intermediate accompanied with site-specific fluorescence study suggests that the N-terminal and central portions mainly participate in the helix-rich intermediate formation while the C-terminus remained in an extended conformation. However, significant conformational transitions occur at the middle and at the C-terminus during helix to β-sheet transition as evident from Trp fluorescence study. Since partial helix-rich intermediates were also observed for other amyloidogenic proteins such as Aβ and IAPP, we hypothesize that this class of intermediates may be one of the important intermediates for amyloid formation pathway by many natively unstructured protein/peptides and represent a potential target for drug development against amyloid diseases. PMID:25784353

Quantifying polypeptide conformational space: sensitivity to conformation and ensemble definition.

PubMed

Sullivan, David C; Lim, Carmay

2006-08-24

Quantifying the density of conformations over phase space (the conformational distribution) is needed to model important macromolecular processes such as protein folding. In this work, we quantify the conformational distribution for a simple polypeptide (N-mer polyalanine) using the cumulative distribution function (CDF), which gives the probability that two randomly selected conformations are separated by less than a "conformational" distance and whose inverse gives conformation counts as a function of conformational radius. An important finding is that the conformation counts obtained by the CDF inverse depend critically on the assignment of a conformation's distance span and the ensemble (e.g., unfolded state model): varying ensemble and conformation definition (1 --> 2 A) varies the CDF-based conformation counts for Ala(50) from 10(11) to 10(69). In particular, relatively short molecular dynamics (MD) relaxation of Ala(50)'s random-walk ensemble reduces the number of conformers from 10(55) to 10(14) (using a 1 A root-mean-square-deviation radius conformation definition) pointing to potential disconnections in comparing the results from simplified models of unfolded proteins with those from all-atom MD simulations. Explicit waters are found to roughen the landscape considerably. Under some common conformation definitions, the results herein provide (i) an upper limit to the number of accessible conformations that compose unfolded states of proteins, (ii) the optimal clustering radius/conformation radius for counting conformations for a given energy and solvent model, (iii) a means of comparing various studies, and (iv) an assessment of the applicability of random search in protein folding.

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.

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.

Localized conformational changes trigger the pH-induced fibrillogenesis of an amyloidogenic λ light chain protein.

PubMed

Velázquez-López, Isabel; Valdés-García, Gilberto; Romero Romero, Sergio; Maya Martínez, Roberto; Leal-Cervantes, Ana I; Costas, Miguel; Sánchez-López, Rosana; Amero, Carlos; Pastor, Nina; Fernández Velasco, D Alejandro

2018-07-01

Solvent conditions modulate the expression of the amyloidogenic potential of proteins. In this work the effect of pH on the fibrillogenic behavior and the conformational properties of 6aJL2, a model protein of the highly amyloidogenic variable light chain λ6a gene segment, was examined. Ordered aggregates showing the ultrastructural and spectroscopic properties observed in amyloid fibrils were formed in the 2.0-8.0 pH range. At pH <3.0 a drastic decrease in lag time and an increase in fibril formation rate were found. In the 4.0-8.0 pH range there was no spectroscopic evidence for significant conformational changes in the native state. Likewise, heat capacity measurements showed no evidence for residual structure in the unfolded state. However, at pH <3.0 stability is severely decreased and the protein suffers conformational changes as detected by circular dichroism, tryptophan and ANS fluorescence, as well as by NMR spectroscopy. Molecular dynamics simulations indicate that acid-induced conformational changes involve the exposure of the loop connecting strands E and F. These results are compatible with pH-induced changes in the NMR spectra. Overall, the results indicate that the mechanism involved in the acid-induced increase in the fibrillogenic potential of 6aJL2 is profoundly different to that observed in κ light chains, and is promoted by localized conformational changes in a region of the protein that was previously not known to be involved in acid-induced light chain fibril formation. The identification of this region opens the potential for the design of specific inhibitors. Copyright © 2018 Elsevier B.V. All rights reserved.

Modeling activated states of GPCRs: the rhodopsin template.

PubMed

Niv, Masha Y; Skrabanek, Lucy; Filizola, Marta; Weinstein, Harel

2006-01-01

Activation of G Protein-Coupled Receptors (GPCRs) is an allosteric mechanism triggered by ligand binding and resulting in conformational changes transduced by the transmembrane domain. Models of the activated forms of GPCRs have become increasingly necessary for the development of a clear understanding of signal propagation into the cell. Experimental evidence points to a multiplicity of conformations related to the activation of the receptor, rendered important physiologically by the suggestion that different conformations may be responsible for coupling to different signaling pathways. In contrast to the inactive state of rhodopsin (RHO) for which several high quality X-ray structures are available, the structure-related information for the active states of rhodopsin and all other GPCRs is indirect. We have collected and stored such information in a repository we maintain for activation-specific structural data available for rhodopsin-like GPCRs, http://www.physiology.med.cornell.edu/GPCRactivation/gpcrindex.html . Using these data as structural constraints, we have applied Simulated Annealing Molecular Dynamics to construct a number of different active state models of RHO starting from the known inactive structure. The common features of the models indicate that TM3 and TM5 play an important role in activation, in addition to the well-established rearrangement of TM6. Some of the structural changes observed in these models occur in regions that were not involved in the constraints, and have not been previously tested experimentally; they emerge as interesting candidates for further experimental exploration of the conformational space of activated GPCRs. We show that none of the normal modes calculated from the inactive structure has a dominant contribution along the path of conformational rearrangement from inactive to the active forms of RHO in the models. This result may differentiate rhodopsin from other GPCRs, and the reasons for this difference are discussed in the context of the structural properties and the physiological function of the protein.

Single-Point Mutation with a Rotamer Library Toolkit: Toward Protein Engineering.

PubMed

Pottel, Joshua; Moitessier, Nicolas

2015-12-28

Protein engineers have long been hard at work to harness biocatalysts as a natural source of regio-, stereo-, and chemoselectivity in order to carry out chemistry (reactions and/or substrates) not previously achieved with these enzymes. The extreme labor demands and exponential number of mutation combinations have induced computational advances in this domain. The first step in our virtual approach is to predict the correct conformations upon mutation of residues (i.e., rebuilding side chains). For this purpose, we opted for a combination of molecular mechanics and statistical data. In this work, we have developed automated computational tools to extract protein structural information and created conformational libraries for each amino acid dependent on a variable number of parameters (e.g., resolution, flexibility, secondary structure). We have also developed the necessary tool to apply the mutation and optimize the conformation accordingly. For side-chain conformation prediction, we obtained overall average root-mean-square deviations (RMSDs) of 0.91 and 1.01 Å for the 18 flexible natural amino acids within two distinct sets of over 3000 and 1500 side-chain residues, respectively. The commonly used dihedral angle differences were also evaluated and performed worse than the state of the art. These two metrics are also compared. Furthermore, we generated a family-specific library for kinases that produced an average 2% lower RMSD upon side-chain reconstruction and a residue-specific library that yielded a 17% improvement. Ultimately, since our protein engineering outlook involves using our docking software, Fitted/Impacts, we applied our mutation protocol to a benchmarked data set for self- and cross-docking. Our side-chain reconstruction does not hinder our docking software, demonstrating differences in pose prediction accuracy of approximately 2% (RMSD cutoff metric) for a set of over 200 protein/ligand structures. Similarly, when docking to a set of over 100 kinases, side-chain reconstruction (using both general and biased conformation libraries) had minimal detriment to the docking accuracy.

Hierarchical Modeling of Activation Mechanisms in the ABL and EGFR Kinase Domains: Thermodynamic and Mechanistic Catalysts of Kinase Activation by Cancer Mutations

PubMed Central

Dixit, Anshuman; Verkhivker, Gennady M.

2009-01-01

Structural and functional studies of the ABL and EGFR kinase domains have recently suggested a common mechanism of activation by cancer-causing mutations. However, dynamics and mechanistic aspects of kinase activation by cancer mutations that stimulate conformational transitions and thermodynamic stabilization of the constitutively active kinase form remain elusive. We present a large-scale computational investigation of activation mechanisms in the ABL and EGFR kinase domains by a panel of clinically important cancer mutants ABL-T315I, ABL-L387M, EGFR-T790M, and EGFR-L858R. We have also simulated the activating effect of the gatekeeper mutation on conformational dynamics and allosteric interactions in functional states of the ABL-SH2-SH3 regulatory complexes. A comprehensive analysis was conducted using a hierarchy of computational approaches that included homology modeling, molecular dynamics simulations, protein stability analysis, targeted molecular dynamics, and molecular docking. Collectively, the results of this study have revealed thermodynamic and mechanistic catalysts of kinase activation by major cancer-causing mutations in the ABL and EGFR kinase domains. By using multiple crystallographic states of ABL and EGFR, computer simulations have allowed one to map dynamics of conformational fluctuations and transitions in the normal (wild-type) and oncogenic kinase forms. A proposed multi-stage mechanistic model of activation involves a series of cooperative transitions between different conformational states, including assembly of the hydrophobic spine, the formation of the Src-like intermediate structure, and a cooperative breakage and formation of characteristic salt bridges, which signify transition to the active kinase form. We suggest that molecular mechanisms of activation by cancer mutations could mimic the activation process of the normal kinase, yet exploiting conserved structural catalysts to accelerate a conformational transition and the enhanced stabilization of the active kinase form. The results of this study reconcile current experimental data with insights from theoretical approaches, pointing to general mechanistic aspects of activating transitions in protein kinases. PMID:19714203

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

The evolution of conformist transmission in social learning when the environment changes periodically.

PubMed

Nakahashi, Wataru

2007-08-01

Conformity is often observed in human social learning. Social learners preferentially imitate the majority or most common behavior in many situations, though the strength of conformity varies with the situation. Why has such a psychological tendency evolved? I investigate this problem by extending a standard model of social learning evolution with infinite environmental states (Feldman, M.W., Aoki, K., Kumm, J., 1996. Individual versus social learning: evolutionary analysis in a fluctuating environment. Anthropol. Sci. 104, 209-231) to include conformity bias. I mainly focus on the relationship between the strength of conformity bias that evolves and environmental stability, which is one of the most important factors in the evolution of social learning. Using the evolutionarily stable strategy (ESS) approach, I show that conformity always evolves when environmental stability and the cost of adopting a wrong behavior are small, though environmental stability and the cost of individual learning both negatively affect the strength of conformity.

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

NASA Astrophysics Data System (ADS)

Unan, Hulya; Yildirim, Ahmet; Tekpinar, Mustafa

2015-07-01

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

Conformational and vibrational reassessment of solid paracetamol

NASA Astrophysics Data System (ADS)

Amado, Ana M.; Azevedo, Celeste; Ribeiro-Claro, Paulo J. A.

2017-08-01

This work provides an answer to the urge for a more detailed and accurate knowledge of the vibrational spectrum of the widely used analgesic/antipyretic drug commonly known as paracetamol. A comprehensive spectroscopic analysis - including infrared, Raman, and inelastic neutron scattering (INS) - is combined with a computational approach which takes account for the effects of intermolecular interactions in the solid state. This allows a full reassessment of the vibrational assignments for Paracetamol, thus preventing the propagation of incorrect data analysis and misassignments already found in the literature. In particular, the vibrational modes involving the hydrogen-bonded Nsbnd H and Osbnd H groups are correctly reallocated to bands shifted by up to 300 cm- 1 relatively to previous assignments.

77 FR 429 - Clarification and Further Guidance on the Fireworks Approvals Policy

Federal Register 2010, 2011, 2012, 2013, 2014

2012-01-05

...) and the American Pyrotechnic Association (APA) Standard 87-1. PHMSA understands that it is a common... approval is requested conforms to the APA Standard 87-1, and the descriptions and technical information... manufacturer must sign and certify that the device for which the approval is requested conforms to the APA...

Beyond basins: φ,ψ preferences of a residue depend heavily on the φ,ψ values of its neighbors.

PubMed

Hollingsworth, Scott A; Lewis, Matthew C; Karplus, P Andrew

2016-09-01

The Ramachandran plot distributions of nonglycine residues from experimentally determined structures are routinely described as grouping into one of six major basins: β, PII , α, αL , ξ and γ'. Recent work describing the most common conformations adopted by pairs of residues in folded proteins [i.e., (φ,ψ)2 -motifs] showed that commonly described major basins are not true single thermodynamic basins, but are composed of distinct subregions that are associated with various conformations of either the preceding or following neighbor residue. Here, as documentation of the extent to which the conformational preferences of a central residue are influenced by the conformations of its two neighbors, we present a set of φ,ψ-plots that are delimited simultaneously by the φ,ψ-angles of its neighboring residues on both sides. The level of influence seen here is typically greater than the influence associated with considering the identities of neighboring residues, implying that the use of this heretofore untapped information can improve the accuracy of structure prediction algorithms and low resolution protein structure refinement. © 2016 The Protein Society.

Conformational transitions of uracil transporter UraA from Escherichia coli: a molecular simulation study.

PubMed

Yang, Liu; Yang, Lianjuan; Yu, Hui; Liu, Lu; Zhao, Xi; Huang, Xuri

2017-10-26

The Escherichia coli uracil/H + symporter UraA, known as the representative nucleobase/cation symporter 2(NCS2) protein, gets involved in several crucial physiological processes for most living organisms on Earth, such as the uptake of nucleobases and transport of vitamin C. Some experiments proposed a working model to explain proton-coupling and uracil transporting process of UraA on the basis of the crystal structure of NCS2 protein, but the details of conformational changes remained unknown. Thus, in order to make clear conformational changes caused by the protonation and deprotonation process of some conserved proton-coupled residues, the molecular dynamics simulation was used to study the conformation of UraA complexes in different protonation states. The results demonstrated that the protonation of residue Glu241 and Glu290 resulted in the whole conformational transition from the inward-open to the outward-open state. It can be concluded that Glu290 was crucial in a network of hydrogen-bonds in the middle of the core domain involving another essential residue, mainly including tyr288 in TM8, Tyr342, Ser338 in TM12, and the network of hydrogen-bonds was the key to maintain the stability of conformation. Protonation of Glu290 affects the stability of network of H-bond and changed the domains TM3 TM10 TM12. Thus, Glu290 may play a vital role as a 'proton trigger' that affects spatial structural of amino and residues near substrate binding side leading to an outward-open conformation transition.

Nekrasov and Argyres-Douglas theories in spherical Hecke algebra representation

NASA Astrophysics Data System (ADS)

Rim, Chaiho; Zhang, Hong

2017-06-01

AGT conjecture connects Nekrasov instanton partition function of 4D quiver gauge theory with 2D Liouville conformal blocks. We re-investigate this connection using the central extension of spherical Hecke algebra in q-coordinate representation, q being the instanton expansion parameter. Based on AFLT basis together with intertwiners we construct gauge conformal state and demonstrate its equivalence to the Liouville conformal state, with careful attention to the proper scaling behavior of the state. Using the colliding limit of regular states, we obtain the formal expression of irregular conformal states corresponding to Argyres-Douglas theory, which involves summation of functions over Young diagrams.

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.

Molecular modelling indicates that the pathological conformations of prion proteins might be beta-helical.

PubMed Central

Downing, D T; Lazo, N D

1999-01-01

Creutzfeldt-Jakob disease, kuru, scrapie and bovine spongiform encephalopathy are diseases of the mammalian central nervous system that involve the conversion of a cellular protein into an insoluble extracellular isoform. Spectroscopic studies have shown that the precursor protein contains mainly alpha-helical and random-coil conformations, whereas the prion isoform is largely in the beta conformation. The pathogenic prion is resistant to denaturation and protease digestion and can promote the conversion of the precursor protein to the pathogenic form. These properties have yet to be explained in terms of the structural conformations of the proteins. In the present study, molecular modelling showed that prion proteins could adopt the beta-helical conformation, which has been established for a number of fibrous proteins and has been suggested previously as the basis of amyloid fibrils. The beta-helical conformation provides explanations for the biophysical and biochemical stability of prions, their ability to form templates for the transmission of pathological conformation, and the existence of phenotypical strains of the prion diseases. PMID:10510313

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

Characterizing Conformational Dynamics of Proteins Using Evolutionary Couplings.

PubMed

Feng, Jiangyan; Shukla, Diwakar

2018-01-25

Understanding of protein conformational dynamics is essential for elucidating molecular origins of protein structure-function relationship. Traditionally, reaction coordinates, i.e., some functions of protein atom positions and velocities have been used to interpret the complex dynamics of proteins obtained from experimental and computational approaches such as molecular dynamics simulations. However, it is nontrivial to identify the reaction coordinates a priori even for small proteins. Here, we evaluate the power of evolutionary couplings (ECs) to capture protein dynamics by exploring their use as reaction coordinates, which can efficiently guide the sampling of a conformational free energy landscape. We have analyzed 10 diverse proteins and shown that a few ECs are sufficient to characterize complex conformational dynamics of proteins involved in folding and conformational change processes. With the rapid strides in sequencing technology, we expect that ECs could help identify reaction coordinates a priori and enhance the sampling of the slow dynamical process associated with protein folding and conformational change.

From spinning conformal blocks to matrix Calogero-Sutherland models

NASA Astrophysics Data System (ADS)

Schomerus, Volker; Sobko, Evgeny

2018-04-01

In this paper we develop further the relation between conformal four-point blocks involving external spinning fields and Calogero-Sutherland quantum mechanics with matrix-valued potentials. To this end, the analysis of [1] is extended to arbitrary dimensions and to the case of boundary two-point functions. In particular, we construct the potential for any set of external tensor fields. Some of the resulting Schrödinger equations are mapped explicitly to the known Casimir equations for 4-dimensional seed conformal blocks. Our approach furnishes solutions of Casimir equations for external fields of arbitrary spin and dimension in terms of functions on the conformal group. This allows us to reinterpret standard operations on conformal blocks in terms of group-theoretic objects. In particular, we shall discuss the relation between the construction of spinning blocks in any dimension through differential operators acting on seed blocks and the action of left/right invariant vector fields on the conformal group.

Direct observation of fast protein conformational switching.

PubMed

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

2008-06-24

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

Mapping transiently formed and sparsely populated conformations on a complex energy landscape.

PubMed

Wang, Yong; Papaleo, Elena; Lindorff-Larsen, Kresten

2016-08-23

Determining the structures, kinetics, thermodynamics and mechanisms that underlie conformational exchange processes in proteins remains extremely difficult. Only in favourable cases is it possible to provide atomic-level descriptions of sparsely populated and transiently formed alternative conformations. Here we benchmark the ability of enhanced-sampling molecular dynamics simulations to determine the free energy landscape of the L99A cavity mutant of T4 lysozyme. We find that the simulations capture key properties previously measured by NMR relaxation dispersion methods including the structure of a minor conformation, the kinetics and thermodynamics of conformational exchange, and the effect of mutations. We discover a new tunnel that involves the transient exposure towards the solvent of an internal cavity, and show it to be relevant for ligand escape. Together, our results provide a comprehensive view of the structural landscape of a protein, and point forward to studies of conformational exchange in systems that are less characterized experimentally.

Conformational selection in protein binding and function

PubMed Central

Weikl, Thomas R; Paul, Fabian

2014-01-01

Protein binding and function often involves conformational changes. Advanced nuclear magnetic resonance (NMR) experiments indicate that these conformational changes can occur in the absence of ligand molecules (or with bound ligands), and that the ligands may “select” protein conformations for binding (or unbinding). In this review, we argue that this conformational selection requires transition times for ligand binding and unbinding that are small compared to the dwell times of proteins in different conformations, which is plausible for small ligand molecules. Such a separation of timescales leads to a decoupling and temporal ordering of binding/unbinding events and conformational changes. We propose that conformational-selection and induced-change processes (such as induced fit) are two sides of the same coin, because the temporal ordering is reversed in binding and unbinding direction. Conformational-selection processes can be characterized by a conformational excitation that occurs prior to a binding or unbinding event, while induced-change processes exhibit a characteristic conformational relaxation that occurs after a binding or unbinding event. We discuss how the ordering of events can be determined from relaxation rates and effective on- and off-rates determined in mixing experiments, and from the conformational exchange rates measured in advanced NMR or single-molecule fluorescence resonance energy transfer experiments. For larger ligand molecules such as peptides, conformational changes and binding events can be intricately coupled and exhibit aspects of conformational-selection and induced-change processes in both binding and unbinding direction. PMID:25155241

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

PubMed

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

2009-02-20

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

Dimerization effects on coacervation property of an elastin-derived synthetic peptide (FPGVG)5.

PubMed

Suyama, Keitaro; Taniguchi, Suguru; Tatsubo, Daiki; Maeda, Iori; Nose, Takeru

2016-04-01

Elastin, a core protein of the elastic fibers, exhibits the coacervation (temperature-dependent reversible association/dissociation) under physiological conditions. Because of this characteristic, elastin and elastin-derived peptides have been considered to be useful as base materials for developing various biomedical products, skin substitutes, synthetic vascular grafts, and drug delivery systems. Although elastin-derived polypeptide (Val-Pro-Gly-Val-Gly)n also has been known to demonstrate coacervation property, a sufficiently high (VPGVG)n repetition number (n>40) is required for coacervation. In the present study, a series of elastin-derived peptide (Phe-Pro-Gly-Val-Gly)5 dimers possessing high coacervation potential were newly developed. These novel dimeric peptides exhibited coacervation at significantly lower concentrations and temperatures than the commonly used elastin-derived peptide analogs; this result suggests that the coacervation ability of the peptides is enhanced by dimerization. Circular dichroism (CD) measurements indicate that the dimers undergo similar temperature-dependent and reversible conformational changes when coacervation occurs. The molecular dynamics calculation results reveal that the sheet-turn-sheet motif involving a type II β-turn-like structure commonly observed among the dimers and caused formation of globular conformation of them. These synthesized peptide dimers may be useful not only as model peptides for structural analysis of elastin and elastin-derived peptides, but also as base materials for developing various temperature-sensitive biomedical and industrial products. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Functional characterization of a competitive peptide antagonist of p65 in human macrophage-like cells suggests therapeutic potential for chronic inflammation

PubMed Central

Srinivasan, Mythily; Blackburn, Corinne; Lahiri, Debomoy K

2014-01-01

Glucocorticoid-induced leucine zipper (GILZ) is a glucocorticoid responsive protein that links the nuclear factor-kappa B (NFκB) and the glucocorticoid signaling pathways. Functional and binding studies suggest that the proline-rich region at the carboxy terminus of GILZ binds the p65 subunit of NFκB and suppresses the immunoinflammatory response. A widely-used strategy in the discovery of peptide drugs involves exploitation of the complementary surfaces of naturally occurring binding partners. Previously, we observed that a synthetic peptide (GILZ-P) derived from the proline-rich region of GILZ bound activated p65 and ameliorated experimental encephalomyelitis. Here we characterize the secondary structure of GILZ-P by circular dichroic analysis. GILZ-P adopts an extended polyproline type II helical conformation consistent with the structural conformation commonly observed in interfaces of transient intermolecular interactions. To determine the potential application of GILZ-P in humans, we evaluated the toxicity and efficacy of the peptide drug in mature human macrophage-like THP-1 cells. Treatment with GILZ-P at a wide range of concentrations commonly used for peptide drugs was nontoxic as determined by cell viability and apoptosis assays. Functionally, GILZ-P suppressed proliferation and glutamate secretion by activated macrophages by inhibiting nuclear translocation of p65. Collectively, our data suggest that the GILZ-P has therapeutic potential in chronic CNS diseases where persistent inflammation leads to neurodegeneration such as multiple sclerosis and Alzheimer’s disease. PMID:25584020

Structural analysis of Arabidopsis thaliana nucleoside diphosphate kinase-2 for phytochrome-mediated light signaling.

PubMed

Im, Young Jun; Kim, Jeong-Il; Shen, Yu; Na, Young; Han, Yun-Jeong; Kim, Seong-Hee; Song, Pill-Soon; Eom, Soo Hyun

2004-10-22

In plants, nucleoside diphosphate kinases (NDPKs) play a key role in the signaling of both stress and light. However, little is known about the structural elements involved in their function. Of the three NDPKs (NDPK1-NDPK3) expressed in Arabidopsis thaliana, NDPK2 is involved in phytochrome-mediated signal transduction. In this study, we found that the binding of dNDP or NTP to NDPK2 strengthens the interaction significantly between activated phytochrome and NDPK2. To better understand the structural basis of the phytochrome-NDPK2 interaction, we determined the X-ray structures of NDPK1, NDPK2, and dGTP-bound NDPK2 from A.thaliana at 1.8A, 2.6A, and 2.4A, respectively. The structures showed that nucleotide binding caused a slight conformational change in NDPK2 that was confined to helices alphaA and alpha2. This suggests that the presence of nucleotide in the active site and/or the evoked conformational change contributes to the recognition of NDPK2 by activated phytochrome. In vitro binding assays showed that only NDPK2 interacted specifically with the phytochrome and the C-terminal regulatory domain of phytochrome is involved in the interaction. A domain swap experiment between NDPK1 and NDPK2 showed that the variable C-terminal region of NDPK2 is important for the activation by phytochrome. The structure of Arabidopsis NDPK1 and NDPK2 showed that the isoforms share common electrostatic surfaces at the nucleotide-binding site, but the variable C-terminal regions have distinct electrostatic charge distributions. These findings suggest that the binding of nucleotide to NDPK2 plays a regulatory role in phytochrome signaling and that the C-terminal extension of NDPK2 provides a potential binding surface for the specific interaction with phytochromes.

Analysis of the Effects of Polymorphism on Pollen Profilin Structural Functionality and the Generation of Conformational, T- and B-Cell Epitopes

PubMed Central

Jimenez-Lopez, Jose C.; Rodríguez-García, María I.; Alché, Juan D.

2013-01-01

An extensive polymorphism analysis of pollen profilin, a fundamental regulator of the actin cytoskeleton dynamics, has been performed with a major focus in 3D-folding maintenance, changes in the 2-D structural elements, surface residues involved in ligands-profilin interactions and functionality, and the generation of conformational and lineal B- and T-cell epitopes variability. Our results revealed that while the general fold is conserved among profilins, substantial structural differences were found, particularly affecting the special distribution and length of different 2-D structural elements (i.e. cysteine residues), characteristic loops and coils, and numerous micro-heterogeneities present in fundamental residues directly involved in the interacting motifs, and to some extension these residues nearby to the ligand-interacting areas. Differential changes as result of polymorphism might contribute to generate functional variability among the plethora of profilin isoforms present in the olive pollen from different genetic background (olive cultivars), and between plant species, since biochemical interacting properties and binding affinities to natural ligands may be affected, particularly the interactions with different actin isoforms and phosphoinositides lipids species. Furthermore, conspicuous variability in lineal and conformational epitopes was found between profilins belonging to the same olive cultivar, and among different cultivars as direct implication of sequences polymorphism. The variability of the residues taking part of IgE-binding epitopes might be the final responsible of the differences in cross-reactivity among olive pollen cultivars, among pollen and plant-derived food allergens, as well as between distantly related pollen species, leading to a variable range of allergy reactions among atopic patients. Identification and analysis of commonly shared and specific epitopes in profilin isoforms is essential to gain knowledge about the interacting surface of these epitopes, and for a better understanding of immune responses, helping design and development of rational and effective immunotherapy strategies for the treatment of allergy diseases. PMID:24146818

Innovation and restrictive conformity among hospital employees: individual outcomes and organizational considerations.

PubMed

Jansen, E; Eccles, D; Chandler, G N

1994-01-01

Two factors characterize innovative organizational climates as perceived by nonmanagerial hospital employees. The first, innovation, comprises perceptions about the consequences or contingencies (e.g., punished, ignored, rewarded) of such proactive activities as implementing new ideas, questioning established methods, and communicating with other departments and the supervisor. The second, restrictive conformity, comprises perceptions about the consequences of risk-aversive, conflict-avoidant activities that suggest dysfunctional conformity (e.g., "sticking to the rules no matter what"). Positive personal outcomes--greater role clarity, organizational involvement, and satisfaction, and lower role conflict and willingness to leave the organization--are associated with innovation; negative personal outcomes are associated with restrictive conformity. The dialectical tension between conformity and innovation is discussed in terms of loose coupling and a reward systems perspective.

Multicritical scalar theories with higher-derivative kinetic terms: A perturbative RG approach with the ɛ -expansion

NASA Astrophysics Data System (ADS)

Safari, M.; Vacca, G. P.

2018-02-01

We employ perturbative renormalization group and ɛ -expansion to study multicritical single-scalar field theories with higher derivative kinetic terms of the form ϕ (-□)kϕ . We focus on those with a Z2-symmetric critical point which are characterized by an upper critical dimension dc=2 n k /(n -1 ) accumulating at even integers. We distinguish two types of theories depending on whether or not the numbers k and n -1 are relatively prime. When they are, the critical theory involves a marginal powerlike interaction ϕ2 n and the deformations admit a derivative expansion that at leading order involves only the potential. In this case we present the beta functional of the potential and use this to calculate some anomalous dimensions and operator product expansion coefficients. These confirm some conformal field theory data obtained using conformal-block techniques, while giving new results. In the second case where k and n -1 have a common divisor, the theories show a much richer structure induced by the presence of marginal derivative operators at criticality. We study the case k =2 with odd values of n , which fall in the second class, and calculate the functional flows and spectrum. These theories have a phase diagram characterized at leading order in ɛ by four fixed points which apart from the Gaussian UV fixed point include an IR fixed point with a purely derivative interaction.

Prosthetic Rehabilitation Following Socket Reconstruction with Blair-Brown Graft and Conformer Therapy for Management of Severe Post-Enucleation Socket Syndrome--A Clinical Report.

PubMed

Aggarwal, Himanshi; Singh, Saumyendra V; Kumar, Pradeep; Kumar Singh, Arun

2015-06-01

One of the most common tumors of the eye diagnosed in childhood is retinoblastoma, which mandates enucleation with adjunctive chemotherapy and radiotherapy to save the patient's life. The most common late enucleation complication is post-enucleation socket syndrome (PESS), which poses a management dilemma for the prosthodontist and surgeon, along with being a major esthetic concern for the patient. The reconstruction of such sockets is complex. The purpose of this clinical report is to describe the rehabilitation of such a pediatric patient with severe PESS. The patient was successfully rehabilitated by presurgical conformer therapy, socket reconstruction surgery with non-meshed intermediate split thickness skin graft (STSG)/Blair-Brown graft, and postsurgical conformer stent. This was followed by fabrication of a custom ocular prosthesis, to achieve favorable functional, physical, and psychological effects. © 2014 by the American College of Prosthodontists.

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

PubMed Central

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

2010-01-01

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

pH-induced conformational change of the rotavirus VP4 spike: implications for cell entry and antibody neutralization.

PubMed

Pesavento, Joseph B; Crawford, Sue E; Roberts, Ed; Estes, Mary K; Prasad, B V Venkataram

2005-07-01

The rotavirus spike protein, VP4, is a major determinant of infectivity and neutralization. Previously, we have shown that trypsin-enhanced infectivity of rotavirus involves a transformation of the VP4 spike from a flexible to a rigid bilobed structure. Here we show that at elevated pH the spike undergoes a drastic, irreversible conformational change and becomes stunted, with a pronounced trilobed appearance. These particles with altered spikes, at a normal pH of 7.5, despite the loss of infectivity and the ability to hemagglutinate, surprisingly exhibit sialic acid (SA)-independent cell binding in contrast to the SA-dependent cell binding exhibited by native virions. Remarkably, a neutralizing monoclonal antibody that remains bound to spikes throughout the pH changes (pH 7 to 11 and back to pH 7) completely prevents this conformational change, preserving the SA-dependent cell binding and hemagglutinating functions of the virion. A hypothesis that emerges from the present study is that high-pH treatment triggers a conformational change that mimics a post-SA-attachment step to expose an epitope recognized by a downstream receptor in the rotavirus cell entry process. This process involves sequential interactions with multiple receptors, and the mechanism by which the antibody neutralizes is by preventing this conformational change.

Sodium ion effect on silk fibroin conformation characterized by solid-state NMR and generalized 2D NMR NMR correlation

NASA Astrophysics Data System (ADS)

Ruan, Qing-Xia; Zhou, Ping

2008-07-01

In the present work, we investigated Na + ion effect on the silk fibroin (SF) conformation. Samples are Na +-involved regenerated silk fibroin films. 13C CP-MAS NMR demonstrates that as added [Na +] increases, partial silk fibroin conformation transit from helix-form to β-form at certain Na + ion concentration which is much higher than that in Bombyx mori silkworm gland. The generalized two-dimensional NMR-NMR correlation analysis reveals that silk fibroin undergoes several intermediate states during its conformation transition process as [Na +] increase. The appearance order of the intermediates is followed as: helix and/or random coil → helix-like → β-sheet-like → β-sheet, which is the same as that produced by pH decrease from 6.8 to 4.8 in the resultant regenerated silk fibroin films. The binding sites of Na + to silk fibroin might involve the carbonyl oxygen atom of certain amino acids sequence which could promote the formation of β-sheet conformation. Since the Na +sbnd O bond is weak, the ability of Na + inducing the secondary structure transition is weaker than those of Ca 2+, Cu 2+ and even K +. It is maybe a reason why the sodium content is much lower than potassium in the silkworm gland.

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.

Effects of oncogenic mutations on the conformational free-energy landscape of EGFR kinase

PubMed Central

Sutto, Ludovico; Gervasio, Francesco Luigi

2013-01-01

Activating mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase are frequently found in many cancers. It has been suggested that changes in the equilibrium between its active and inactive conformations are linked to its oncogenic potential. Here, we quantify the effects of some of the most common single (L858R and T790M) and double (T790M-L858R) oncogenic mutations on the conformational free-energy landscape of the EGFR kinase domain by using massive molecular dynamics simulations together with parallel tempering, metadynamics, and one of the best force-fields available. Whereas the wild-type EGFR catalytic domain monomer is mostly found in an inactive conformation, our results show a clear shift toward the active conformation for all of the mutants. The L858R mutation stabilizes the active conformation at the expense of the inactive conformation and rigidifies the αC-helix. The T790M gatekeeper mutant favors activation by stabilizing a hydrophobic cluster. Finally, T790M with L858R shows a significant positive epistasis effect. This combination not only stabilizes the active conformation, but in nontrivial ways changes the free-energy landscape lowering the transition barriers. PMID:23754386

Effects of oncogenic mutations on the conformational free-energy landscape of EGFR kinase.

PubMed

Sutto, Ludovico; Gervasio, Francesco Luigi

2013-06-25

Activating mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase are frequently found in many cancers. It has been suggested that changes in the equilibrium between its active and inactive conformations are linked to its oncogenic potential. Here, we quantify the effects of some of the most common single (L858R and T790M) and double (T790M-L858R) oncogenic mutations on the conformational free-energy landscape of the EGFR kinase domain by using massive molecular dynamics simulations together with parallel tempering, metadynamics, and one of the best force-fields available. Whereas the wild-type EGFR catalytic domain monomer is mostly found in an inactive conformation, our results show a clear shift toward the active conformation for all of the mutants. The L858R mutation stabilizes the active conformation at the expense of the inactive conformation and rigidifies the αC-helix. The T790M gatekeeper mutant favors activation by stabilizing a hydrophobic cluster. Finally, T790M with L858R shows a significant positive epistasis effect. This combination not only stabilizes the active conformation, but in nontrivial ways changes the free-energy landscape lowering the transition barriers.

NMR conformational studies of micelle-bound orexin-B: a neuropeptide involved in the sleep/awake cycle and feeding regulation.

PubMed

Miskolzie, Mark; Lucyk, Scott; Kotovych, George

2003-12-01

The preferred conformation of orexin-B, an orphan G-protein coupled receptor agonist (the human sequence is RSGPPGLQGRLQRLLQASGNHAAGILTM-NH(2)) has been determined by (1)H and (13)C 2D NMR spectroscopy and molecular modeling. Orexin-B has been implicated in sleep-wakefulness and feeding regulation. The membrane mimetic, sodium dodecylsulphate-d(25) (SDS), was used to mimic a physiological environment for the peptide. The secondary structure of orexin-B in SDS consists of two helical sections; helix I spans Leu(7) to Ser(18) and helix II spans Ala(22) to Leu(26). Helices I and II are believed to be involved in membrane binding, as is supported by the results of the spin label studies with 5-doxylstearic acid. Lee et al. (Eur. J. Biochem. 266, 831-839 (1999)) determined the [Phe(1)]-orexin-B conformation in water solution by NMR and showed that helix II extends from Ala(23) to Met(28). The C-terminal dipeptide, Thr(27)-Met(28), is unstructured is SDS, whereas in water it forms the end of helix II. The lack of apparent structure for Thr(27)-Met(28) in SDS allows the dipeptide to have conformational freedom to interact with the receptor. The conformation of orexin-B can now be used to explain the Ala substitution mutagenesis experiments and the D-amino acid substitution experiments (S. Asahi et al., Bioorg. Med. Chem. Lett. 13, 111-113, 2003). Asahi et al. have shown that Ala substitution from Gly(24) to Met(28) or D-amino acid substitution from Ala(23) to Met(28) causes a significant reduction in the potency of orexin-B for both OX(1)R and OX(2)R receptors. We postulate that helix II is involved in membrane recognition, and its binding to the membrane is essential for Thr(27)-Met(28) to adopt the correct receptor-binding conformation.

Similarity Measures for Protein Ensembles

PubMed Central

Lindorff-Larsen, Kresten; Ferkinghoff-Borg, Jesper

2009-01-01

Analyses of similarities and changes in protein conformation can provide important information regarding protein function and evolution. Many scores, including the commonly used root mean square deviation, have therefore been developed to quantify the similarities of different protein conformations. However, instead of examining individual conformations it is in many cases more relevant to analyse ensembles of conformations that have been obtained either through experiments or from methods such as molecular dynamics simulations. We here present three approaches that can be used to compare conformational ensembles in the same way as the root mean square deviation is used to compare individual pairs of structures. The methods are based on the estimation of the probability distributions underlying the ensembles and subsequent comparison of these distributions. We first validate the methods using a synthetic example from molecular dynamics simulations. We then apply the algorithms to revisit the problem of ensemble averaging during structure determination of proteins, and find that an ensemble refinement method is able to recover the correct distribution of conformations better than standard single-molecule refinement. PMID:19145244

Cyclo-biphenalenyl Biradicaloid Molecular Materials: Conformation, Tautomerization, Magnetism, and Thermochromism

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

Huang, Jingsong; Meunier, Vincent; Tian, Yong-Hui

2010-01-01

Phenalenyl and its derivatives have recently attracted a great deal of interest as a result of a two-electron multicenter (2e/mc) - bonding between two -stacked phenalenyl units. The 2e/mc bonded -dimers are close in energy to the -dimers of phenalenyl and therefore fickle properties may emerge from bond fluctuation, yielding smart -functional materials. Here, we examine the valence tautomerization of two cyclo-biphenalenyl biradicaloid molecular materials with chair and boat conformations by spin-restricted (R) and unrestricted (U) DFT using the M06 and B3LYP functionals. We found that the chair conformation involves a 2e/4c - bonded structure, whereas the boat conformation involvesmore » a 2e/12c - bonded structure on their potential energy surfaces. The global minimum for the chair conformation is the -bonded structure, whereas it is the - bonded structure for the boat conformation. The chair conformation exhibits a stepwise [3,3]-sigmatropic rearrangement, and calculations predict a negligible paramagnetic susceptibility near room temperature. In comparison, the paramagnetism of the boat conformation should be observable by SQUID and ESR. According to the energy differences of the respective - and -dimers of the two conformations and the UV-vis calculations, the color of the chair conformation is expected to become darker, whereas that of the boat conformation should become lighter with increasing temperature.« less

Dissecting the large-scale galactic conformity

NASA Astrophysics Data System (ADS)

Seo, Seongu

2018-01-01

Galactic conformity is an observed phenomenon that galaxies located in the same region have similar properties such as star formation rate, color, gas fraction, and so on. The conformity was first observed among galaxies within in the same halos (“one-halo conformity”). The one-halo conformity can be readily explained by mutual interactions among galaxies within a halo. Recent observations however further witnessed a puzzling connection among galaxies with no direct interaction. In particular, galaxies located within a sphere of ~5 Mpc radius tend to show similarities, even though the galaxies do not share common halos with each other ("two-halo conformity" or “large-scale conformity”). Using a cosmological hydrodynamic simulation, Illustris, we investigate the physical origin of the two-halo conformity and put forward two scenarios. First, back-splash galaxies are likely responsible for the large-scale conformity. They have evolved into red galaxies due to ram-pressure stripping in a given galaxy cluster and happen to reside now within a ~5 Mpc sphere. Second, galaxies in strong tidal field induced by large-scale structure also seem to give rise to the large-scale conformity. The strong tides suppress star formation in the galaxies. We discuss the importance of the large-scale conformity in the context of galaxy evolution.

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

A comparative study of structural and conformational properties of casein kinase-1 isoforms: insights from molecular dynamics and principal component analysis.

PubMed

Singh, Surya Pratap; Gupta, Dwijendra K

2015-04-21

Wnt signaling pathway regulates several developmental processes in human; however recently this pathway has been associated with development of different types of cancers. Casein kinase-1 (CK1) constitutes a family of serine-threonine protein kinase; various members of this family participate in Wnt signal transduction pathway and serve as molecular switch to this pathway. Among the known six isoforms of CK1, in human, at least three isoforms (viz. alpha, delta and epsilon) have been reported as oncogenic. The development of common therapeutics against these kinases is an arduous task; unless we have the detailed information of their tertiary structures and conformational properties. In the present work, the dynamical and conformational properties for each of three isoforms of CK1 are explored through molecular dynamics (MD) simulations. The conformational space distribution of backbone atoms is evaluated using principal component analysis of MD data, which are further validated on the basis of potential energy surface. Based on these analytics, it is suggested that conformational subspace shifts upon binding to ligands and guides the kinase action of CK1 isoforms. Further, this paper as a first effort to concurrently study all the three isoforms of CK1 provides structural basis for development of common anticancer therapeutics against three isoforms of CK1. Copyright © 2015 Elsevier Ltd. All rights reserved.

A functional selectivity mechanism at the serotonin-2A GPCR involves ligand-dependent conformations of intracellular loop 2

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

Perez-Aguilar, Jose Manuel; Shan, Jufang; LeVine, Michael V.

With recent progress in determination of G protein-coupled receptor (GPCR) structure with crystallography, a variety of other experimental approaches (e.g., NMR spectroscopy, fluorescent-based assays, mass spectrometry techniques) are also being used to characterize state-specific and ligand-specific conformational states. MD simulations offer a powerful complementary approach to elucidate the dynamic features associated with ligand-specific GPCR conformations. To shed light on the conformational elements and dynamics of the important aspect of GPCR functional selectivity, we carried out unbiased microsecond-length MD simulations of the human serotonin 2A receptor (5-HT 2AR) in the absence of ligand and bound to four distinct serotonergic agonists. Themore » 5-HT 2AR is a suitable system to study the structural features involved in the ligand-dependent conformational heterogeneity of GPCRs because it is well-characterized experimentally and exhibits a strong agonist-specific phenotype in that some 5-HT 2AR agonists induce LSD-like hallucinations, while others lack this psychoactive property entirely. Here we report evidence for structural and dynamic differences in 5-HT 2AR interacting with such pharmacologically distinct ligands, hallucinogens, and nonhallucinogens obtained from all-atom MD simulations. Differential ligand binding contacts were identified for structurally similar hallucinogens and nonhallucinogens and found to correspond to different conformations in the intracellular loop 2 (ICL2). From the different ICL2 conformations, functional selective phenotypes are suggested through effects on dimerization and/or distinct direct interaction with effector proteins. Lastly, the findings are presented in the context of currently proposed hallucinogenesis mechanisms, and ICL2 is proposed as a fine-tuning selective switch that can differentiates modes of 5-HT 2AR activation.« less

A functional selectivity mechanism at the serotonin-2A GPCR involves ligand-dependent conformations of intracellular loop 2

DOE PAGES

Perez-Aguilar, Jose Manuel; Shan, Jufang; LeVine, Michael V.; ...

2014-10-14

With recent progress in determination of G protein-coupled receptor (GPCR) structure with crystallography, a variety of other experimental approaches (e.g., NMR spectroscopy, fluorescent-based assays, mass spectrometry techniques) are also being used to characterize state-specific and ligand-specific conformational states. MD simulations offer a powerful complementary approach to elucidate the dynamic features associated with ligand-specific GPCR conformations. To shed light on the conformational elements and dynamics of the important aspect of GPCR functional selectivity, we carried out unbiased microsecond-length MD simulations of the human serotonin 2A receptor (5-HT 2AR) in the absence of ligand and bound to four distinct serotonergic agonists. Themore » 5-HT 2AR is a suitable system to study the structural features involved in the ligand-dependent conformational heterogeneity of GPCRs because it is well-characterized experimentally and exhibits a strong agonist-specific phenotype in that some 5-HT 2AR agonists induce LSD-like hallucinations, while others lack this psychoactive property entirely. Here we report evidence for structural and dynamic differences in 5-HT 2AR interacting with such pharmacologically distinct ligands, hallucinogens, and nonhallucinogens obtained from all-atom MD simulations. Differential ligand binding contacts were identified for structurally similar hallucinogens and nonhallucinogens and found to correspond to different conformations in the intracellular loop 2 (ICL2). From the different ICL2 conformations, functional selective phenotypes are suggested through effects on dimerization and/or distinct direct interaction with effector proteins. Lastly, the findings are presented in the context of currently proposed hallucinogenesis mechanisms, and ICL2 is proposed as a fine-tuning selective switch that can differentiates modes of 5-HT 2AR activation.« less

Replacement of Ser108 in Plasmodium falciparum enolase results in weak Mg(II) binding: role of a parasite-specific pentapeptide insert in stabilizing the active conformation of the enzyme.

PubMed

Dutta, Sneha; Mukherjee, Debanjan; Jarori, Gotam K

2015-06-01

A distinct structural feature of Plasmodium falciparum enolase (Pfeno) is the presence of a five amino acid insert -104EWGWS108- that is not found in host enolases. Its conservation among apicomplexan enolases has raised the possibility of its involvement in some important physiological function(s). Deletion of this sequence is known to lower k(cat)/K(m), increase K(a) for Mg(II) and convert dimer into monomers (Vora HK, Shaik FR, Pal-Bhowmick I, Mout R & Jarori GK (2009) Arch Biochem Biophys 485, 128-138). These authors also raised the possibility of the formation of an H-bond between Ser108 and Leu49 that could stabilize the apo-Pfeno in an active closed conformation that has high affinity for Mg(II). Here, we examined the effect of replacement of Ser108 with Gly/Ala/Thr on enzyme activity, Mg(II) binding affinity, conformational states and oligomeric structure and compared it with native recombinant Pfeno. The results obtained support the view that Ser108 is likely to be involved in the formation of certain crucial H-bonds with Leu49. The presence of these interactions can stabilize apo-Pfeno in an active closed conformation similar to that of Mg(II) bound yeast enolase. As predicted, S108G/A-Pfeno variants (where Ser108-Leu49 H-bonds are likely to be disrupted) were found to exist in an open conformation and had low affinity for Mg(II). They also required Mg(II) induced conformational changes to acquire the active closed conformational state essential for catalysis. The possible physiological relevance of apo-Pfeno being in such an active state is discussed. © 2015 FEBS.

Protonation States in molecular dynamics simulations of peptide folding and binding.

PubMed

Ben-Shimon, Avraham; Shalev, Deborah E; Niv, Masha Y

2013-01-01

Peptides are important signaling modules, acting both as individual hormones and as parts of larger molecules, mediating their protein-protein interactions. Many peptidic and peptidomimetic drugs have reached the marketplace and opportunities for peptide-based drug discovery are on the rise. pH-dependent behavior of peptides is well documented in the context of misfolding diseases and peptide translocation. Changes in the protonation states of peptide residues often have a crucial effect on a peptide's structure, dynamics and function, which may be exploited for biotechnological applications. The current review surveys the increasing levels of sophistication in the treatment of protonation states in computational studies involving peptides. Specifically we describe I) the common practice of assigning a single protonation state and using it throughout the dynamic simulation, II) approaches that consider multiple protonation states and compare computed observables to experimental ones, III) constant pH molecular dynamics methods that couple changes in protonation states with conformational dynamics "on the fly". Applications of conformational dynamics treatment of peptides in the context of binding, folding and interactions with the membrane are presented, illustrating the growing body of work in this field and highlighting the importance of careful handling of protonation states of peptidic residues.

Mechanism of Positive Allosteric Modulators Acting on AMPA Receptors

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

Jin,R.; Clark, S.; Weeks, A.

2005-01-01

Ligand-gated ion channels involved in the modulation of synaptic strength are the AMPA, kainate, and NMDA glutamate receptors. Small molecules that potentiate AMPA receptor currents relieve cognitive deficits caused by neurodegenerative diseases such as Alzheimer's disease and show promise in the treatment of depression. Previously, there has been limited understanding of the molecular mechanism of action for AMPA receptor potentiators. Here we present cocrystal structures of the glutamate receptor GluR2 S1S2 ligand-binding domain in complex with aniracetam [1-(4-methoxybenzoyl)-2-pyrrolidinone] or CX614 (pyrrolidino-1, 3-oxazino benzo-1, 4-dioxan-10-one), two AMPA receptor potentiators that preferentially slow AMPA receptor deactivation. Both potentiators bind within the dimermore » interface of the nondesensitized receptor at a common site located on the twofold axis of molecular symmetry. Importantly, the potentiator binding site is adjacent to the 'hinge' in the ligand-binding core 'clamshell' that undergoes conformational rearrangement after glutamate binding. Using rapid solution exchange, patch-clamp electrophysiology experiments, we show that point mutations of residues that interact with potentiators in the cocrystal disrupt potentiator function. We suggest that the potentiators slow deactivation by stabilizing the clamshell in its closed-cleft, glutamate-bound conformation.« less

Microwave Spectroscopic Study of the Atmospheric Oxidation Product m-TOLUIC Acid and its Monohydrate

NASA Astrophysics Data System (ADS)

Al-Jabiri, Mohamad; Schnitzler, Elijah G.; Seifert, Nathan A.; Jäger, Wolfgang

2017-06-01

m-Toluic acid is a photo-oxidation product of m-xylene, a chemical byproduct of the oil and gas industry, and is a common component of secondary atmospheric aerosol. Organic acids, such as m-toluic acid, are also thought to play an important role in the initial steps of aerosol formation, which involves formation of hydrogen bonded clusters with molecular species, such as water, ammonia, and sulfuric acid. Somewhat surprisingly, the rotational spectrum of the m-toluic acid monomer has not been studied before. We have identified four stable conformers using ab initio calculations at the MP2/6-311++G(2df,2pd) level of theory. The two lowest energy conformers are rather close in energy and their rotational spectra were measured using a Balle-Flygare type microwave spectrometer. The structures and barriers to methyl internal rotation were determined. We have identified four isomers of the monohydrate of m-toluic acid using ab initio calculations. Measurements of the microwave spectra of the two lowest energy isomers are underway with a newly constructed chirped pulse microwave Fourier transform spectrometer in the frequency range from 2 to 6 GHz. The spectra and analyses will be presented.

Spacecraft materials guide. [including: encapsulants and conformal coatings; optical materials; lubrication; and, bonding and joining processes

NASA Technical Reports Server (NTRS)

Staugaitis, C. L. (Editor)

1975-01-01

Materials which have demonstrated their suitability for space application are summarized. Common, recurring problems in encapsulants and conformal coatings, optical materials, lubrication, and bonding and joining are noted. The subjects discussed include: low density and syntactic foams, electrical encapsulants; optical glasses, interference filter, mirrors; oils, greases, lamillar lubricants; and, soldering and brazing processes.

Unitary subsector of generalized minimal models

NASA Astrophysics Data System (ADS)

Behan, Connor

2018-05-01

We revisit the line of nonunitary theories that interpolate between the Virasoro minimal models. Numerical bootstrap applications have brought about interest in the four-point function involving the scalar primary of lowest dimension. Using recent progress in harmonic analysis on the conformal group, we prove the conjecture that global conformal blocks in this correlator appear with positive coefficients. We also compute many such coefficients in the simplest mixed correlator system. Finally, we comment on the status of using global conformal blocks to isolate the truly unitary points on this line.

Theoretical calculation of enthalpy of formation of multiconformational molecules: 1,2-ethanediol, propanediols, and glycerol

NASA Astrophysics Data System (ADS)

Dorofeeva, Olga V.; Suchkova, Taisiya A.

2018-04-01

The gas-phase enthalpies of formation of four molecules with high flexibility, which leads to the existence of a large number of low-energy conformers, were calculated with the G4 method to see whether the lowest energy conformer is sufficient to achieve high accuracy in the computed values. The calculated values were in good agreement with the experiment, whereas adding the correction for conformer distribution makes the agreement worse. The reason for this effect is a large anharmonicity of low-frequency torsional motions, which is ignored in the calculation of ZPVE and thermal enthalpy. It was shown that the approximate correction for anharmonicity estimated using a free rotor model is of very similar magnitude compared with the conformer correction but has the opposite sign, and thus almost fully compensates for it. Therefore, the common practice of adding only the conformer correction is not without problems.

The butyrophilin 3A1 intracellular domain undergoes a conformational change involving the juxtamembrane region.

PubMed

Nguyen, Khiem; Li, Jin; Puthenveetil, Robbins; Lin, Xiaochen; Poe, Michael M; Hsiao, Chia-Hung Christine; Vinogradova, Olga; Wiemer, Andrew J

2017-11-01

Small isoprenoid diphosphates, such as ( E )-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), are ligands of the internal domain of BTN3A1. Ligand binding in target cells promotes activation of Vγ9Vδ2 T cells. We demonstrate by small-angle X-ray scattering (SAXS) that HMBPP binding to the internal domain of BTN3A1 induces a conformational change in the position of the B30.2 domain relative to the juxtamembrane (JM) region. To better understand the molecular details of this conformational rearrangement, NMR spectroscopy was used to discover that the JM region interacts with HMBPP, specifically at the diphosphate. The spectral location of the affected amide peaks, partial NMR assignments, and JM mutants (ST 296 AA or T 304 A) investigated, confirm that the backbone amide of at least one Thr (Thr 304 ), adjacent to conserved Ser, comes close to the HMBPP diphosphate, whereas double mutation of nonconserved residues (Ser/Thr 296/297 ) may perturb the local fold. Cellular mutation of either of the identified Thr residues reduces the activation of Vγ9Vδ2 T cells by HMBPP, zoledronate, and POM 2 -C-HMBP, but not by a partial agonist BTN3 antibody. Taken together, our results show that ligand binding to BTN3A1 induces a conformational change within the intracellular domain that involves the JM region and is required for full activation.-Nguyen, K., Li, J., Puthenveetil, R., Lin, X., Poe, M. M., Hsiao, C.-H. C., Vinogradova, O., Wiemer, A. J. The butyrophilin 3A1 intracellular domain undergoes a conformational change involving the juxtamembrane region. © FASEB.

Higher Curvature Gravity from Entanglement in Conformal Field Theories.

PubMed

Haehl, Felix M; Hijano, Eliot; Parrikar, Onkar; Rabideau, Charles

2018-05-18

By generalizing different recent works to the context of higher curvature gravity, we provide a unifying framework for three related results: (i) If an asymptotically anti-de Sitter (AdS) spacetime computes the entanglement entropies of ball-shaped regions in a conformal field theory using a generalized Ryu-Takayanagi formula up to second order in state deformations around the vacuum, then the spacetime satisfies the correct gravitational equations of motion up to second order around the AdS background. (ii) The holographic dual of entanglement entropy in higher curvature theories of gravity is given by the Wald entropy plus a particular correction term involving extrinsic curvatures. (iii) Conformal field theory relative entropy is dual to gravitational canonical energy (also in higher curvature theories of gravity). Especially for the second point, our novel derivation of this previously known statement does not involve the Euclidean replica trick.

Higher Curvature Gravity from Entanglement in Conformal Field Theories

NASA Astrophysics Data System (ADS)

Haehl, Felix M.; Hijano, Eliot; Parrikar, Onkar; Rabideau, Charles

2018-05-01

By generalizing different recent works to the context of higher curvature gravity, we provide a unifying framework for three related results: (i) If an asymptotically anti-de Sitter (AdS) spacetime computes the entanglement entropies of ball-shaped regions in a conformal field theory using a generalized Ryu-Takayanagi formula up to second order in state deformations around the vacuum, then the spacetime satisfies the correct gravitational equations of motion up to second order around the AdS background. (ii) The holographic dual of entanglement entropy in higher curvature theories of gravity is given by the Wald entropy plus a particular correction term involving extrinsic curvatures. (iii) Conformal field theory relative entropy is dual to gravitational canonical energy (also in higher curvature theories of gravity). Especially for the second point, our novel derivation of this previously known statement does not involve the Euclidean replica trick.

Conformational Flexibility of Human Casein Kinase Catalytic Subunit Explored by Metadynamics

PubMed Central

Gouron, Aurélie; Milet, Anne; Jamet, Helene

2014-01-01

Casein kinase CK2 is an essential enzyme in higher organisms, catalyzing the transfer of the γ phosphate from ATP to serine and threonine residues on protein substrates. In a number of animal tumors, CK2 activity has been shown to escape normal cellular control, making it a potential target for cancer therapy. Several crystal structures of human CK2 have been published with different conformations for the CK2α catalytic subunit. This variability reflects a high flexibility for two regions of CK2α: the interdomain hinge region, and the glycine-rich loop (p-loop). Here, we present a computational study simulating the equilibrium between three conformations involving these regions. Simulations were performed using well-tempered metadynamics combined with a path collective variables approach. This provides a reference pathway describing the conformational changes being studied, based on analysis of free energy surfaces. The free energies of the three conformations were found to be close and the paths proposed had low activation barriers. Our results indicate that these conformations can exist in water. This information should be useful when designing inhibitors specific to one conformation. PMID:24606937

Protein Allostery and Conformational Dynamics.

PubMed

Guo, Jingjing; Zhou, Huan-Xiang

2016-06-08

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

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.

Mapping transiently formed and sparsely populated conformations on a complex energy landscape

PubMed Central

Wang, Yong; Papaleo, Elena; Lindorff-Larsen, Kresten

2016-01-01

Determining the structures, kinetics, thermodynamics and mechanisms that underlie conformational exchange processes in proteins remains extremely difficult. Only in favourable cases is it possible to provide atomic-level descriptions of sparsely populated and transiently formed alternative conformations. Here we benchmark the ability of enhanced-sampling molecular dynamics simulations to determine the free energy landscape of the L99A cavity mutant of T4 lysozyme. We find that the simulations capture key properties previously measured by NMR relaxation dispersion methods including the structure of a minor conformation, the kinetics and thermodynamics of conformational exchange, and the effect of mutations. We discover a new tunnel that involves the transient exposure towards the solvent of an internal cavity, and show it to be relevant for ligand escape. Together, our results provide a comprehensive view of the structural landscape of a protein, and point forward to studies of conformational exchange in systems that are less characterized experimentally. DOI: http://dx.doi.org/10.7554/eLife.17505.001 PMID:27552057

Generalized conformal structure, dilaton gravity and SYK

NASA Astrophysics Data System (ADS)

Taylor, Marika

2018-01-01

A theory admits generalized conformal structure if the only scale in the quantum theory is set by a dimensionful coupling. SYK is an example of a theory with generalized conformal structure and in this paper we investigate the consequences of this structure for correlation functions and for the holographic realization of SYK. The Ward identities associated with the generalized conformal structure of SYK are implemented holographically in gravity/multiple scalar theories, which always have a parent AdS3 origin. For questions involving only the graviton/running scalar sector, one can always describe the bulk running in terms of a single scalar but multiple running scalars are in general needed once one includes the bulk fields corresponding to all SYK operators. We then explore chaos in holographic theories with generalized conformal structure. The four point function explored by Maldacena, Shenker and Stanford exhibits exactly the same chaotic behaviour in any such theory as in holographic realizations of conformal theories i.e. the dimensionful coupling scale does not affect the chaotic exponential growth.

tRNA Shifts the G-quadruplex-Hairpin Conformational Equilibrium in RNA towards the Hairpin Conformer.

PubMed

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

2016-11-07

Non-coding RNAs play important roles in cellular homeostasis and are involved in many human diseases including cancer. Intermolecular RNA-RNA interactions are the basis for the diverse functions of many non-coding RNAs. Herein, we show how the presence of tRNA influences the equilibrium between hairpin and G-quadruplex conformations in the 5' untranslated regions of oncogenes and model sequences. Kinetic and equilibrium analyses of the hairpin to G-quadruplex conformational transition of purified RNA as well as during co-transcriptional folding indicate that tRNA significantly shifts the equilibrium toward the hairpin conformer. The enhancement of relative translation efficiency in a reporter gene assay is shown to be due to the tRNA-mediated shift in hairpin-G-quadruplex equilibrium of oncogenic mRNAs. Our findings suggest that tRNA is a possible therapeutic target in diseases in which RNA conformational equilibria is dysregulated. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fourier transform vibrational circular dichroism of small pharmaceutical molecules

NASA Astrophysics Data System (ADS)

Long, Fujin; Freedman, Teresa B.; Nafie, Laurence A.

1998-06-01

Fourier transform vibrational circular dichroism (FT-VCD) spectra of the small pharmaceutical molecules propanolol, ibuprofen and naproxen have been measured in the hydrogen stretching and mid-infrared regions to obtain information on solution conformation and to identify markers for absolute configuration determination. Ab initio molecular orbital calculations of low energy conformations, vibrational frequencies and VCD intensities for fragments of the drugs were utilized in interpreting the spectra. Features characteristic of five conformers of propranolol were identified. The weak positive CH stretching VCD signal in ibuprofen and naproxen is characteristic of the S-configuration of the chiral center common to these two analgesics.

Organization in memory and behavior1

PubMed Central

Shimp, Charles P.

1976-01-01

Some common reinforcement contingencies make the delivery of a reinforcer depend on the occurrence of behavior lacking significant temporal structure: a reinforcer may be contingent on nearly instantaneous responses such as a pigeon's key peck, a rat's lever press, a human's button press or brief verbal utterance, and so on. Such a reinforcement contingency conforms much more closely to the functionalist tradition in experimental psychology than to the structuralist tradition. Until recently, the functionalist tradition, in the form of a kind of associationism, typified most research on human learning and memory. Recently, however, research on human memory has focused more on structural issues: now the basic unit of analysis often involves an organized temporal pattern of behavior. A focus on the interrelations between the function and structure of behavior identifies a set of independent and dependent variables different from those identified by certain common kinds of “molar” behavioral analyses. In so doing, such a focus redefines some of the significant issues in the experimental analysis of behavior. PMID:16811925

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.

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

SU-F-P-52: A Meta-Analysis of Controlled Clinical Trials Comparing Elective Nodal Irradiation with Involved-Field Irradiation for Conformal Or Intensity-Modulated Radiotherapy in Patients with Esophageal Cancer

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

Bai, W; Zhang, R; Zhou, Z

Purpose: To compare elective nodal irradiation with involved-field irradiation for three-dimensional conformal radiotherapy or intensity-modulated radiotherapy in patients with esophageal cancer by a metaanalysis. Methods: Wanfang, CNKI, VIP, CBM databases, PubMed, Embase and Cochrane Library were searched to identify the controlled clinical trials of elective nodal irradiation with involved-field irradiation for three-dimensional conformal radiotherapy or intensity-modulated radiotherapy in patients with esophageal cancer. The obtained data were analyzed using Stata 11.0. The difference between two groups was estimated by calculating the odds ratio (OR) with 95% confidence interval (95% CI). Results: A total of 12 controlled clinical trials involving 1095 esophagealmore » cancer patients, which were selected according to inclusion and exclusion criteria, were included in this meta-analysis. The meta-analysis showed that the elective nodal irradiation group reduced the rates of out-field failure comparing with involved-field irradiation group (OR=3.727, P=0.007). However, the rates of ≥grades 3 acute radiation pneumonitis and esophagitis were significantly higher in the elective nodal irradiation group than in the involved-field irradiation group (OR=0.348, P=0.001, OR=0.385, P=0.000). 1-, 2-, 3-year local control rates (OR=0.966, P=0.837, OR=0.946, P=0.781; OR=0.732P=0.098) and 1-, 3-, 5-year survival rates were similar in the two groups ( OR=0.966, P=0.837; OR=0.946, P=0.781; OR=0.732, P=0.098; OR=0.952, P=0.756; OR=1.149, P=0.422; OR=0.768, P=0.120). It is the same with the rates of distant metastasis (OR=0.986, P=0.937). Conclusion: Compared with involved-field irradiation, the elective nodal irradiation can reduce the rates of out-field failure for three-dimensional conformal radiotherapy or intensity-modulated radiotherapy in patients with esophageal cancer. However, its advantage of local control and survival rates is not obvious and it increases the incidence of toxicities.« less

The Conformational Change in Elongation Factor Tu Involves Separation of Its Domains

DOE PAGES

Lai, Jonathan; Ghaemi, Zhaleh; Luthey-Schulten, Zaida

2017-10-18

Elongation factor Tu (EF-Tu) is a highly conserved GTPase that is responsible for supplying the aminoacylated tRNA to the ribosome. Upon binding to the ribosome, EF-Tu undergoes GTP hydrolysis, which drives a major conformational change, triggering the release of aminoacylated tRNA to the ribosome. Using a combination of molecular simulation techniques, we studied the transition between the pre- and post-hydrolysis structures through two distinct pathways. Here, we show that the transition free energy is minimal along a non-intuitive pathway that involves “separation” of the GTP binding domain (domain 1) from the OB folds (domains 2 and 3), followed by domainmore » 1 rotation, and, eventually, locking the EF-Tu conformation in the post-hydrolysis state. The domain separation also leads to a slight extension of the linker connecting domain 1 to domain 2. Using docking tools and correlation-based analysis, we identified and characterized the EF-Tu conformations that release the tRNA. These calculations suggest that EF-Tu can release the tRNA before the domains separate and after domain 1 rotates by 25°. Lastly, we also examined the EF-Tu conformations in the context of the ribosome. Given the high degrees of sequence similarity with other translational GTPases, we predict a similar separation mechanism is followed.« less

The Conformational Change in Elongation Factor Tu Involves Separation of Its Domains

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

Lai, Jonathan; Ghaemi, Zhaleh; Luthey-Schulten, Zaida

Elongation factor Tu (EF-Tu) is a highly conserved GTPase that is responsible for supplying the aminoacylated tRNA to the ribosome. Upon binding to the ribosome, EF-Tu undergoes GTP hydrolysis, which drives a major conformational change, triggering the release of aminoacylated tRNA to the ribosome. Using a combination of molecular simulation techniques, we studied the transition between the pre- and post-hydrolysis structures through two distinct pathways. Here, we show that the transition free energy is minimal along a non-intuitive pathway that involves “separation” of the GTP binding domain (domain 1) from the OB folds (domains 2 and 3), followed by domainmore » 1 rotation, and, eventually, locking the EF-Tu conformation in the post-hydrolysis state. The domain separation also leads to a slight extension of the linker connecting domain 1 to domain 2. Using docking tools and correlation-based analysis, we identified and characterized the EF-Tu conformations that release the tRNA. These calculations suggest that EF-Tu can release the tRNA before the domains separate and after domain 1 rotates by 25°. Lastly, we also examined the EF-Tu conformations in the context of the ribosome. Given the high degrees of sequence similarity with other translational GTPases, we predict a similar separation mechanism is followed.« less

Impact of mutations on the allosteric conformational equilibrium

PubMed Central

Weinkam, Patrick; Chen, Yao Chi; Pons, Jaume; Sali, Andrej

2012-01-01

Allostery in a protein involves effector binding at an allosteric site that changes the structure and/or dynamics at a distant, functional site. In addition to the chemical equilibrium of ligand binding, allostery involves a conformational equilibrium between one protein substate that binds the effector and a second substate that less strongly binds the effector. We run molecular dynamics simulations using simple, smooth energy landscapes to sample specific ligand-induced conformational transitions, as defined by the effector-bound and unbound protein structures. These simulations can be performed using our web server: http://salilab.org/allosmod/. We then develop a set of features to analyze the simulations and capture the relevant thermodynamic properties of the allosteric conformational equilibrium. These features are based on molecular mechanics energy functions, stereochemical effects, and structural/dynamic coupling between sites. Using a machine-learning algorithm on a dataset of 10 proteins and 179 mutations, we predict both the magnitude and sign of the allosteric conformational equilibrium shift by the mutation; the impact of a large identifiable fraction of the mutations can be predicted with an average unsigned error of 1 kBT. With similar accuracy, we predict the mutation effects for an 11th protein that was omitted from the initial training and testing of the machine-learning algorithm. We also assess which calculated thermodynamic properties contribute most to the accuracy of the prediction. PMID:23228330

Stereoselective synthesis of conformationally constrained omega-amino acid analogues from pyroglutamic acid.

PubMed

Bentz, Emilie L; Goswami, Rajesh; Moloney, Mark G; Westaway, Susan M

2005-08-07

Bicyclic lactams derived from pyroglutamic acid provide a useful scaffold for synthesis of conformationally restricted analogues of lysine, ornithine and glutamine, as well as an Ala-Ala dipeptide analogue. Amino alcohol and carboxylic acid derivatives are accessible from a common intermediate. In this strategy, the bicyclic lactam system not only controls, but also facilitates the determination of the stereochemistry of the synthetic intermediates.

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

Predictive Sampling of Rare Conformational Events in Aqueous Solution: Designing a Generalized Orthogonal Space Tempering Method.

PubMed

Lu, Chao; Li, Xubin; Wu, Dongsheng; Zheng, Lianqing; Yang, Wei

2016-01-12

In aqueous solution, solute conformational transitions are governed by intimate interplays of the fluctuations of solute-solute, solute-water, and water-water interactions. To promote molecular fluctuations to enhance sampling of essential conformational changes, a common strategy is to construct an expanded Hamiltonian through a series of Hamiltonian perturbations and thereby broaden the distribution of certain interactions of focus. Due to a lack of active sampling of configuration response to Hamiltonian transitions, it is challenging for common expanded Hamiltonian methods to robustly explore solvent mediated rare conformational events. The orthogonal space sampling (OSS) scheme, as exemplified by the orthogonal space random walk and orthogonal space tempering methods, provides a general framework for synchronous acceleration of slow configuration responses. To more effectively sample conformational transitions in aqueous solution, in this work, we devised a generalized orthogonal space tempering (gOST) algorithm. Specifically, in the Hamiltonian perturbation part, a solvent-accessible-surface-area-dependent term is introduced to implicitly perturb near-solute water-water fluctuations; more importantly in the orthogonal space response part, the generalized force order parameter is generalized as a two-dimension order parameter set, in which essential solute-solvent and solute-solute components are separately treated. The gOST algorithm is evaluated through a molecular dynamics simulation study on the explicitly solvated deca-alanine (Ala10) peptide. On the basis of a fully automated sampling protocol, the gOST simulation enabled repetitive folding and unfolding of the solvated peptide within a single continuous trajectory and allowed for detailed constructions of Ala10 folding/unfolding free energy surfaces. The gOST result reveals that solvent cooperative fluctuations play a pivotal role in Ala10 folding/unfolding transitions. In addition, our assessment analysis suggests that because essential conformational events are mainly driven by the compensating fluctuations of essential solute-solvent and solute-solute interactions, commonly employed "predictive" sampling methods are unlikely to be effective on this seemingly "simple" system. The gOST development presented in this paper illustrates how to employ the OSS scheme for physics-based sampling method designs.

Co-existence of Distinct Prion Types Enables Conformational Evolution of Human PrPSc by Competitive Selection*

PubMed Central

Haldiman, Tracy; Kim, Chae; Cohen, Yvonne; Chen, Wei; Blevins, Janis; Qing, Liuting; Cohen, Mark L.; Langeveld, Jan; Telling, Glenn C.; Kong, Qingzhong; Safar, Jiri G.

2013-01-01

The unique phenotypic characteristics of mammalian prions are thought to be encoded in the conformation of pathogenic prion proteins (PrPSc). The molecular mechanism responsible for the adaptation, mutation, and evolution of prions observed in cloned cells and upon crossing the species barrier remains unsolved. Using biophysical techniques and conformation-dependent immunoassays in tandem, we isolated two distinct populations of PrPSc particles with different conformational stabilities and aggregate sizes, which frequently co-exist in the most common human prion disease, sporadic Creutzfeldt-Jakob disease. The protein misfolding cyclic amplification replicates each of the PrPSc particle types independently and leads to the competitive selection of those with lower initial conformational stability. In serial propagation with a nonglycosylated mutant PrPC substrate, the dominant PrPSc conformers are subject to further evolution by natural selection of the subpopulation with the highest replication rate due to its lowest stability. Cumulatively, the data show that sporadic Creutzfeldt-Jakob disease PrPSc is not a single conformational entity but a dynamic collection of two distinct populations of particles. This implies the co-existence of different prions, whose adaptation and evolution are governed by the selection of progressively less stable, faster replicating PrPSc conformers. PMID:23974118

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.

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

DOE PAGES

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

2015-09-30

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

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

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

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

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

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

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

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

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

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…

Analysis of a Clonal Lineage of HIV-1 Envelope V2/V3 Conformational Epitope-Specific Broadly Neutralizing Antibodies and Their Inferred Unmutated Common Ancestors ▿ †

PubMed Central

Bonsignori, Mattia; Hwang, Kwan-Ki; Chen, Xi; Tsao, Chun-Yen; Morris, Lynn; Gray, Elin; Marshall, Dawn J.; Crump, John A.; Kapiga, Saidi H.; Sam, Noel E.; Sinangil, Faruk; Pancera, Marie; Yongping, Yang; Zhang, Baoshan; Zhu, Jiang; Kwong, Peter D.; O'Dell, Sijy; Mascola, John R.; Wu, Lan; Nabel, Gary J.; Phogat, Sanjay; Seaman, Michael S.; Whitesides, John F.; Moody, M. Anthony; Kelsoe, Garnett; Yang, Xinzhen; Sodroski, Joseph; Shaw, George M.; Montefiori, David C.; Kepler, Thomas B.; Tomaras, Georgia D.; Alam, S. Munir; Liao, Hua-Xin; Haynes, Barton F.

2011-01-01

V2/V3 conformational epitope antibodies that broadly neutralize HIV-1 (PG9 and PG16) have been recently described. Since an elicitation of previously known broadly neutralizing antibodies has proven elusive, the induction of antibodies with such specificity is an important goal for HIV-1 vaccine development. A critical question is which immunogens and vaccine formulations might be used to trigger and drive the development of memory B cell precursors with V2/V3 conformational epitope specificity. In this paper we identified a clonal lineage of four V2/V3 conformational epitope broadly neutralizing antibodies (CH01 to CH04) from an African HIV-1-infected broad neutralizer and inferred their common reverted unmutated ancestor (RUA) antibodies. While conformational epitope antibodies rarely bind recombinant Env monomers, a screen of 32 recombinant envelopes for binding to the CH01 to CH04 antibodies showed monoclonal antibody (MAb) binding to the E.A244 gp120 Env and to chronic Env AE.CM243; MAbs CH01 and CH02 also bound to transmitted/founder Env B.9021. CH01 to CH04 neutralized 38% to 49% of a panel of 91 HIV-1 tier 2 pseudoviruses, while the RUAs neutralized only 16% of HIV-1 isolates. Although the reverted unmutated ancestors showed restricted neutralizing activity, they retained the ability to bind to the E.A244 gp120 HIV-1 envelope with an affinity predicted to trigger B cell development. Thus, E.A244, B.9021, and AE.CM243 Envs are three potential immunogen candidates for studies aimed at defining strategies to induce V2/V3 conformational epitope-specific antibodies. PMID:21795340

Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS

PubMed Central

Bosco, Daryl A.; Morfini, Gerardo; Karabacak, N. Murat; Song, Yuyu; Gros-Louis, Francois; Pasinelli, Piera; Goolsby, Holly; Fontaine, Benjamin A.; Lemay, Nathan; McKenna-Yasek, Diane; Frosch, Matthew P.; Agar, Jeffery N.; Julien, Jean-Pierre; Brady, Scott T.; Brown, Robert H.

2010-01-01

Many mutations confer upon copper/zinc superoxide dismutase-1 (SOD1) one or more toxic function(s) that impair motor neuron viability and cause familial amyotrophic lateral sclerosis (FALS). Using a conformation-specific antibody that detects misfolded SOD1 (C4F6), we demonstrate that oxidized WT-SOD1 and mutant-SOD1 share a conformational epitope that is not present in normal WT-SOD1. In a subset of human sporadic ALS (SALS) cases, motor neurons in the lumbosacral spinal cord displayed striking C4F6 immunoreactivity, denoting the presence of aberrant WT-SOD1 species. Recombinant, oxidized WT-SOD1 and WT-SOD1 immunopurified from SALS tissues inhibited kinesin-based fast axonal transport in a manner similar to FALS-linked mutant SOD1. Studies here suggest that WT-SOD1 can be pathogenic in SALS and identifies an SOD1-dependent pathogenic mechanism common to FALS and SALS. PMID:20953194

Three-step Channel Conformational Changes Common to DNA Packaging Motors of Bacterial Viruses T3, T4, SPP1, and Phi29

PubMed Central

Wang, Shaoying; Ji, Zhouxiang; Yan, Erfu; Haque, Farzin; Guo, Peixuan

2016-01-01

The DNA packaging motor of dsDNA bacterial viruses contains a head-tail connector with a channel for genome to enter during assembly and to exit during host infection. The DNA packaging motor of bacterial virus phi29 was recently reported to use the “One-way Revolution” mechanism for DNA packaging. This raises a question of how dsDNA is ejected during infection if the channel acts as a one-way inward valve. Here we report a three step conformational change of the portal channel that is common among DNA translocation motors of bacterial viruses T3, T4, SPP1, and phi29. The channels of these motors exercise three discrete steps of gating, as revealed by electrophysiological assays. It is proposed that the three step channel conformational changes occur during DNA entry process, resulting in a structural transition in preparation of DNA movement in the reverse direction during ejection. PMID:27181501

We are Family: the Conformations of 1-FLUOROALKANES, C_nH2n+1F (n = 2,3,4,5,6,7,8)

NASA Astrophysics Data System (ADS)

Obenchain, Daniel A.; Orellana, W.; Cooke, S. A.

2016-06-01

he pure rotational spectra of the n = 5, 6, 7, and 8 members of the 1-fluoroalkane family have been recorded between 7 GHz and 14 GHz using chirped pulse Fourier transform microwave spectroscopy. The spectra have been analyzed and results will be presented and compared with previous work on the n= 2, 3, and 4 members. The lowest energy conformer for all family members has the common feature that the fluorine is in a gauche position relative to the alkyl tail for which all other heavy atom dihedral angles, where appropriate, are 180 degrees. For the n = 3 and higher family members the second lowest energy conformer has all heavy atom dihedral angles equal to 180 degrees. For each family member transitions carried by both low energy conformers were observed in the collected rotational spectra. Quantum chemical calculations were performed and trends in the energy separations between these two common conformers will be presented as a function of chain length. Furthermore, longer chain lengths have been examined using only quantum chemical calculations and results will be presented. M. Hayashi, M. Fujitake, T. Inagusa, S. Miyazaki, J.Mol.Struct., 216, 9-26, 1990 W. Caminati, A. C. Fantoni, F. Manescalchi, F. Scappini, Mol.Phys., 64, 1089 ,1988 L. B. Favero, A. Maris, A. Degli Esposti, P. G. Favero, W. Caminati, G. Pawelke, Chem.Eur.J., 6(16), 3018-3025, 2000

Ramachandran analysis of conserved glycyl residues in homologous proteins of known structure.

PubMed

Lakshmi, Balasubramanian; Sinduja, Chandrasekaran; Archunan, Govind; Srinivasan, Narayanaswamy

2014-06-01

High conservation of glycyl residues in homologous proteins is fairly frequent. It is commonly understood that glycine tends to be highly conserved either because of its unique Ramachandran angles or to avoid steric clash that would arise with a larger side chain. Using a database of aligned 3D structures of homologous proteins we identified conserved Gly in 288 alignment positions from 85 families. Ninety-six of these alignment positions correspond to conserved Gly residue with (φ, ψ) values allowed for non-glycyl residues. Reasons for this observation were investigated by in-silico mutation of these glycyl residues to Ala. We found in 94% of the cases a short contact exists between the C(β) atom of the introduced Ala with the atoms which are often distant in the primary structure. This suggests the lack of space even for a short side chain thereby explaining high conservation of glycyl residues even when they adopt (φ, ψ) values allowed for Ala. In 189 alignment positions, the conserved glycyl residues adopt (φ, ψ) values which are disallowed for Ala. In-silico mutation of these Gly residues to Ala almost always results in steric hindrance involving C(β) atom of Ala as one would expect by comparing Ramachandran maps for Ala and Gly. Rare occurrence of the disallowed glycyl conformations even in ultrahigh resolution protein structures are accompanied by short contacts in the crystal structures and such disallowed conformations are not conserved in the homologues. These observations raise the doubt on the accuracy of such glycyl conformations in proteins. © 2014 The Protein Society.

The feasibility and benefits of using volumetric arc therapy in patients with brain metastases: a systematic review.

PubMed

Andrevska, Adriana; Knight, Kellie A; Sale, Charlotte A

2014-12-01

Radiotherapy management of patients with brain metastases most commonly involve a whole-brain radiation therapy (WBRT) regime, as well as newer techniques such as stereotactic radiosurgery (SRS) and intensity modulated radiotherapy (IMRT). The long treatment times incurred by these techniques indicates the need for a novel technique that has shorter treatment times, whilst still producing highly conformal treatment with the potential to deliver escalated doses to the target area. Volumetric modulated arc therapy (VMAT) is a dynamic, highly conformal technique that may deliver high doses of radiation through a single gantry arc and reduce overall treatment times. The aim of this systematic review is to determine the feasibility and benefits of VMAT treatment in regard to overall survival rates and local control in patients with brain metastases, in comparison with patients treated with WBRT, SRS and IMRT. A search of the literature identified 23 articles for the purpose of this review. Articles were included on the basis they were human-based studies, with sample sizes of more than five patients who were receiving treatment for 1-10 metastatic brain lesions. VMAT was found to be highly conformal, have a reduced treatment delivery time and incurred no significant toxicities in comparison with WBRT, SRS and IMRT. Compared to other treatment techniques, VMAT proved to have fewer toxicities than conventional WBRT, shorter treatment times than SRS and similar dose distributions to IMRT plans. Future prospective studies are needed to accurately assess the prognostic benefits of VMAT as well as the occurrence of late toxicities.

Conformational dynamics of the molecular chaperone Hsp90

PubMed Central

Krukenberg, Kristin A.; Street, Timothy O.; Lavery, Laura A.; Agard, David A.

2016-01-01

The molecular chaperone Hsp90 is an essential eukaryotic protein that makes up 1–2% of all cytosolic proteins. Hsp90 is vital for the maturation and maintenance of a wide variety of substrate proteins largely involved in signaling and regulatory processes. Many of these substrates have also been implicated in cancer and other diseases making Hsp90 an attractive target for therapeutics. Hsp90 is a highly dynamic and flexible molecule that can adapt its conformation to the wide variety of substrate proteins with which it acts. Large conformational rearrangements are also required for the activation of these client proteins. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis shifts the equilibrium between a pre-existing set of conformational states in an organism-dependent manner. In vivo Hsp90 functions as part of larger heterocomplexes. The binding partners of Hsp90, co-chaperones, assist in the recruitment and activation of substrates, and many co-chaperones further regulate the conformational dynamics of Hsp90 by shifting the conformational equilibrium towards a particular state. Studies have also suggested alternative mechanisms for the regulation of Hsp90’s conformation. In this review, we discuss the structural and biochemical studies leading to our current understanding of the conformational dynamics of Hsp90 and the role that nucleotide, co-chaperones, post-translational modification and clients play in regulating Hsp90’s conformation. We also discuss the effects of current Hsp90 inhibitors on conformation and the potential for developing small molecules that inhibit Hsp90 by disrupting the conformational dynamics. PMID:21414251

Intramolecular interactions in the polar headgroup of sphingosine: serinol† †Electronic supplementary information (ESI) available: Ab initio parameters for serinol conformers within 1000 cm–1, measured transition frequencies, typical a-type transition for conformer aa1, interconversion barriers and possible tunnelling pathways. See DOI: 10.1039/c5cc09423b Click here for additional data file.

PubMed Central

Loru, Donatella; Peña, Isabel; Alonso, José L.

2016-01-01

The intramolecular interactions in the lipid sphingosine have been elucidated through the investigation of the amino alcohol serinol which mimics its polar headgroup. Intricate networks of intramolecular hydrogen bonds involving the hydroxyl groups and the amino group contribute to the stabilisation of five different conformations observed in the broadband rotational spectrum. PMID:26727395

Effect of mutation of Phe 2436.44 of the histamine H2 receptor on cimetidine and ranitidine mechanism of action.

PubMed

Granja-Galeano, Gina; Zappia, Carlos Daniel; Fabián, Lucas; Davio, Carlos; Shayo, Carina; Fernández, Natalia; Monczor, Federico

2017-12-15

Despite the pivotal role GPCRs play in cellular signaling, it is only in the recent years that structural biology has begun to elucidate how GPCRs function and to provide a platform for structure-based drug design. It is postulated that GPCR activation involves the movement of transmembrane helices. The finding that many residues, which have been shown to be critical for receptor activation and are highly conserved among different GPCRs, are clustered in particular positions of transmembrane helices suggests that activation of GPCRs may involve common molecular mechanisms. In particular, phenylalanine 6.44, located in the upper half of TMVI, is highly conserved among almost all GPCRs. We generated Phe 243 6.44 Ala/Ser mutants of histamine H 2 receptor and found that while the substitutions do not affect receptor expression or ligand signaling, are able to specifically alter cimetidine and ranitidine mechanisms of action from simply inactivating the receptor to produce a ligand-induced G-protein sequestering conformation, that interferes with the signaling of β2-adrenoceptor. Taking advantage of the cubic ternary complex model, and mathematically modeling our results, we hypothesize that this alteration in ligand mechanism of action is consequence of a change in ligand-induced conformational rearrangement of receptor and its effect on G-protein coupling. Our results show that receptor point mutations can not only alter receptor behavior, as shown for activating/inactivating mutations, but also can have more subtle effects changing ligand mechanism of action. Copyright © 2017 Elsevier Inc. All rights reserved.

Structural Refinement of Proteins by Restrained Molecular Dynamics Simulations with Non-interacting Molecular Fragments.

PubMed

Shen, Rong; Han, Wei; Fiorin, Giacomo; Islam, Shahidul M; Schulten, Klaus; Roux, Benoît

2015-10-01

The knowledge of multiple conformational states is a prerequisite to understand the function of membrane transport proteins. Unfortunately, the determination of detailed atomic structures for all these functionally important conformational states with conventional high-resolution approaches is often difficult and unsuccessful. In some cases, biophysical and biochemical approaches can provide important complementary structural information that can be exploited with the help of advanced computational methods to derive structural models of specific conformational states. In particular, functional and spectroscopic measurements in combination with site-directed mutations constitute one important source of information to obtain these mixed-resolution structural models. A very common problem with this strategy, however, is the difficulty to simultaneously integrate all the information from multiple independent experiments involving different mutations or chemical labels to derive a unique structural model consistent with the data. To resolve this issue, a novel restrained molecular dynamics structural refinement method is developed to simultaneously incorporate multiple experimentally determined constraints (e.g., engineered metal bridges or spin-labels), each treated as an individual molecular fragment with all atomic details. The internal structure of each of the molecular fragments is treated realistically, while there is no interaction between different molecular fragments to avoid unphysical steric clashes. The information from all the molecular fragments is exploited simultaneously to constrain the backbone to refine a three-dimensional model of the conformational state of the protein. The method is illustrated by refining the structure of the voltage-sensing domain (VSD) of the Kv1.2 potassium channel in the resting state and by exploring the distance histograms between spin-labels attached to T4 lysozyme. The resulting VSD structures are in good agreement with the consensus model of the resting state VSD and the spin-spin distance histograms from ESR/DEER experiments on T4 lysozyme are accurately reproduced.

Structure of the torque ring of the flagellar motor and the molecular basis for rotational switching.

PubMed

Lee, Lawrence K; Ginsburg, Michael A; Crovace, Claudia; Donohoe, Mhairi; Stock, Daniela

2010-08-19

The flagellar motor drives the rotation of flagellar filaments at hundreds of revolutions per second, efficiently propelling bacteria through viscous media. The motor uses the potential energy from an electrochemical gradient of cations across the cytoplasmic membrane to generate torque. A rapid switch from anticlockwise to clockwise rotation determines whether a bacterium runs smoothly forward or tumbles to change its trajectory. A protein called FliG forms a ring in the rotor of the flagellar motor that is involved in the generation of torque through an interaction with the cation-channel-forming stator subunit MotA. FliG has been suggested to adopt distinct conformations that induce switching but these structural changes and the molecular mechanism of switching are unknown. Here we report the molecular structure of the full-length FliG protein, identify conformational changes that are involved in rotational switching and uncover the structural basis for the formation of the FliG torque ring. This allows us to propose a model of the complete ring and switching mechanism in which conformational changes in FliG reverse the electrostatic charges involved in torque generation.

Conformational Transition of Key Structural Features Involved in Activation of ALK Induced by Two Neuroblastoma Mutations and ATP Binding: Insight from Accelerated Molecular Dynamics Simulations.

PubMed

He, Mu-Yang; Li, Wei-Kang; Zheng, Qing-Chuan; Zhang, Hong-Xing

2018-04-17

Deregulated kinase activity of anaplastic lymphoma kinase (ALK) has been observed to be implicated in the development of tumor progression. The activation mechanism of ALK is proposed to be similar to other receptor tyrosine kinases (RTKs), but the distinct static X-ray crystal conformation of ALK suggests its unique conformational transition. Herein, we have illustrated the dynamic conformational property of wild-type ALK as well as the kinase activation equilibrium variation induced by two neuroblastoma mutations (R1275Q and Y1278S) and ATP binding by performing enhanced sampling accelerated Molecular Dynamics (aMD) simulations. The results suggest that the wild-type ALK is mostly favored in the inactive state, whereas the mutations and ATP binding promote a clear shift toward the active-like conformation. The R1275Q mutant stabilizes the active conformation by rigidifying the αC-in conformation. The Y1278S mutant promotes activation at the expense of a π-stacking hydrophobic cluster, which plays a critical role in the stabilization of the inactive conformation of native ALK. ATP produces a more compact active site and thereby facilitates the activation of ALK. Taken together, these findings not only elucidate the diverse conformations in different ALKs but can also shed light on new strategies for protein engineering and structural-based drug design for ALK.

Conformational flexibility of human casein kinase catalytic subunit explored by metadynamics.

PubMed

Gouron, Aurélie; Milet, Anne; Jamet, Helene

2014-03-04

Casein kinase CK2 is an essential enzyme in higher organisms, catalyzing the transfer of the γ phosphate from ATP to serine and threonine residues on protein substrates. In a number of animal tumors, CK2 activity has been shown to escape normal cellular control, making it a potential target for cancer therapy. Several crystal structures of human CK2 have been published with different conformations for the CK2α catalytic subunit. This variability reflects a high flexibility for two regions of CK2α: the interdomain hinge region, and the glycine-rich loop (p-loop). Here, we present a computational study simulating the equilibrium between three conformations involving these regions. Simulations were performed using well-tempered metadynamics combined with a path collective variables approach. This provides a reference pathway describing the conformational changes being studied, based on analysis of free energy surfaces. The free energies of the three conformations were found to be close and the paths proposed had low activation barriers. Our results indicate that these conformations can exist in water. This information should be useful when designing inhibitors specific to one conformation. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

An Experimental and Computational Study of the Gas-Phase Acidities of the Common Amino Acid Amides.

PubMed

Plummer, Chelsea E; Stover, Michele L; Bokatzian, Samantha S; Davis, John T M; Dixon, David A; Cassady, Carolyn J

2015-07-30

Using proton-transfer reactions in a Fourier transform ion cyclotron resonance mass spectrometer and correlated molecular orbital theory at the G3(MP2) level, gas-phase acidities (GAs) and the associated structures for amides corresponding to the common amino acids have been determined for the first time. These values are important because amino acid amides are models for residues in peptides and proteins. For compounds whose most acidic site is the C-terminal amide nitrogen, two ions populations were observed experimentally with GAs that differ by 4-7 kcal/mol. The lower energy, more acidic structure accounts for the majority of the ions formed by electrospray ionization. G3(MP2) calculations predict that the lowest energy anionic conformer has a cis-like orientation of the [-C(═O)NH](-) group whereas the higher energy, less acidic conformer has a trans-like orientation of this group. These two distinct conformers were predicted for compounds with aliphatic, amide, basic, hydroxyl, and thioether side chains. For the most acidic amino acid amides (tyrosine, cysteine, tryptophan, histidine, aspartic acid, and glutamic acid amides) only one conformer was observed experimentally, and its experimental GA correlates with the theoretical GA related to side chain deprotonation.

The Neuropeptide Oxytocin Enhances Information Sharing and Group Decision Making Quality.

PubMed

De Wilde, Tim R W; Ten Velden, Femke S; De Dreu, Carsten K W

2017-01-11

Groups can make better decisions than individuals when members cooperatively exchange and integrate their uniquely held information and insights. However, under conformity pressures group members are biased towards exchanging commonly known information, and away from exchanging unique information, thus undermining group decision-making quality. At the neurobiological level, conformity associates with the neuropeptide oxytocin. A double-blind placebo controlled study found no evidence for oxytocin induced conformity. Compared to placebo groups, three-person groups whose members received intranasal oxytocin, focused more on unique information (i) and repeated this information more often (ii). These findings reveal oxytocin as a neurobiological driver of group decision-making processes.

The Neuropeptide Oxytocin Enhances Information Sharing and Group Decision Making Quality

PubMed Central

De Wilde, Tim R. W.; Ten Velden, Femke S.; De Dreu, Carsten K. W.

2017-01-01

Groups can make better decisions than individuals when members cooperatively exchange and integrate their uniquely held information and insights. However, under conformity pressures group members are biased towards exchanging commonly known information, and away from exchanging unique information, thus undermining group decision-making quality. At the neurobiological level, conformity associates with the neuropeptide oxytocin. A double-blind placebo controlled study found no evidence for oxytocin induced conformity. Compared to placebo groups, three-person groups whose members received intranasal oxytocin, focused more on unique information (i) and repeated this information more often (ii). These findings reveal oxytocin as a neurobiological driver of group decision-making processes. PMID:28074896

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

Molecular dynamics studies of the protein-protein interactions in inhibitor of κB kinase-β.

PubMed

Jones, Michael R; Liu, Cong; Wilson, Angela K

2014-02-24

Activation of the inhibitor of κB kinase subunit β (IKKβ) oligomer initiates a cascade that results in the translocation of transcription factors involved in mediating immune responses. Dimerization of IKKβ is required for its activation. Coarse-grained and atomistic molecular dynamics simulations were used to investigate the conformation-activity and structure-activity relationships within the oligomer assembly of IKKβ that are impacted upon activation, mutation, and binding of ATP. Intermolecular interactions, free energies, and conformational changes were compared among several conformations, including a monomer, two different dimers, and the tetramer. Modifications to the activation segment induce conformational changes that disrupt dimerization and suggest that the multimeric assembly mediates a global stability for the enzyme that influences the activity of IKKβ.

IMP Dehydrogenase: Structural Schizophrenia and an Unusual Base

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

Hedstrom,L.; Gan, L.

2006-01-01

Textbooks describe enzymes as relatively rigid templates for the transition state of a chemical reaction, and indeed an enzyme such as chymotrypsin, which catalyzes a relatively simple hydrolysis reaction, is reasonably well described by this model. Inosine monophosphate dehydrogenase (IMPDH) undergoes a remarkable array of conformational transitions in the course of a complicated catalytic cycle, offering a dramatic counterexample to this view. IMPDH displays several other unusual mechanistic features, including an Arg residue that may act as a general base catalyst and a dynamic monovalent cation site. Further, IMPDH appears to be involved in 'moon-lighting' functions that may require additionalmore » conformational states. How the balance between conformational states is maintained and how the various conformational states interconvert is only beginning to be understood.« less

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.

Increased reliability of nuclear magnetic resonance protein structures by consensus structure bundles.

PubMed

Buchner, Lena; Güntert, Peter

2015-02-03

Nuclear magnetic resonance (NMR) structures are represented by bundles of conformers calculated from different randomized initial structures using identical experimental input data. The spread among these conformers indicates the precision of the atomic coordinates. However, there is as yet no reliable measure of structural accuracy, i.e., how close NMR conformers are to the "true" structure. Instead, the precision of structure bundles is widely (mis)interpreted as a measure of structural quality. Attempts to increase precision often overestimate accuracy by tight bundles of high precision but much lower accuracy. To overcome this problem, we introduce a protocol for NMR structure determination with the software package CYANA, which produces, like the traditional method, bundles of conformers in agreement with a common set of conformational restraints but with a realistic precision that is, throughout a variety of proteins and NMR data sets, a much better estimate of structural accuracy than the precision of conventional structure bundles. Copyright © 2015 Elsevier Ltd. All rights reserved.

18 CFR 1309.9 - How does TVA provide financial assistance in conformity with the Act?

Code of Federal Regulations, 2010 CFR

2010-04-01

... involving the provision of similar services or benefits. Where the financial assistance involves the... contain a covenant running with the land assuring compliance with this part and the guidelines contained... assistance is extended or for another purpose involving the provision of similar services or benefits. Where...

Bag molding processes

NASA Astrophysics Data System (ADS)

Slobodzinsky, A.

Features, materials, and techniques of vacuum, pressure, and autoclave FRP bag molding processes are described. The bags are used in sealed environments, inflated to flexibly force a curing FRP laminate to conform to a stiff mold form which defines the shape of the finished product. Densification is achieved as the bag presses out the voids and excess resin from the laminate, and consolidation occurs as the plies and adherends are bonded by the bag pressure. Curing techniques nominally involved room temperature or high temperature, and investigations of alternative techniques, such as induction, dielectric, microwave, xenon flash, UV, electron beam, and gamma radiation heating are proceeding. Polysulfone is the most common thermoplastic. Details are given of mold preparations, peel plies or release films and fabrics, bagging techniques, and reusable venting blankets and silicone rubber bags.

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

PubMed Central

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

2015-01-01

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

Epoxide Hydrolase Conformational Heterogeneity for the Resolution of Bulky Pharmacologically Relevant Epoxide Substrates.

PubMed

Serrano-Hervás, Eila; Casadevall, Guillem; Garcia-Borràs, Marc; Feixas, Ferran; Osuna, Sílvia

2018-04-06

The conformational landscape of Bacillus megaterium epoxide hydrolase (BmEH) and how it is altered by mutations that confer the enzyme the ability to accept bulky epoxide substrates has been investigated. Extensive molecular dynamics (MD) simulations coupled to active site volume calculations have unveiled relevant features of the enzyme conformational dynamics and function. Our long-timescale MD simulations identify key conformational states not previously observed by means of X-ray crystallography and short MD simulations that present the loop containing one of the catalytic residues, Asp239, in a wide-open conformation, which is likely involved in the binding of the epoxide substrate. Introduction of mutations M145S and F128A dramatically alters the conformational landscape of the enzyme. These singly mutated variants can accept bulky epoxide substrates due to the disorder induced by mutation in the α-helix containing the catalytic Tyr144 and some parts of the lid domain. These changes impact the enzyme active site, which is substantially wider and more complementary to the bulky pharmacologically relevant epoxide substrates. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural analyses of human thymidylate synthase reveal a site that may control conformational switching between active and inactive states.

PubMed

Chen, Dan; Jansson, Anna; Sim, Daniel; Larsson, Andreas; Nordlund, Pär

2017-08-11

Thymidylate synthase (TS) is the sole enzyme responsible for de novo biosynthesis of thymidylate (TMP) and is essential for cell proliferation and survival. Inhibition of human TS (hTS) has been extensively investigated for cancer chemotherapy, but several aspects of its activity and regulation are still uncertain. In this study, we performed comprehensive structural and biophysical studies of hTS using crystallography and thermal shift assay and provided the first detailed structural information on the conformational changes induced by ligand binding to the hTS active site. We found that upon binding of the antifolate agents raltitrexed and nolatrexed, the two insert regions in hTS, the functions of which are unclear, undergo positional shifts toward the catalytic center. We investigated the inactive conformation of hTS and found that the two insert regions are also involved in the conformational transition between the active and inactive state of hTS. Moreover, we identified a ligand-binding site in the dimer interface, suggesting that the cavity in the dimer interface could serve as an allosteric site of hTS to regulate the conformational switching between the active and inactive states. On the basis of these findings, we propose a regulatory mechanism of hTS activity that involves allosteric regulation of interactions of hTS with its own mRNA depending on cellular demands for TMP. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

Local Structural Differences in Homologous Proteins: Specificities in Different SCOP Classes

PubMed Central

Joseph, Agnel Praveen; Valadié, Hélène; Srinivasan, Narayanaswamy; de Brevern, Alexandre G.

2012-01-01

The constant increase in the number of solved protein structures is of great help in understanding the basic principles behind protein folding and evolution. 3-D structural knowledge is valuable in designing and developing methods for comparison, modelling and prediction of protein structures. These approaches for structure analysis can be directly implicated in studying protein function and for drug design. The backbone of a protein structure favours certain local conformations which include α-helices, β-strands and turns. Libraries of limited number of local conformations (Structural Alphabets) were developed in the past to obtain a useful categorization of backbone conformation. Protein Block (PB) is one such Structural Alphabet that gave a reasonable structure approximation of 0.42 Å. In this study, we use PB description of local structures to analyse conformations that are preferred sites for structural variations and insertions, among group of related folds. This knowledge can be utilized in improving tools for structure comparison that work by analysing local structure similarities. Conformational differences between homologous proteins are known to occur often in the regions comprising turns and loops. Interestingly, these differences are found to have specific preferences depending upon the structural classes of proteins. Such class-specific preferences are mainly seen in the all-β class with changes involving short helical conformations and hairpin turns. A test carried out on a benchmark dataset also indicates that the use of knowledge on the class specific variations can improve the performance of a PB based structure comparison approach. The preference for the indel sites also seem to be confined to a few backbone conformations involving β-turns and helix C-caps. These are mainly associated with short loops joining the regular secondary structures that mediate a reversal in the chain direction. Rare β-turns of type I’ and II’ are also identified as preferred sites for insertions. PMID:22745680

The Role of Flexibility and Conformational Selection in the Binding Promiscuity of PDZ Domains

PubMed Central

Münz, Márton; Hein, Jotun; Biggin, Philip C.

2012-01-01

In molecular recognition, it is often the case that ligand binding is coupled to conformational change in one or both of the binding partners. Two hypotheses describe the limiting cases involved; the first is the induced fit and the second is the conformational selection model. The conformational selection model requires that the protein adopts conformations that are similar to the ligand-bound conformation in the absence of ligand, whilst the induced-fit model predicts that the ligand-bound conformation of the protein is only accessible when the ligand is actually bound. The flexibility of the apo protein clearly plays a major role in these interpretations. For many proteins involved in signaling pathways there is the added complication that they are often promiscuous in that they are capable of binding to different ligand partners. The relationship between protein flexibility and promiscuity is an area of active research and is perhaps best exemplified by the PDZ domain family of proteins. In this study we use molecular dynamics simulations to examine the relationship between flexibility and promiscuity in five PDZ domains: the human Dvl2 (Dishevelled-2) PDZ domain, the human Erbin PDZ domain, the PDZ1 domain of InaD (inactivation no after-potential D protein) from fruit fly, the PDZ7 domain of GRIP1 (glutamate receptor interacting protein 1) from rat and the PDZ2 domain of PTP-BL (protein tyrosine phosphatase) from mouse. We show that despite their high structural similarity, the PDZ binding sites have significantly different dynamics. Importantly, the degree of binding pocket flexibility was found to be closely related to the various characteristics of peptide binding specificity and promiscuity of the five PDZ domains. Our findings suggest that the intrinsic motions of the apo structures play a key role in distinguishing functional properties of different PDZ domains and allow us to make predictions that can be experimentally tested. PMID:23133356

Salt bridge dynamics control substrate-induced conformational change in the membrane transporter GlpT

PubMed Central

Law, Christopher J.; Almqvist, Jonas; Bernstein, Adam; Goetz, Regina M.; Huang, Yafei; Soudant, Celine; Laaksonen, Aatto; Hovmöller, Sven; Wang, Da-Neng

2008-01-01

Summary Active transport of substrates across cytoplasmic membranes is of great physiological, medical and pharmaceutical importance. The glycerol-3-phosphate (G3P) transporter (GlpT) of the E. coli inner membrane is a secondary active antiporter from the ubiquitous major facilitator superfamily that couples the import of G3P to the efflux of inorganic phosphate (Pi) down its concentration gradient. Integrating information from a novel combination of structural, molecular dynamics simulations and biochemical studies, we identify the residues involved directly in binding of substrate to the inward-facing conformation of GlpT, thus defining the structural basis for the substrate-specificity of this transporter. The substrate binding mechanism involves protonation of a histidine residue at the binding site. Furthermore, our data suggest that the formation and breaking of inter- and intradomain salt bridges control the conformational change of the transporter that accompanies substrate translocation across the membrane. The mechanism we propose may be a paradigm for organophosphate/phosphate antiporters. PMID:18395745

Involvement of the glucose moiety in the molecular recognition of methyl beta-lactoside by ricin: synthesis, conformational analysis, and binding studies of different derivatives at the C-3 region.

PubMed

Fernández, P; Jiménez-Barbero, J; Martín-Lomas, M; Solís, D; Díaz-Mauriño, T

1994-04-01

Syntheses of the 3-aminodeoxy (4), 3-deoxy-3-methyl (5), and 3-epi (6) derivatives of methyl beta-lactoside (1) have been achieved from 1 in a straightforward way, and their solution conformations in water and dimethyl sulfoxide analysed through molecular mechanics and dynamics calculations and nuclear magnetic resonance data. The overall shape of all the compounds studied is fairly similar and may be described by conformers included in a low energy region with phi = 15 +/- 45 degrees and psi = -25 +/- 30 degrees, that is ca. 5% of the total potential energy surface for the glycosidic linkages of the disaccharides. The binding of the different compounds to ricin, the galactose-specific toxin from Ricinus communis, has been investigated. The results confirm the involvement of the C-3 region in a nonpolar interaction with the protein at the periphery of the combining site.

Tick receptor for outer surface protein A from Ixodes ricinus — the first intrinsically disordered protein involved in vector-microbe recognition

NASA Astrophysics Data System (ADS)

Urbanowicz, Anna; Lewandowski, Dominik; Szpotkowski, Kamil; Figlerowicz, Marek

2016-04-01

The tick receptor for outer surface protein A (TROSPA) is the only identified factor involved in tick gut colonization by various Borrelia species. TROSPA is localized in the gut epithelium and can recognize and bind the outer surface bacterial protein OspA via an unknown mechanism. Based on earlier reports and our latest observations, we considered that TROSPA would be the first identified intrinsically disordered protein (IDP) involved in the interaction between a vector and a pathogenic microbe. To verify this hypothesis, we performed structural studies of a TROSPA mutant from Ixodes ricinus using both computational and experimental approaches. Irrespective of the method used, we observed that the secondary structure content of the TROSPA polypeptide chain is low. In addition, the collected SAXS data indicated that this protein is highly extended and exists in solution as a set of numerous conformers. These features are all commonly considered hallmarks of IDPs. Taking advantage of our SAXS data, we created structural models of TROSPA and proposed a putative mechanism for the TROSPA-OspA interaction. The disordered nature of TROSPA may explain the ability of a wide spectrum of Borrelia species to colonize the tick gut.

Benford’s Law: Textbook Exercises and Multiple-Choice Testbanks

PubMed Central

Slepkov, Aaron D.; Ironside, Kevin B.; DiBattista, David

2015-01-01

Benford’s Law describes the finding that the distribution of leading (or leftmost) digits of innumerable datasets follows a well-defined logarithmic trend, rather than an intuitive uniformity. In practice this means that the most common leading digit is 1, with an expected frequency of 30.1%, and the least common is 9, with an expected frequency of 4.6%. Currently, the most common application of Benford’s Law is in detecting number invention and tampering such as found in accounting-, tax-, and voter-fraud. We demonstrate that answers to end-of-chapter exercises in physics and chemistry textbooks conform to Benford’s Law. Subsequently, we investigate whether this fact can be used to gain advantage over random guessing in multiple-choice tests, and find that while testbank answers in introductory physics closely conform to Benford’s Law, the testbank is nonetheless secure against such a Benford’s attack for banal reasons. PMID:25689468

Benford's Law: textbook exercises and multiple-choice testbanks.

PubMed

Slepkov, Aaron D; Ironside, Kevin B; DiBattista, David

2015-01-01

Benford's Law describes the finding that the distribution of leading (or leftmost) digits of innumerable datasets follows a well-defined logarithmic trend, rather than an intuitive uniformity. In practice this means that the most common leading digit is 1, with an expected frequency of 30.1%, and the least common is 9, with an expected frequency of 4.6%. Currently, the most common application of Benford's Law is in detecting number invention and tampering such as found in accounting-, tax-, and voter-fraud. We demonstrate that answers to end-of-chapter exercises in physics and chemistry textbooks conform to Benford's Law. Subsequently, we investigate whether this fact can be used to gain advantage over random guessing in multiple-choice tests, and find that while testbank answers in introductory physics closely conform to Benford's Law, the testbank is nonetheless secure against such a Benford's attack for banal reasons.

Rock-weathering rates as functions of time

USGS Publications Warehouse

Colman, Steven M.

1981-01-01

The scarcity of documented numerical relations between rock weathering and time has led to a common assumption that rates of weathering are linear. This assumption has been strengthened by studies that have calculated long-term average rates. However, little theoretical or empirical evidence exists to support linear rates for most chemical-weathering processes, with the exception of congruent dissolution processes. The few previous studies of rock-weathering rates that contain quantitative documentation of the relation between chemical weathering and time suggest that the rates of most weathering processes decrease with time. Recent studies of weathering rinds on basaltic and andesitic stones in glacial deposits in the western United States also clearly demonstrate that rock-weathering processes slow with time. Some weathering processes appear to conform to exponential functions of time, such as the square-root time function for hydration of volcanic glass, which conforms to the theoretical predictions of diffusion kinetics. However, weathering of mineralogically heterogeneous rocks involves complex physical and chemical processes that generally can be expressed only empirically, commonly by way of logarithmic time functions. Incongruent dissolution and other weathering processes produce residues, which are commonly used as measures of weathering. These residues appear to slow movement of water to unaltered material and impede chemical transport away from it. If weathering residues impede weathering processes then rates of weathering and rates of residue production are inversely proportional to some function of the residue thickness. This results in simple mathematical analogs for weathering that imply nonlinear time functions. The rate of weathering becomes constant only when an equilibrium thickness of the residue is reached. Because weathering residues are relatively stable chemically, and because physical removal of residues below the ground surface is slight, many weathering features require considerable time to reach constant rates of change. For weathering rinds on volcanic stones in the western United States, this time is at least 0.5 my. ?? 1981.

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

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

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

Insights into bunyavirus architecture from electron cryotomography of Uukuniemi virus

PubMed Central

Överby, A. K.; Pettersson, R. F.; Grünewald, K.; Huiskonen, J. T.

2008-01-01

Bunyaviridae is a large family of viruses that have gained attention as “emerging viruses” because many members cause serious disease in humans, with an increasing number of outbreaks. These negative-strand RNA viruses possess a membrane envelope covered by glycoproteins. The virions are pleiomorphic and thus have not been amenable to structural characterization using common techniques that involve averaging of electron microscopic images. Here, we determined the three-dimensional structure of a member of the Bunyaviridae family by using electron cryotomography. The genome, incorporated as a complex with the nucleoprotein inside the virions, was seen as a thread-like structure partially interacting with the viral membrane. Although no ordered nucleocapsid was observed, lateral interactions between the two membrane glycoproteins determine the structure of the viral particles. In the most regular particles, the glycoprotein protrusions, or “spikes,” were seen to be arranged on an icosahedral lattice, with T = 12 triangulation. This arrangement has not yet been proven for a virus. Two distinctly different spike conformations were observed, which were shown to depend on pH. This finding is reminiscent of the fusion proteins of alpha-, flavi-, and influenza viruses, in which conformational changes occur in the low pH of the endosome to facilitate fusion of the viral and host membrane during viral entry. PMID:18272496

Eating disorders during the adolescence: personality characteristics associated with anorexia and bulimia nervosa.

PubMed

Barajas Iglesias, Belén; Jáuregui Lobera, Ignacio; Laporta Herrero, Isabel; Santed Germán, Miguel Ángel

2017-10-24

Previous studies provide relevant information about the relationship between personality and eating disorders (ED). The involvement of personality factors in the etiology and maintenance of ED indicates the need of emphasizing the study of the adolescent's personality when diagnosed of ED. The aims of this study were to analyze the adolescent's personality profiles that differ significantly in anorexia nervosa (AN) and bulimia nervosa (BN), and to explore the most common profiles and their associations with those subtypes of eating disorders (ED). A total of 104 patients with AN and BN were studied by means of the Millon Adolescent Clinical Inventory (MACI). The personality profiles that differ significantly in both AN and BN were submissive, egotistic, unruly, forceful, conforming, oppositional, self-demeaning and borderline. The most frequent profiles in AN were conforming (33.33%), egotistic (22.72%) and dramatizing (18.18%) while in the case of BN those profiles were unruly (18.42%), submissive (18.42%) and borderline (15.78%). We did not find any associations between the diagnostic subgroup (AN, BN) and the fact of having personality profiles that could become dysfunctional. Bearing in mind these results, it may be concluded that there are relevant differences between personality profiles associated with AN and BN during adolescence, so tailoring therapeutic interventions for this specific population would be important.

Modeling side-chains using molecular dynamics improve recognition of binding region in CAPRI targets.

PubMed

Camacho, Carlos J

2005-08-01

The CAPRI-II experiment added an extra level of complexity to the problem of predicting protein-protein interactions by including 5 targets for which participants had to build or complete the 3-dimensional (3D) structure of either the receptor or ligand based on the structure of a close homolog. In this article, we describe how modeling key side-chains using molecular dynamics (MD) in explicit solvent improved the recognition of the binding region of a free energy- based computational docking method. In particular, we show that MD is able to predict with relatively high accuracy the rotamer conformation of the anchor side-chains important for molecular recognition as suggested by Rajamani et al. (Proc Natl Acad Sci USA 2004;101:11287-11292). As expected, the conformations are some of the most common rotamers for the given residue, while latch side-chains that undergo induced fit upon binding are forced into less common conformations. Using these models as starting conformations in conjunction with the rigid-body docking server ClusPro and the flexible docking algorithm SmoothDock, we produced valuable predictions for 6 of the 9 targets in CAPRI-II, missing only the 3 targets that underwent significant structural rearrangements upon binding. We also show that our free energy- based scoring function, consisting of the sum of van der Waals, Coulombic electrostatic with a distance-dependent dielectric, and desolvation free energy successfully discriminates the nativelike conformation of our submitted predictions. The latter emphasizes the critical role that thermodynamics plays on our methodology, and validates the generality of the algorithm to predict protein interactions.

Sweet Structural Signatures Unveiled in Ketohexoses.

PubMed

Bermúdez, Celina; Peña, Isabel; Mata, Santiago; Alonso, José L

2016-11-14

The conformational behaviour of naturally occurring ketohexoses has been revealed in a supersonic expansion by Fourier transform microwave spectroscopy coupled with a laser ablation source. Three, two and one conformers of d-tagatose, d-psicose and l-sorbose, respectively, have been identified by their rotational constants extracted from the analysis of the spectra. Singular structural signatures involving the hydroxyl groups OH (1) and OH (2) have been disentangled from the intricate intramolecular hydrogen bond networks stabilising the most abundant conformers. The present results place the old Shallenberger and Kier sweetness theories on a firmer footing. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Conformational relaxation dynamics in the excited electronic states of benzil in solution

NASA Astrophysics Data System (ADS)

Singh, Ajay K.; Palit, Dipak K.; Mittal, Jai P.

2002-07-01

Relaxation dynamics in the excited singlet (S1) state of benzil have been studied in solution using pico and subpicosecond transient absorption spectroscopic techniques. The triple exponential decay dynamics of the S1 state indicates that the process of conformational change from the cis-skewed to the trans-planar form takes place via the formation of a meta-stable intermediate conformer resulting the involvement of two consequent barrier crossing processes. The barrier crossing dynamics is governed by both the polarity of the solvent, which alters the barrier heights by `static' interactions, as well as the viscosity of the solvent via `dynamical' interactions.

System and method for forming synthetic protein crystals to determine the conformational structure by crystallography

DOEpatents

Craig, George D.; Glass, Robert; Rupp, Bernhard

1997-01-01

A method for forming synthetic crystals of proteins in a carrier fluid by use of the dipole moments of protein macromolecules that self-align in the Helmholtz layer adjacent to an electrode. The voltage gradients of such layers easily exceed 10.sup.6 V/m. The synthetic protein crystals are subjected to x-ray crystallography to determine the conformational structure of the protein involved.

Conformational stability of the epidermal growth factor (EGF) receptor as influenced by glycosylation, dimerization and EGF hormone binding.

PubMed

Taylor, Eric S; Pol-Fachin, Laercio; Lins, Roberto D; Lower, Steven K

2017-04-01

The epidermal growth factor receptor (EGFR) is an important transmembrane glycoprotein kinase involved the initiation or perpetuation of signal transduction cascades within cells. These processes occur after EGFR binds to a ligand [epidermal growth factor (EGF)], thus inducing its dimerization and tyrosine autophosphorylation. Previous publications have highlighted the importance of glycosylation and dimerization for promoting proper function of the receptor and conformation in membranes; however, the effects of these associations on the protein conformational stability have not yet been described. Molecular dynamics simulations were performed to characterize the conformational preferences of the monomeric and dimeric forms of the EGFR extracellular domain upon binding to EGF in the presence and absence of N-glycan moieties. Structural stability analyses revealed that EGF provides the most conformational stability to EGFR, followed by glycosylation and dimerization, respectively. The findings also support that EGF-EGFR binding takes place through a large-scale induced-fitting mechanism. Proteins 2017; 85:561-570. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

PubMed

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

2014-10-27

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

Localized frustration and binding-induced conformational change in recognition of 5S RNA by TFIIIA zinc finger.

PubMed

Tan, Cheng; Li, Wenfei; Wang, Wei

2013-12-19

Protein TFIIIA is composed of nine tandemly arranged Cys2His2 zinc fingers. It can bind either to the 5S RNA gene as a transcription factor or to the 5S RNA transcript as a chaperone. Although structural and biochemical data provided valuable information on the recognition between the TFIIIIA and the 5S DNA/RNA, the involved conformational motions and energetic factors contributing to the binding affinity and specificity remain unclear. In this work, we conducted MD simulations and MM/GBSA calculations to investigate the binding-induced conformational changes in the recognition of the 5S RNA by the central three zinc fingers of TFIIIA and the energetic factors that influence the binding affinity and specificity at an atomistic level. Our results revealed drastic interdomain conformational changes between these three zinc fingers, involving the exposure/burial of several crucial DNA/RNA binding residues, which can be related to the competition between DNA and RNA for the binding of TFIIIA. We also showed that the specific recognition between finger 4/finger 6 and the 5S RNA introduces frustrations to the nonspecific interactions between finger 5 and the 5S RNA, which may be important to achieve optimal binding affinity and specificity.

Quantum chemical calculations in the structural analysis of phloretin

NASA Astrophysics Data System (ADS)

Gómez-Zavaglia, Andrea

2009-07-01

In this work, a conformational search on the molecule of phloretin [2',4',6'-Trihydroxy-3-(4-hydroxyphenyl)-propiophenone] has been performed. The molecule of phloretin has eight dihedral angles, four of them taking part in the carbon backbone and the other four, related with the orientation of the hydroxyl groups. A systematic search involving a random variation of the dihedral angles has been used to generate input structures for the quantum chemical calculations. Calculations at the DFT(B3LYP)/6-311++G(d,p) level of theory permitted the identification of 58 local minima belonging to the C 1 symmetry point group. The molecular structures of the conformers have been analyzed using hierarchical cluster analysis. This method allowed us to group conformers according to their similarities, and thus, to correlate the conformers' stability with structural parameters. The dendrogram obtained from the hierarchical cluster analysis depicted two main clusters. Cluster I included all the conformers with relative energies lower than 25 kJ mol -1 and cluster II, the remaining conformers. The possibility of forming intramolecular hydrogen bonds resulted the main factor contributing for the stability. Accordingly, all conformers depicting intramolecular H-bonds belong to cluster I. These conformations are clearly favored when the carbon backbone is as planar as possible. The values of the νC dbnd O and νOH vibrational modes were compared among all the conformers of phloretin. The redshifts associated with intramolecular H-bonds were correlated with the H-bonds distances and energies.

Solvent, Temperature And Concentration Effects on the Optical Activity of Chiral FIVE-And-SIX Membered Ring Ketones Conformers

NASA Astrophysics Data System (ADS)

Al-Basheer, Watheq

2017-06-01

Chiral five-and-six membered ring ketones are important molecules that are found in many biological systems and can exist in many possible conformers. In this talk, experimental and computational investigation of solvent, temperature and concentration effects on the circular dichroism (CD) and optical rotation (OR) of (R)-3 -methylcyclohexanone (R3MCH), (R)-3-methylcyclopentanone (R3MCP) and carvone conformers will be discussed. CD and OR measurements of these ketones gaseous samples and in ten common solvents of wide polarity range for different concentrations and sample temperatures were recorded and related to molecular conformation. Density functional theoretical calculations were performed using Gaussian09 at B3LYP functions with aug-cc-pVDZ level of theory. Also, CD and OR spectra for the optimized geometries of the ketones dominant conformers were computed over the ultraviolet and visible region in the gas phase as well as in ten solvents of varying polarity range, and under the umbrella of the polarizable continuum model (PCM). By comparing theoretical and experimental results, few thermodynamic parameters were deduced for the individual equatorial and axial conformers of each molecule in gas phase and in solvation.

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

NMR investigation and theoretical calculations of the solvent effect on the conformation of valsartan

NASA Astrophysics Data System (ADS)

Chashmniam, Saeed; Tafazzoli, Mohsen

2017-11-01

Structure and conformational properties of valsartan were studied by advanced NMR techniques and quantum calculation methods. Potential energy scanning using B3LYP/6-311++g** and B3LYP-D3/6-311++g** methods were performed and four conformers (V1-V4) at minimum points of PES diagram were observed. According to the NMR spectra in acetone-d6, there are two conformers (M and m) with m/M = 0.52 ratio simultaneously and energy barriers of the two conformers were predicted from chemical shifts and multiplicities. While, intramolecular hydrogen bond at tetrazole ring and carboxylic groups prevent the free rotation on N6sbnd C11 bond in M-conformer, this bond rotates freely in m-conformer. On the other hand, intramolecular hydrogen bond at carbonyl and carboxylic acid can be observed at m-conformer. So, different intramolecular hydrogen bond is the reason for the stability of both M and m structures. Quite interestingly, 1H NMR spectra in CDCl3 show two distinct conformers (N and n) with unequal ratio which are differ from M-m conformers. Also, intramolecular hydrogen bond seven-member ring involving five-membered tetrazole ring and carboxylic acid group observed in both N and n-conformers Solvent effect, by using a set of polar and non-polar solvents including DMSO-d6, methanol-d4, benzene-d6, THF-d8, nitromethane-d3, methylene chloride-d2 and acetonitrile-d3 were investigated. NMR parameters include chemical shifts and spin-spin coupling constants were obtained from a set of 2D NMR spectra (H-H COSY, HMQC and HMBC). For this purpose, several DFT functionals from LDA, GGA and hybrid categories were used which the hybrid method showed better agreement with experiment values.

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.

Proline Residues as Switches in Conformational Changes Leading to Amyloid Fibril Formation

PubMed Central

Taler-Verčič, Ajda; Hasanbašić, Samra; Berbić, Selma; Stoka, Veronika; Turk, Dušan; Žerovnik, Eva

2017-01-01

Here we discuss studies of the structure, folding, oligomerization and amyloid fibril formation of several proline mutants of human stefin B, which is a protein inhibitor of lysosomal cysteine cathepsins and a member of the cystatin family. The structurally important prolines in stefin B are responsible for the slow folding phases and facilitate domain swapping (Pro 74) and loop swapping (Pro 79). Moreover, our findings are compared to β2-microglobulin, a protein involved in dialysis-related amyloidosis. The assessment of the contribution of proline residues to the process of amyloid fibril formation may shed new light on the critical molecular events involved in conformational disorders. PMID:28272335

Chiral organosilica particles and their use as inducers of conformational deracemization of liquid crystal phases

NASA Astrophysics Data System (ADS)

Cohen, Orit; Ferris, Andrew J.; Adkins, Raymond; Lemieux, Robert P.; Avnir, David; Gelman, Dmitri; Rosenblatt, Charles

2018-03-01

Chiral organosilica particles of size ∼200 nm were synthesized from an enantio-pure multi-armed chiral D-maltose organosilane precursor in the absence of co-condensation with an achiral monomer. Two distinct experiments were performed to demonstrate the particles' ability to induce conformational deracemization of a host liquid crystal. The first involves an electric field-induced tilt of the liquid crystal director in the deracemized smectic-A phase. The other involves domain wall curvature separating left- and right-handed liquid crystal helical pitch domains imposed by the cells' substrates. The results demonstrate unequivocally that enantio-pure organosilica nanoparticles can be synthesized and can induce chirality in a host.

Conformational sampling with stochastic proximity embedding and self-organizing superimposition: establishing reasonable parameters for their practical use.

PubMed

Tresadern, Gary; Agrafiotis, Dimitris K

2009-12-01

Stochastic proximity embedding (SPE) and self-organizing superimposition (SOS) are two recently introduced methods for conformational sampling that have shown great promise in several application domains. Our previous validation studies aimed at exploring the limits of these methods and have involved rather exhaustive conformational searches producing a large number of conformations. However, from a practical point of view, such searches have become the exception rather than the norm. The increasing popularity of virtual screening has created a need for 3D conformational search methods that produce meaningful answers in a relatively short period of time and work effectively on a large scale. In this work, we examine the performance of these algorithms and the effects of different parameter settings at varying levels of sampling. Our goal is to identify search protocols that can produce a diverse set of chemically sensible conformations and have a reasonable probability of sampling biologically active space within a small number of trials. Our results suggest that both SPE and SOS are extremely competitive in this regard and produce very satisfactory results with as few as 500 conformations per molecule. The results improve even further when the raw conformations are minimized with a molecular mechanics force field to remove minor imperfections and any residual strain. These findings provide additional evidence that these methods are suitable for many everyday modeling tasks, both high- and low-throughput.

The conformational free-energy landscape of β-D-mannopyranose: evidence for a (1)S(5) → B(2,5) → (O)S(2) catalytic itinerary in β-mannosidases.

PubMed

Ardèvol, Albert; Biarnés, Xevi; Planas, Antoni; Rovira, Carme

2010-11-17

The mechanism of glycosidic bond cleavage by glycosidases involves substrate ring distortions in the Michaelis complex that favor catalysis. Retaining β-mannosidases bind the substrate in a (1)S(5) conformation, and recent experiments have proposed an unusual substrate conformational pathway ((1)S(5) → B(2,5) → (O)S(2)) for the hydrolysis reaction. By means of Car-Parrinello metadynamics simulations, we have obtained the conformational free-energy surface (FES) of a β-d-mannopyranose molecule associated with the ideal Stoddart conformational diagram. We have found that (1)S(5) is among the most stable conformers and simultaneously is the most preactivated conformation in terms of elongation/shortening of the C1-O1/C1-O5 bonds, C1-O1 orientation, and charge development at the anomeric carbon. Analysis of the computed FES gives support to the proposed (1)S(5) → B(2,5) → (O)S(2) catalytic itinerary, showing that the degree of preactivation of the substrate in glycoside hydrolases (GHs) is related to the properties of an isolated sugar ring. We introduce a simple preactivation index integrating several structural, electronic, and energetic properties that can be used to predict the conformation of the substrate in the Michaelis complex of any GH.

Solving the Nonlocality Riddle by Conformal Quantum Geometrodynamics

NASA Astrophysics Data System (ADS)

Santamato, Enrico; de Martini, Francesco

2012-01-01

Since the 1935 proposal by Einstein, Podolsky and Rosen the riddle of nonlocality, today demonstrated by the violation of Bell's inequalities within innumerable experiments, has been a cause of concern and confusion within the debate over the foundations of quantum mechanics. The present paper tackles the problem by a nonrelativistic approach based on conformal differential geometry applied to the solution of the dynamical problem of two entangled spin 1/2 particles. It is found that the quantum nonlocality may be understood on the basis of a conformal quantum geometrodynamics acting necessarily on the full "configuration space" of the entangled particles. At the end, the violation of the Bell inequalities is demonstrated without making recourse to the common nonlocality paradigm.

Distinguishing Signatures of Multipathway Conformational Transitions

NASA Astrophysics Data System (ADS)

Pierse, Christopher A.; Dudko, Olga K.

2017-02-01

The folding and binding of biomolecules into functional conformations are thought to be commonly mediated by multiple pathways rather than a unique route. Yet even in experiments where one can "see" individual conformational transitions, their stochastic nature generally precludes one from determining whether the transitions occurred through one or multiple pathways. We establish model-free, observable signatures in the response of macromolecules to force that unambiguously identify multiple pathways—even when the pathways themselves cannot be resolved. The unified analytical description reveals that, through multiple pathways, the response of molecules to external forces can be shaped in diverse ways, resulting in a rich design space for a tailored biological function already at the single-molecule level.

Sex differences on a measure of conformity in automated teller machine lines.

PubMed

Reysen, Stephen; Reysen, Matthew B

2004-10-01

Sex differences in conformity were examined as participants approached two ATMs, one of which was occupied by three confederates and the other immediately available. The number of men and women in the line in front of one of the ATMs was manipulated (3 men or 3 women), and an unobtrusive observer recorded the sex of each participant. The results indicated that women were more likely than men to wait in line to use the ATM regardless of the makeup of the line. Thus, the present study provides evidence in favor of the idea that sex differences in conformity are evident on a common task performed in a natural setting.

Conformational equilibria of light-activated rhodopsin in nanodiscs

PubMed Central

Van Eps, Ned; Caro, Lydia N.; Morizumi, Takefumi; Kusnetzow, Ana Karin; Szczepek, Michal; Hofmann, Klaus Peter; Bayburt, Timothy H.; Sligar, Stephen G.; Ernst, Oliver P.; Hubbell, Wayne L.

2017-01-01

Conformational equilibria of G-protein–coupled receptors (GPCRs) are intimately involved in intracellular signaling. Here conformational substates of the GPCR rhodopsin are investigated in micelles of dodecyl maltoside (DDM) and in phospholipid nanodiscs by monitoring the spatial positions of transmembrane helices 6 and 7 at the cytoplasmic surface using site-directed spin labeling and double electron–electron resonance spectroscopy. The photoactivated receptor in DDM is dominated by one conformation with weak pH dependence. In nanodiscs, however, an ensemble of pH-dependent conformational substates is observed, even at pH 6.0 where the MIIbH+ form defined by proton uptake and optical spectroscopic methods is reported to be the sole species present in native disk membranes. In nanodiscs, the ensemble of substates in the photoactivated receptor spontaneously decays to that characteristic of the inactive state with a lifetime of ∼16 min at 20 °C. Importantly, transducin binding to the activated receptor selects a subset of the ensemble in which multiple substates are apparently retained. The results indicate that in a native-like lipid environment rhodopsin activation is not analogous to a simple binary switch between two defined conformations, but the activated receptor is in equilibrium between multiple conformers that in principle could recognize different binding partners. PMID:28373559

Intrinsic Conformational Preferences of Cα,α-Dibenzylglycine

PubMed Central

Casanovas, Jordi; Nussinov, Ruth; Alemán, Carlos

2009-01-01

The intrinsic conformational preferences of Cα,α-dibenzylglycine, a symmetric α,α-dialkylated amino acid bearing two benzyl substituents on the α-carbon atom, have been determined using quantum chemical calculations at the B3LYP/6-31+G(d,p) level. A total of 46 minimum energy conformations were found for the N-acetyl-N'-methylamide derivative, even though only 9 of them showed a relative energy lower than 5.0 kcal/mol. The latter involves C7, C5 and α' backbone conformations stabilized by intramolecular hydrogen bonds and/or N-H…π interactions. Calculation of the conformational free energies in different environments (gas-phase, carbon tetrachloride, chloroform, methanol and water solutions) indicates that four different minima (two C5 and two C7) are energetically accessible at room temperature in the gas-phase, while in methanol and aqueous solutions one such minimum (C5) becomes the only significant conformation. Comparison with results recently reported for Cα,α-diphenylglycine indicates that substitution of phenyl side groups by benzyl enhances the conformational flexibility leading to (i) a reduction of the strain of the peptide backbone; and (ii) alleviating the repulsive interactions between the π electron density of the phenyl groups and the lone pairs of the carbonyl oxygen atoms. PMID:18465898

Mechanochemical Modeling of Dynamic Microtubule Growth Involving Sheet-to-Tube Transition

PubMed Central

Ji, Xiang-Ying; Feng, Xi-Qiao

2011-01-01

Microtubule dynamics is largely influenced by nucleotide hydrolysis and the resultant tubulin configuration changes. The GTP cap model has been proposed to interpret the stabilizing mechanisms of microtubule growth from the view of hydrolysis effects. Besides, the growth of a microtubule involves the closure of a curved sheet at its growing end. The curvature conversion from the longitudinal direction to the circumferential direction also helps to stabilize the successive growth, and the curved sheet is referred to as the conformational cap. However, there still lacks theoretical investigation on the mechanical–chemical coupling growth process of microtubules. In this paper, we study the growth mechanisms of microtubules by using a coarse-grained molecular method. First, the closure process involving a sheet-to-tube transition is simulated. The results verify the stabilizing effect of the sheet structure and predict that the minimum conformational cap length that can stabilize the growth is two dimers. Then, we show that the conformational cap and the GTP cap can function independently and harmoniously, signifying the pivotal role of mechanical factors. Furthermore, based on our theoretical results, we describe a Tetris-like growth style of microtubules: the stochastic tubulin assembly is regulated by energy and harmonized with the seam zipping such that the sheet keeps a practically constant length during growth. PMID:22205994

Collision cross sections of high-mannose N-glycans in commonly observed adduct states--identification of gas-phase conformers unique to [M-H](-) ions.

PubMed

Struwe, W B; Benesch, J L; Harvey, D J; Pagel, K

2015-10-21

We report collision cross sections (CCS) of high-mannose N-glycans as [M + Na](+), [M + K](+), [M + H](+), [M + Cl](-), [M + H2PO4](-) and [M - H](-) ions, measured by drift tube (DT) ion mobility-mass spectrometry (IM-MS) in helium and nitrogen gases. Further analysis using traveling wave (TW) IM-MS reveal the existence of distinct conformers exclusive to [M - H](-) ions.

Measurement Assurance for End-Item Users

NASA Technical Reports Server (NTRS)

Mimbs, Scott M.

2008-01-01

The goal of a Quality Management System (QMS) as specified in ISO 9001 and AS9100 is to assure the end product meets specifications and customer requirements. Measuring devices, often called measuring and test equipments (MTE), provide the evidence of product conformity to the prescribed requirements. Therefore the processes which employ MTE can become a weak link to the overall QMS if proper attention is not given to development and execution of these processes. Traditionally, calibration of MTE is given more focus in industry standards and process control efforts than the equally important proper usage of the same equipment. It is a common complaint of calibration laboratory personnel that MTE users are only interested in "a sticker." If the QMS requires the MTE "to demonstrate conformity of the product," then the quality of the measurement process must be adequate for the task. This leads to an ad hoc definition; measurement assurance is a discipline that assures that all processes, activities, environments, standards, and procedures involved in making a measurement produce a result that can be rigorously evaluated for validity and accuracy. To evaluate that the existing measurement processes are providing an adequate level of quality to support the decisions based upon this measurement data, an understanding of measurement assurance basics is essential. This topic is complimentary to the calibration standard, ANSI/NCSL Z540.3-2006, which targets the calibration of MTE at the organizational level. This paper will discuss general measurement assurance when MTE is used to provide evidence of product conformity, therefore the target audience of this paper is end item users of MTE. A central focus of the paper will be the verification of tolerances and the associated risks, so calibration professionals may find the paper useful in communication with their customers, MTE users.

Relaxation mode analysis and Markov state relaxation mode analysis for chignolin in aqueous solution near a transition temperature

NASA Astrophysics Data System (ADS)

Mitsutake, Ayori; Takano, Hiroshi

2015-09-01

It is important to extract reaction coordinates or order parameters from protein simulations in order to investigate the local minimum-energy states and the transitions between them. The most popular method to obtain such data is principal component analysis, which extracts modes of large conformational fluctuations around an average structure. We recently applied relaxation mode analysis for protein systems, which approximately estimates the slow relaxation modes and times from a simulation and enables investigations of the dynamic properties underlying the structural fluctuations of proteins. In this study, we apply this relaxation mode analysis to extract reaction coordinates for a system in which there are large conformational changes such as those commonly observed in protein folding/unfolding. We performed a 750-ns simulation of chignolin protein near its folding transition temperature and observed many transitions between the most stable, misfolded, intermediate, and unfolded states. We then applied principal component analysis and relaxation mode analysis to the system. In the relaxation mode analysis, we could automatically extract good reaction coordinates. The free-energy surfaces provide a clearer understanding of the transitions not only between local minimum-energy states but also between the folded and unfolded states, even though the simulation involved large conformational changes. Moreover, we propose a new analysis method called Markov state relaxation mode analysis. We applied the new method to states with slow relaxation, which are defined by the free-energy surface obtained in the relaxation mode analysis. Finally, the relaxation times of the states obtained with a simple Markov state model and the proposed Markov state relaxation mode analysis are compared and discussed.

Probing Vitamine C, Aspirin and Paracetamol in the Gas Phase: High Resolution Rotational Studies

NASA Astrophysics Data System (ADS)

Mata, S.; Cabezas, C.; Varela, M.; Pena, I.; Nino, A.; López, J. C.; Alonso, J. L.; Grabow, J.-U.

2011-06-01

A solid sample of Vitamin C (m.p. 190°C) vaporized by laser ablation has been investigated in gas phase and characterized through their rotational spectra. Two spectroscopy techniques has been used to obtain the spectra: a new design of broadband chirped pulse Fourier transform microwave spectroscopy with in-phase/quadrature-phase-modulation passage-acquired-coherence technique (IMPACT) and conventional laser ablation molecular beam Fourier transform microwave spectroscopy (LA-MB-FTMW). Up to now, two low-energy conformer have been observed and their rotational constants determined. Ab initio calculations at the MP2/6-311++G (d,p) level of theory predicted rotational constants which helped us to identify these conformers unequivocally. Among the molecules to benefit from the LA-MB-FTMW technique there are common important drugs never observed in the gas phase through rotational spectroscopy. We present here the results on acetyl salicylic acid and acetaminophen (m.p. 136°C), commonly known as aspirin and paracetamol respectively. We have observed two stable conformers of aspirin and two for paracetamol. The internal rotation barrier of the methyl group in aspirin has been determined for both conformers from the analysis of the A-E splittings due to the coupling of internal and overall rotation. J. L. Alonso, C. Pérez, M. E. Sanz, J. C. López, S. Blanco, Phys. Chem. Chem. Phys. 11,617-627 (2009)and references therein

Using 1H and 13C NMR chemical shifts to determine cyclic peptide conformations: a combined molecular dynamics and quantum mechanics approach.

PubMed

Nguyen, Q Nhu N; Schwochert, Joshua; Tantillo, Dean J; Lokey, R Scott

2018-05-10

Solving conformations of cyclic peptides can provide insight into structure-activity and structure-property relationships, which can help in the design of compounds with improved bioactivity and/or ADME characteristics. The most common approaches for determining the structures of cyclic peptides are based on NMR-derived distance restraints obtained from NOESY or ROESY cross-peak intensities, and 3J-based dihedral restraints using the Karplus relationship. Unfortunately, these observables are often too weak, sparse, or degenerate to provide unequivocal, high-confidence solution structures, prompting us to investigate an alternative approach that relies only on 1H and 13C chemical shifts as experimental observables. This method, which we call conformational analysis from NMR and density-functional prediction of low-energy ensembles (CANDLE), uses molecular dynamics (MD) simulations to generate conformer families and density functional theory (DFT) calculations to predict their 1H and 13C chemical shifts. Iterative conformer searches and DFT energy calculations on a cyclic peptide-peptoid hybrid yielded Boltzmann ensembles whose predicted chemical shifts matched the experimental values better than any single conformer. For these compounds, CANDLE outperformed the classic NOE- and 3J-coupling-based approach by disambiguating similar β-turn types and also enabled the structural elucidation of the minor conformer. Through the use of chemical shifts, in conjunction with DFT and MD calculations, CANDLE can help illuminate conformational ensembles of cyclic peptides in solution.

Transmutation of planar media singularities in a conformal cloak.

PubMed

Liu, Yichao; Mukhtar, Musawwadah; Ma, Yungui; Ong, C K

2013-11-01

Invisibility cloaking based on optical transformation involves materials singularity at the branch cut points. Many interesting optical devices, such as the Eaton lens, also require planar media index singularities in their implementation. We show a method to transmute two singularities simultaneously into harmless topological defects formed by anisotropic permittivity and permeability tensors. Numerical simulation is performed to verify the functionality of the transmuted conformal cloak consisting of two kissing Maxwell fish eyes.

System and method for forming synthetic protein crystals to determine the conformational structure by crystallography

DOEpatents

Craig, G.D.; Glass, R.; Rupp, B.

1997-01-28

A method is disclosed for forming synthetic crystals of proteins in a carrier fluid by use of the dipole moments of protein macromolecules that self-align in the Helmholtz layer adjacent to an electrode. The voltage gradients of such layers easily exceed 10{sup 6}V/m. The synthetic protein crystals are subjected to x-ray crystallography to determine the conformational structure of the protein involved. 2 figs.

Multiplex PCR method for use in real-time PCR for identification of fish fillets from grouper (Epinephelus and Mycteroperca species) and common substitute species.

PubMed

Trotta, Michele; Schönhuth, Susana; Pepe, Tiziana; Cortesi, M Luisa; Puyet, Antonio; Bautista, José M

2005-03-23

Mitochondrial 16S rRNA sequences from morphological validated grouper (Epinephelus aeneus, E. caninus, E. costae, and E. marginatus; Mycteroperca fusca and M. rubra), Nile perch (Lates niloticus), and wreck fish (Polyprion americanus) were used to develop an analytical system for group diagnosis based on two alternative Polymerase Chain Reaction (PCR) approaches. The first includes conventional multiplex PCR in which electrophoretic migration of different sizes of bands allowed identification of the fish species. The second approach, involving real-time PCR, produced a single amplicon from each species that showed different Tm values allowing the fish groups to be directly identified. Real-time PCR allows the quick differential diagnosis of the three groups of species and high-throughput screening of multiple samples. Neither PCR system cross-reacted with DNA samples from 41 common marketed fish species, thus conforming to standards for species validation. The use of these two PCR-based methods makes it now possible to discriminate grouper from substitute fish species.

Multiple loop conformations of peptides predicted by molecular dynamics simulations are compatible with nuclear magnetic resonance.

PubMed

Carstens, Heiko; Renner, Christian; Milbradt, Alexander G; Moroder, Luis; Tavan, Paul

2005-03-29

The affinity and selectivity of protein-protein interactions can be fine-tuned by varying the size, flexibility, and amino acid composition of involved surface loops. As a model for such surface loops, we study the conformational landscape of an octapeptide, whose flexibility is chemically steered by a covalent ring closure integrating an azobenzene dye into and by a disulfide bridge additionally constraining the peptide backbone. Because the covalently integrated azobenzene dyes can be switched by light between a bent cis state and an elongated trans state, six cyclic peptide models of strongly different flexibilities are obtained. The conformational states of these peptide models are sampled by NMR and by unconstrained molecular dynamics (MD) simulations. Prototypical conformations and the free-energy landscapes in the high-dimensional space spanned by the phi/psi angles at the peptide backbone are obtained by clustering techniques from the MD trajectories. Multiple open-loop conformations are shown to be predicted by MD particularly in the very flexible cases and are shown to comply with the NMR data despite the fact that such open-loop conformations are missing in the refined NMR structures.

Slow ligand-induced conformational switch increases the catalytic rate in Plasmodium falciparum hypoxanthine guanine xanthine phosphoribosyltransferase.

PubMed

Roy, Sourav; Karmakar, Tarak; Prahlada Rao, Vasudeva S; Nagappa, Lakshmeesha K; Balasubramanian, Sundaram; Balaram, Hemalatha

2015-05-01

P. falciparum (Pf) hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) exhibits a unique mechanism of activation where the enzyme switches from a low activity (unactivated) to a high activity (activated) state upon pre-incubation with substrate/products. Xanthine phosphoribosylation by unactivated PfHGXPRT exhibits a lag phase, the duration of which reduces with an increase in concentration of the enzyme or substrate, PRPP·Mg(2+). Activated PfHGXPRT does not display the lag phase and exhibits a ten-fold drop in the Km value for PRPP·Mg(2+). These observations suggest the involvement of ligand-mediated oligomerization and conformational changes in the process of activation. The dipeptide Leu-Lys in the PPi binding site of human and T. gondii HG(X)PRT that facilitates PRPP·Mg(2+) binding by isomerization from trans to cis conformation is conserved in PfHGXPRT. Free energy calculations using the well-tempered metadynamics technique show the ligand-free enzyme to be more stable when this dipeptide is in the trans conformation than in the cis conformation. The high rotational energy barrier observed for the conformational change from experimental and computational studies permits delineation of the activation mechanism.

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.

Thermodynamic free energy methods to investigate shape transitions in bilayer membranes.

PubMed

Ramakrishnan, N; Tourdot, Richard W; Radhakrishnan, Ravi

2016-06-01

The conformational free energy landscape of a system is a fundamental thermodynamic quantity of importance particularly in the study of soft matter and biological systems, in which the entropic contributions play a dominant role. While computational methods to delineate the free energy landscape are routinely used to analyze the relative stability of conformational states, to determine phase boundaries, and to compute ligand-receptor binding energies its use in problems involving the cell membrane is limited. Here, we present an overview of four different free energy methods to study morphological transitions in bilayer membranes, induced either by the action of curvature remodeling proteins or due to the application of external forces. Using a triangulated surface as a model for the cell membrane and using the framework of dynamical triangulation Monte Carlo, we have focused on the methods of Widom insertion, thermodynamic integration, Bennett acceptance scheme, and umbrella sampling and weighted histogram analysis. We have demonstrated how these methods can be employed in a variety of problems involving the cell membrane. Specifically, we have shown that the chemical potential, computed using Widom insertion, and the relative free energies, computed using thermodynamic integration and Bennett acceptance method, are excellent measures to study the transition from curvature sensing to curvature inducing behavior of membrane associated proteins. The umbrella sampling and WHAM analysis has been used to study the thermodynamics of tether formation in cell membranes and the quantitative predictions of the computational model are in excellent agreement with experimental measurements. Furthermore, we also present a method based on WHAM and thermodynamic integration to handle problems related to end-point-catastrophe that are common in most free energy methods.

Creative Writing in the Language Arts.

ERIC Educational Resources Information Center

Ediger, Marlow

Pupils need to express themselves in creative processes and products in the language arts curriculum. Too frequently, teachers require behavior which involves conformity on the part of learners. Specific objectives many times delimit pupils' opportunities to express original ideas that come from within the involved learners. Many activities can…

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

PubMed

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

2012-01-01

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

Intramolecular interactions contributing for the conformational preference of bioactive diphenhydramine: Manifestation of the gauche effect

NASA Astrophysics Data System (ADS)

de Rezende, Fátima M. P.; Andrade, Laize A. F.; Freitas, Matheus P.

2015-08-01

Diphenhydramine is an antihistamine used to treat some symptoms of allergies and the common cold. It is usually marketed as the hydrochloride salt, and both the neutral and cation forms have the O-C-C-N fragment. The gauche effect is well known in fluorine-containing chains, because its main origin is hyperconjugative and the σ∗C-F is a low-lying acceptor orbital, allowing electron delocalization in the conformation where F and an adjacent electronegative substituent in an ethane fragment are in the gauche orientation. Our experimental (NMR) and theoretical findings indicate that diphenhydramine exhibits the gauche effect, since the preferential conformations have the O-C-C-N moiety in this orientation due especially to antiperiplanar σC-H → σ∗C-O and σC-H → σ∗C-N interactions. This conformational preference is strengthened in the protonated form due to an incremental electrostatic gauche effect. Because the gauche conformation matches the bioactive structure of diphenhydramine complexed with histamine methyltransferase, it is suggested that intramolecular interactions, and not only induced fit, rule its bioactive form.

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

Use of Phage Display to Generate Conformation-Sensor Recombinant Antibodies

PubMed Central

Haque, Aftabul; Tonks, Nicholas K.

2013-01-01

We describe a phage display approach that we have previously used to generate conformation-sensor antibodies that recognize specifically and stabilize the oxidized, inactive conformation of protein tyrosine phosphatase 1B (PTP1B). We use a solution-based panning and screening strategy conducted in the presence of reduced active PTP1B, which enriches antibodies to epitopes unique to the oxidized form, while excluding antibodies that recognize epitopes common to oxidized and reduced forms of PTP1B. This strategy avoids conventional solid-phase immobilization, with its inherent potential for denaturation of the antigen. In addition, a functional screening strategy selects scFvs directly for their capacity for both specific binding and stabilization of the target enzyme in its inactive conformation. These conformation-specific scFvs illustrate that stabilization of oxidized PTP1B is an effective strategy to inhibit PTP1B function; it is possible that this approach may be applicable to the PTP family as a whole. Using this protocol, isolation and characterization of specific scFvs from immune responsive animals should take ~6 weeks. PMID:23154784

Veterinary Safety's Conflicts in the EAEU

ERIC Educational Resources Information Center

Kalymbek, Bakytzhan; Shulanbekova, Gulmira K.; Madiyarova, Ainur S.; Mirambaeva, Gulnaz Zh.

2016-01-01

This article is devoted to the problem of veterinary safety of the countries under the Eurasian Economic Union. Animal health's measures are provided in order to prevent the entry and spread of infectious animal diseases, including common to humans and animals, as well as goods not conforming to the common veterinary and sanitary requirements.…

Witten diagrams revisited: the AdS geometry of conformal blocks

DOE PAGES

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

2016-01-25

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

Allosteric control of transcription in GntR family of transcription regulators: A structural overview.

PubMed

Jain, Deepti

2015-07-01

The GntR family of transcription regulators constitutes one of the most abundant family of transcription factors. These modulators are involved in a variety of mechanisms controlling various metabolic processes. GntR family members are typically two domain proteins with a smaller N-terminus domain (NTD) with conserved architecture of winged-helix-turn-helix (wHTH) for DNA binding and a larger C-terminus domain (CTD) or the effector binding domain which is also involved in oligomerization. Interestingly, the CTD shows structural heterogeneity depending upon the type of effector molecule that it binds and displays structural homology to various classes of proteins. Binding of the effector molecule to the CTD brings about a conformational change in the transcription factor such that its affinity for its cognate DNA sequence is altered. This review summarizes the structural information available on the members of GntR family and discusses the common features of the DNA binding and operator recognition within the family. The variation in the allosteric mechanism employed by the members of this family is also discussed. © 2015 International Union of Biochemistry and Molecular Biology.

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

Taneja, Bhupesh; Patel, Asmita; Slesarev, Alexei

Topoisomerases are involved in controlling and maintaining the topology of DNA and are present in all kingdoms of life. Unlike all other types of topoisomerases, similar type IB enzymes have only been identified in bacteria and eukarya. The only putative type IB topoisomerase in archaea is represented by Methanopyrus kandleri topoisomerase V. Despite several common functional characteristics, topoisomerase V shows no sequence similarity to other members of the same type. The structure of the 61 kDa N-terminal fragment of topoisomerase V reveals no structural similarity to other topoisomerases. Furthermore, the structure of the active site region is different, suggesting nomore » conservation in the cleavage and religation mechanism. Additionally, the active site is buried, indicating the need of a conformational change for activity. The presence of a topoisomerase in archaea with a unique structure suggests the evolution of a separate mechanism to alter DNA.« 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.

Can acyclic conformational control be achieved via a sulfur-fluorine gauche effect?

PubMed

Thiehoff, C; Holland, M C; Daniliuc, C; Houk, K N; Gilmour, R

2015-06-01

The gauche conformation of the 1,2-difluoroethane motif is known to involve stabilising hyperconjugative interactions between donor (bonding, σ C-H ) and acceptor (antibonding, σ *C-F) orbitals. This model rationalises the generic conformational preference of F-C β -C α -X systems ( φ FCCX ≈ 60°), where X is an electron deficient substituent containing a Period 2 atom. Little is known about the corresponding Period 3 systems, such as sulfur and phosphorus, where multiple oxidation states are possible. Conformational analyses of β-fluorosulfides, -sulfoxides and -sulfones are disclosed here, thus extending the scope of the fluorine gauche effect to the 3rd Period (F-C-C-S(O) n ; φ FCCS ≈ 60°). Synergy between experiment and computation has revealed that the gauche effect is only pronounced in structures bearing an electropositive vicinal sulfur atom (S + -O - , SO 2 ).

Manipulation of Origin of Life Molecules: Recognizing Single-Molecule Conformations in β-Carotene and Chlorophyll-a/β-Carotene Clusters

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

Ngo, Anh T.; Skeini, Timur; Iancu, Violeta

Carotenoids and chlorophyll are essential parts of plant leaves and are involved in photosynthesis, a vital biological process responsible for the origin of life on Earth. Here, we investigate how beta-carotene and chlorophyll-a form mixed molecular phases On a Au(111) surface using low-temperature scanning tunneling microscopy and molecular manipulation at the single-molecule level supported by density functional theory calculations. By isolating individual molecules from nanoscale molecular clusters with a scanning tunneling microscope tip, we are able to identify five beta-carotene conformations including a structure exhibiting a three-dimensional conformation. Furthermore, molecular resolution images enable direct visualization of beta-carotene/chlorophyll-a clsuters, with intimatemore » structural details highlighting how they pair: beta-carotene preferentially positions next to chlorophyll-a and induces switching of chlorophyll-a from straight to several bent tail conformations in the molecular clusters.« less

Educational Psychologists' Report-Writing: Acts of Justice?

ERIC Educational Resources Information Center

Attard, Sunaina; Mercieca, Daniela; Mercieca, Duncan P.

2016-01-01

One of the major tasks of educational psychologists is the writing of reports. Often, all involvement, assessment and intervention culminate in the production of a report. This paper explores critically the tensions involved in writing reports which are closed down in their conformity to requirements of different bodies, while looking for…

Transformations from an oblate spheroid to a plane and vice versa: The equations used in the cartographic projection program MAP2

NASA Technical Reports Server (NTRS)

Elliott, D. A.; Schwartz, A. A.

1977-01-01

The relationships between the coordinates of a point on the surface on an oblate spheroid and the coordinates of the projection of that point in several common map projections are discussed. Because several of the projections are conformal, the theory of conformally mapping an oblate spheroid to the plane is summarized. For each projection considered, the equations which map the spheroid to the plane and their inverses are given.

Mapping the conformational free energy of aspartic acid in the gas phase and in aqueous solution.

PubMed

Comitani, Federico; Rossi, Kevin; Ceriotti, Michele; Sanz, M Eugenia; Molteni, Carla

2017-04-14

The conformational free energy landscape of aspartic acid, a proteogenic amino acid involved in a wide variety of biological functions, was investigated as an example of the complexity that multiple rotatable bonds produce even in relatively simple molecules. To efficiently explore such a landscape, this molecule was studied in the neutral and zwitterionic forms, in the gas phase and in water solution, by means of molecular dynamics and the enhanced sampling method metadynamics with classical force-fields. Multi-dimensional free energy landscapes were reduced to bi-dimensional maps through the non-linear dimensionality reduction algorithm sketch-map to identify the energetically stable conformers and their interconnection paths. Quantum chemical calculations were then performed on the minimum free energy structures. Our procedure returned the low energy conformations observed experimentally in the gas phase with rotational spectroscopy [M. E. Sanz et al., Phys. Chem. Chem. Phys. 12, 3573 (2010)]. Moreover, it provided information on higher energy conformers not accessible to experiments and on the conformers in water. The comparison between different force-fields and quantum chemical data highlighted the importance of the underlying potential energy surface to accurately capture energy rankings. The combination of force-field based metadynamics, sketch-map analysis, and quantum chemical calculations was able to produce an exhaustive conformational exploration in a range of significant free energies that complements the experimental data. Similar protocols can be applied to larger peptides with complex conformational landscapes and would greatly benefit from the next generation of accurate force-fields.

Mapping the conformational free energy of aspartic acid in the gas phase and in aqueous solution

NASA Astrophysics Data System (ADS)

Comitani, Federico; Rossi, Kevin; Ceriotti, Michele; Sanz, M. Eugenia; Molteni, Carla

2017-04-01

The conformational free energy landscape of aspartic acid, a proteogenic amino acid involved in a wide variety of biological functions, was investigated as an example of the complexity that multiple rotatable bonds produce even in relatively simple molecules. To efficiently explore such a landscape, this molecule was studied in the neutral and zwitterionic forms, in the gas phase and in water solution, by means of molecular dynamics and the enhanced sampling method metadynamics with classical force-fields. Multi-dimensional free energy landscapes were reduced to bi-dimensional maps through the non-linear dimensionality reduction algorithm sketch-map to identify the energetically stable conformers and their interconnection paths. Quantum chemical calculations were then performed on the minimum free energy structures. Our procedure returned the low energy conformations observed experimentally in the gas phase with rotational spectroscopy [M. E. Sanz et al., Phys. Chem. Chem. Phys. 12, 3573 (2010)]. Moreover, it provided information on higher energy conformers not accessible to experiments and on the conformers in water. The comparison between different force-fields and quantum chemical data highlighted the importance of the underlying potential energy surface to accurately capture energy rankings. The combination of force-field based metadynamics, sketch-map analysis, and quantum chemical calculations was able to produce an exhaustive conformational exploration in a range of significant free energies that complements the experimental data. Similar protocols can be applied to larger peptides with complex conformational landscapes and would greatly benefit from the next generation of accurate force-fields.

Single strand conformation polymorphism based SNP and Indel markers for genetic mapping and synteny analysis of common bean (Phaseolus vulgaris L.)

PubMed Central

2009-01-01

Background Expressed sequence tags (ESTs) are an important source of gene-based markers such as those based on insertion-deletions (Indels) or single-nucleotide polymorphisms (SNPs). Several gel based methods have been reported for the detection of sequence variants, however they have not been widely exploited in common bean, an important legume crop of the developing world. The objectives of this project were to develop and map EST based markers using analysis of single strand conformation polymorphisms (SSCPs), to create a transcript map for common bean and to compare synteny of the common bean map with sequenced chromosomes of other legumes. Results A set of 418 EST based amplicons were evaluated for parental polymorphisms using the SSCP technique and 26% of these presented a clear conformational or size polymorphism between Andean and Mesoamerican genotypes. The amplicon based markers were then used for genetic mapping with segregation analysis performed in the DOR364 × G19833 recombinant inbred line (RIL) population. A total of 118 new marker loci were placed into an integrated molecular map for common bean consisting of 288 markers. Of these, 218 were used for synteny analysis and 186 presented homology with segments of the soybean genome with an e-value lower than 7 × 10-12. The synteny analysis with soybean showed a mosaic pattern of syntenic blocks with most segments of any one common bean linkage group associated with two soybean chromosomes. The analysis with Medicago truncatula and Lotus japonicus presented fewer syntenic regions consistent with the more distant phylogenetic relationship between the galegoid and phaseoloid legumes. Conclusion The SSCP technique is a useful and inexpensive alternative to other SNP or Indel detection techniques for saturating the common bean genetic map with functional markers that may be useful in marker assisted selection. In addition, the genetic markers based on ESTs allowed the construction of a transcript map and given their high conservation between species allowed synteny comparisons to be made to sequenced genomes. This synteny analysis may support positional cloning of target genes in common bean through the use of genomic information from these other legumes. PMID:20030833

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

PubMed

Gupta, Sebanti; Bhattacharjya, Surajit

2014-11-01

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

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

Conformational dynamics underlie the activity of the auxin-binding protein, Nt-abp1.

PubMed

David, K; Carnero-Diaz, E; Leblanc, N; Monestiez, M; Grosclaude, J; Perrot-Rechenmann, C

2001-09-14

The auxin-binding protein 1 (ABP1) has been proposed to be involved in the perception of the phytohormone at the plasma membrane. Site-directed mutagenesis was performed on highly conserved residues at the C terminus of ABP1 to investigate their relative importance in protein folding and activation of a functional response at the plasma membrane. Detailed analysis of the dynamic interaction of the wild-type ABP1 and mutated proteins with three distinct monoclonal antibodies recognizing conformation-dependent epitopes was performed by surface plasmon resonance. The influence of auxin on these interactions was also investigated. The Cys(177) as well as Asp(175) and Glu(176) were identified as critical residues for ABP1 folding and action at the plasma membrane. On the contrary, the C-terminal KDEL sequence was demonstrated not to be essential for auxin binding, interaction with the plasma membrane, or activation of the transduction cascade although it does appear to be involved in the stability of ABP1. Taken together, the results confirmed that ABP1 conformational change is the critical step for initiating the signal from the plasma membrane.

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

Techniques used to study the DNA polymerase reaction pathway

PubMed Central

Joyce, Catherine M.

2009-01-01

Summary A minimal reaction pathway for DNA polymerases was established over 20 years ago using chemical quench methods. Since that time there has been considerable interest in noncovalent steps in the reaction pathway, conformational changes involving the polymerase or its DNA substrate that may play a role in substrate specificity. Fluorescence-based assays have been devised in order to study these conformational transitions and the results obtained have added new detail to the reaction pathway. PMID:19665596

DNA-binding protects p53 from interactions with cofactors involved in transcription-independent functions

PubMed Central

Lambrughi, Matteo; De Gioia, Luca; Gervasio, Francesco Luigi; Lindorff-Larsen, Kresten; Nussinov, Ruth; Urani, Chiara; Bruschi, Maurizio; Papaleo, Elena

2016-01-01

Binding-induced conformational changes of a protein at regions distant from the binding site may play crucial roles in protein function and regulation. The p53 tumour suppressor is an example of such an allosterically regulated protein. Little is known, however, about how DNA binding can affect distal sites for transcription factors. Furthermore, the molecular details of how a local perturbation is transmitted through a protein structure are generally elusive and occur on timescales hard to explore by simulations. Thus, we employed state-of-the-art enhanced sampling atomistic simulations to unveil DNA-induced effects on p53 structure and dynamics that modulate the recruitment of cofactors and the impact of phosphorylation at Ser215. We show that DNA interaction promotes a conformational change in a region 3 nm away from the DNA binding site. Specifically, binding to DNA increases the population of an occluded minor state at this distal site by more than 4-fold, whereas phosphorylation traps the protein in its major state. In the minor conformation, the interface of p53 that binds biological partners related to p53 transcription-independent functions is not accessible. Significantly, our study reveals a mechanism of DNA-mediated protection of p53 from interactions with partners involved in the p53 transcription-independent signalling. This also suggests that conformational dynamics is tightly related to p53 signalling. PMID:27604871

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

Yu, Qianlong; Blissard, Gary W.; Liu, Tong-Xian

The Autographa californica multiple nucleopolyhedrovirus GP64 is a class III viral fusion protein. Although the post-fusion structure of GP64 has been solved, its pre-fusion structure and the detailed mechanism of conformational change are unknown. In GP64, domain V is predicted to interact with two domain I segments that flank fusion loop 2. To evaluate the significance of the amino acids involved in these interactions, we examined 24 amino acid positions that represent interacting and conserved residues within domains I and V. In several cases, substitution of a single amino acid involved in a predicted interaction disrupted membrane fusion activity, butmore » no single amino acid pair appears to be absolutely required. We identified 4 critical residues in domain V (G438, W439, T452, and T456) that are important for membrane fusion, and two residues (G438 and W439) that appear to be important for formation or stability of the pre-fusion conformation of GP64. - Highlights: • The baculovirus envelope glycoprotein GP64 is a class III viral fusion protein. • The detailed mechanism of conformational change of GP64 is unknown. • We analyzed 24 positions that might stabilize the post-fusion structure of GP64. • We identified 4 residues in domain V that were critical for membrane fusion. • Two residues are critical for formation of the pre-fusion conformation of GP64.« 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.

Insights into the Photoprotective Switch of the Major Light-harvesting Complex II (LHCII)

PubMed Central

Sunku, Kiran; de Groot, Huub. J. M.; Pandit, Anjali

2013-01-01

Light-harvesting antennae of the LHC family form transmembrane three-helix bundles of which two helices are interlocked by conserved arginine-glutamate (Arg-Glu) ion pairs that form ligation sites for chlorophylls. The antenna proteins of photosystem II have an intriguing dual function. In excess light, they can switch their conformation from a light-harvesting into a photoprotective state, in which the excess and harmful excitation energies are safely dissipated as heat. Here we applied magic angle spinning NMR and selective Arg isotope enrichment as a noninvasive method to analyze the Arg structures of the major light-harvesting complex II (LHCII). The conformations of the Arg residues that interlock helix A and B appear to be preserved in the light-harvesting and photoprotective state. Several Arg residues have very downfield-shifted proton NMR responses, indicating that they stabilize the complex by strong hydrogen bonds. For the Arg Cα chemical shifts, differences are observed between LHCII in the active, light-harvesting and in the photoprotective, quenched state. These differences are attributed to a conformational change of the Arg residue in the stromal loop region. We conclude that the interlocked helices of LHCII form a rigid core. Consequently, the LHCII conformational switch does not involve changes in A/B helix tilting but likely involves rearrangements of the loops and helical segments close to the stromal and lumenal ends. PMID:23629658

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

NASA Astrophysics Data System (ADS)

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

1995-05-01

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

Coherent Conformational Degrees of Freedom as a Structural Basis for Allosteric Communication

PubMed Central

Mitternacht, Simon; Berezovsky, Igor N.

2011-01-01

Conformational changes in allosteric regulation can to a large extent be described as motion along one or a few coherent degrees of freedom. The states involved are inherent to the protein, in the sense that they are visited by the protein also in the absence of effector ligands. Previously, we developed the measure binding leverage to find sites where ligand binding can shift the conformational equilibrium of a protein. Binding leverage is calculated for a set of motion vectors representing independent conformational degrees of freedom. In this paper, to analyze allosteric communication between binding sites, we introduce the concept of leverage coupling, based on the assumption that only pairs of sites that couple to the same conformational degrees of freedom can be allosterically connected. We demonstrate how leverage coupling can be used to analyze allosteric communication in a range of enzymes (regulated by both ligand binding and post-translational modifications) and huge molecular machines such as chaperones. Leverage coupling can be calculated for any protein structure to analyze both biological and latent catalytic and regulatory sites. PMID:22174669

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.

How cholesterol constrains glycolipid conformation for optimal recognition of Alzheimer's beta amyloid peptide (Abeta1-40).

PubMed

Yahi, Nouara; Aulas, Anaïs; Fantini, Jacques

2010-02-05

Membrane lipids play a pivotal role in the pathogenesis of Alzheimer's disease, which is associated with conformational changes, oligomerization and/or aggregation of Alzheimer's beta-amyloid (Abeta) peptides. Yet conflicting data have been reported on the respective effect of cholesterol and glycosphingolipids (GSLs) on the supramolecular assembly of Abeta peptides. The aim of the present study was to unravel the molecular mechanisms by which cholesterol modulates the interaction between Abeta(1-40) and chemically defined GSLs (GalCer, LacCer, GM1, GM3). Using the Langmuir monolayer technique, we show that Abeta(1-40) selectively binds to GSLs containing a 2-OH group in the acyl chain of the ceramide backbone (HFA-GSLs). In contrast, Abeta(1-40) did not interact with GSLs containing a nonhydroxylated fatty acid (NFA-GSLs). Cholesterol inhibited the interaction of Abeta(1-40) with HFA-GSLs, through dilution of the GSL in the monolayer, but rendered the initially inactive NFA-GSLs competent for Abeta(1-40) binding. Both crystallographic data and molecular dynamics simulations suggested that the active conformation of HFA-GSL involves a H-bond network that restricts the orientation of the sugar group of GSLs in a parallel orientation with respect to the membrane. This particular conformation is stabilized by the 2-OH group of the GSL. Correspondingly, the interaction of Abeta(1-40) with HFA-GSLs is strongly inhibited by NaF, an efficient competitor of H-bond formation. For NFA-GSLs, this is the OH group of cholesterol that constrains the glycolipid to adopt the active L-shape conformation compatible with sugar-aromatic CH-pi stacking interactions involving residue Y10 of Abeta(1-40). We conclude that cholesterol can either inhibit or facilitate membrane-Abeta interactions through fine tuning of glycosphingolipid conformation. These data shed some light on the complex molecular interplay between cell surface GSLs, cholesterol and Abeta peptides, and on the influence of this molecular ballet on Abeta-membrane interactions.

Conformational transitions in DNA polymerase I revealed by single-molecule FRET

PubMed Central

Santoso, Yusdi; Joyce, Catherine M.; Potapova, Olga; Le Reste, Ludovic; Hohlbein, Johannes; Torella, Joseph P.; Grindley, Nigel D. F.; Kapanidis, Achillefs N.

2010-01-01

The remarkable fidelity of most DNA polymerases depends on a series of early steps in the reaction pathway which allow the selection of the correct nucleotide substrate, while excluding all incorrect ones, before the enzyme is committed to the chemical step of nucleotide incorporation. The conformational transitions that are involved in these early steps are detectable with a variety of fluorescence assays and include the fingers-closing transition that has been characterized in structural studies. Using DNA polymerase I (Klenow fragment) labeled with both donor and acceptor fluorophores, we have employed single-molecule fluorescence resonance energy transfer to study the polymerase conformational transitions that precede nucleotide addition. Our experiments clearly distinguish the open and closed conformations that predominate in Pol-DNA and Pol-DNA-dNTP complexes, respectively. By contrast, the unliganded polymerase shows a broad distribution of FRET values, indicating a high degree of conformational flexibility in the protein in the absence of its substrates; such flexibility was not anticipated on the basis of the available crystallographic structures. Real-time observation of conformational dynamics showed that most of the unliganded polymerase molecules sample the open and closed conformations in the millisecond timescale. Ternary complexes formed in the presence of mismatched dNTPs or complementary ribonucleotides show unique FRET species, which we suggest are relevant to kinetic checkpoints that discriminate against these incorrect substrates. PMID:20080740

In vivo effects of metal ions on conformation and mechanical performance of silkworm silks.

PubMed

Wang, Xin; Li, Yi; Liu, Qingsong; Chen, Quanmei; Xia, Qingyou; Zhao, Ping

2017-03-01

The mechanism of silk fiber formation is of particular interest. Although in vitro evidence has shown that metal ions affect conformational transitions of silks, the in vivo effects of metal ions on silk conformations and mechanical performance are still unclear. This study explored the effects of metal ions on silk conformations and mechanical properties of silk fibers by adding K + and Cu 2+ into the silk fibroin solutions or injecting them into the silkworms. Aimed by CD analysis, FTIR analysis, and mechanical testing, the conformational and mechanical changes of the silks were estimated. By using BION Web Server, the interactions of K + and N-terminal of silk fibroin were also simulated. We presented that K + and Cu 2+ induced the conformational transitions of silk fibroin by forming β-sheet structures. Moreover, the mechanical parameters of silk fibers, such as strength, toughness and Young's modulus, were also improved after K + or Cu 2+ injection. Using BION Web Server, we found that potassium ions may have strong electrostatic interactions with the negatively charged residues. We suggest that K + and Cu 2+ play crucial roles in the conformation and mechanical performances of silks and they are involved in the silk fiber formation in vivo. Our results are helpful for clarifying the mechanism of silk fiber formation, and provide insights for modifying the mechanical properties of silk fibers. Copyright © 2016 Elsevier B.V. All rights reserved.

Proteomic analysis of common bean stem under drought stress using in-gel stable isotope labeling.

PubMed

Zadražnik, Tanja; Egge-Jacobsen, Wolfgang; Meglič, Vladimir; Šuštar-Vozlič, Jelka

2017-02-01

Drought is an abiotic stress that strongly influences plant growth, development and productivity. Proteome changes in the stem of the drought-tolerant common bean (Phaseolus vulgaris L.) cultivar Tiber have were when the plants were exposed to drought. Five-week-old plants were subjected to water deficit by withholding irrigation for 7, 12 and 17days, whereas control plants were regularly irrigated. Relative water content (RWC) of leaves, as an indicator of the degree of cell and tissue hydration, showed the highest statistically significant differences between control and drought-stressed plants after 17days of treatment, where RWC remained at 90% for control and declined to 45% for stressed plants. Plants exposed to drought for 17days and control plants at the same developmental stage were included in quantitative proteomic analysis using in-gel stable isotope labeling of proteins in combination with mass spectrometry. The quantified proteins were grouped into several functional groups, mainly into energy metabolism, photosynthesis, proteolysis, protein synthesis and proteins related to defense and stress. 70kDa heat shock protein showed the greatest increase in abundance under drought of all the proteins, suggesting its role in protecting plants against stress by re-establishing normal protein conformations and thus cellular homeostasis. The abundance of proteins involved in protein synthesis also increased under drought stress, important for recovery of damaged proteins involved in the plant cell's metabolic activities. Other important proteins in this study were related to proteolysis and folding, which are necessary for maintaining proper cellular protein homeostasis. Taken together, these results reveal the complexity of pathways involved in the drought stress response in common bean stems and enable comparison with the results of proteomic analysis of leaves, thus providing important information to further understand the biochemical and molecular mechanisms of drought response in this important legume. Copyright © 2016 Elsevier GmbH. All rights reserved.

Underlying thermodynamics of pH-dependent allostery.

PubMed

Di Russo, Natali V; Martí, Marcelo A; Roitberg, Adrian E

2014-11-13

Understanding the effects of coupling protein protonation and conformational states is critical to the development of drugs targeting pH sensors and to the rational engineering of pH switches. In this work, we address this issue by performing a comprehensive study of the pH-regulated switch from the closed to the open conformation in nitrophorin 4 (NP4) that determines its pH-dependent activity. Our calculations show that D30 is the only amino acid that has two significantly different pKas in the open and closed conformations, confirming its critical role in regulating pH-dependent behavior. In addition, we describe the free-energy landscape of the conformational change as a function of pH, obtaining accurate estimations of free-energy barriers and equilibrium constants using different methods. The underlying thermodynamic model of the switch workings suggests the possibility of tuning the observed pKa only through the conformational equilibria, keeping the same conformation-specific pKas, as evidenced by the proposed K125L mutant. Moreover, coupling between the protonation and conformational equilibria results in efficient regulation and pH-sensing around physiological pH values only for some combinations of protonation and conformational equilibrium constants, placing constraints on their possible values and leaving a narrow space for protein molecular evolution. The calculations and analysis presented here are of general applicability and provide a guide as to how more complex systems can be studied, offering insight into how pH-regulated allostery works of great value for designing drugs that target pH sensors and for rational engineering of pH switches beyond the common histidine trigger.

Conformational Equilibria in Monomeric α-Synuclein at the Single-Molecule Level

PubMed Central

Tessari, Isabella; Mammi, Stefano; Bergantino, Elisabetta; Musiani, Francesco; Brucale, Marco; Bubacco, Luigi; Samorì, Bruno

2008-01-01

Human α-Synuclein (αSyn) is a natively unfolded protein whose aggregation into amyloid fibrils is involved in the pathology of Parkinson disease. A full comprehension of the structure and dynamics of early intermediates leading to the aggregated states is an unsolved problem of essential importance to researchers attempting to decipher the molecular mechanisms of αSyn aggregation and formation of fibrils. Traditional bulk techniques used so far to solve this problem point to a direct correlation between αSyn's unique conformational properties and its propensity to aggregate, but these techniques can only provide ensemble-averaged information for monomers and oligomers alike. They therefore cannot characterize the full complexity of the conformational equilibria that trigger the aggregation process. We applied atomic force microscopy–based single-molecule mechanical unfolding methodology to study the conformational equilibrium of human wild-type and mutant αSyn. The conformational heterogeneity of monomeric αSyn was characterized at the single-molecule level. Three main classes of conformations, including disordered and “β-like” structures, were directly observed and quantified without any interference from oligomeric soluble forms. The relative abundance of the “β-like” structures significantly increased in different conditions promoting the aggregation of αSyn: the presence of Cu2+, the pathogenic A30P mutation, and high ionic strength. This methodology can explore the full conformational space of a protein at the single-molecule level, detecting even poorly populated conformers and measuring their distribution in a variety of biologically important conditions. To the best of our knowledge, we present for the first time evidence of a conformational equilibrium that controls the population of a specific class of monomeric αSyn conformers, positively correlated with conditions known to promote the formation of aggregates. A new tool is thus made available to test directly the influence of mutations and pharmacological strategies on the conformational equilibrium of monomeric αSyn. PMID:18198943

Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography

NASA Astrophysics Data System (ADS)

Stagno, J. R.; Liu, Y.; Bhandari, Y. R.; Conrad, C. E.; Panja, S.; Swain, M.; Fan, L.; Nelson, G.; Li, C.; Wendel, D. R.; White, T. A.; Coe, J. D.; Wiedorn, M. O.; Knoska, J.; Oberthuer, D.; Tuckey, R. A.; Yu, P.; Dyba, M.; Tarasov, S. G.; Weierstall, U.; Grant, T. D.; Schwieters, C. D.; Zhang, J.; Ferré-D'Amaré, A. R.; Fromme, P.; Draper, D. E.; Liang, M.; Hunter, M. S.; Boutet, S.; Tan, K.; Zuo, X.; Ji, X.; Barty, A.; Zatsepin, N. A.; Chapman, H. N.; Spence, J. C. H.; Woodson, S. A.; Wang, Y.-X.

2017-01-01

Riboswitches are structural RNA elements that are generally located in the 5‧ untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time. Here we use femtosecond X-ray free electron laser (XFEL) pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of ‘mix-and-inject’ time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.

The ion-induced folding of the hammerhead ribozyme: core sequence changes that perturb folding into the active conformation.

PubMed Central

Bassi, G S; Murchie, A I; Lilley, D M

1996-01-01

The hammerhead ribozyme undergoes an ion-dependent folding process into the active conformation. We find that the folding can be blocked at specific stages by changes of sequence or functionality within the core. In the the absence of added metal ions, the global structure of the hammerhead is extended, with a large angle subtended between stems I and II. No core sequence changes appear to alter this geometry, consistent with an unstructured core under these conditions. Upon addition of low concentrations of magnesium ions, the hammerhead folds by an association of stems II and III, to include a large angle between them. This stage is inhibited or altered by mutations within the oligopurine sequence lying between stems II and III, and folding is completely prevented by an A14G mutation. Further increase in magnesium ion concentration brings about a second stage of folding in the natural sequence hammerhead, involving a reorientation of stem I, which rotates around into the same direction of stem II. Because this transition occurs over the same range of magnesium ion concentration over which the hammerhead ribozyme becomes active, it is likely that the final conformation is most closely related to the active form of the structure. Magnesium ion-dependent folding into this conformation is prevented by changes at G5, notably removal of the 2'-hydroxyl group and replacement of the base by cytidine. The ability to dissect the folding process by means of sequence changes suggests that two separate ion-dependent stages are involved in the folding of the hammerhead ribozyme into the active conformation. PMID:8752086

pH-dependent structural change of the extracellular sensor domain of the DraK histidine kinase from Streptomyces coelicolor

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

Yeo, Kwon Joo; Kim, Eun Hye; Hwang, Eunha

2013-02-15

Highlights: ► We described the biochemical and biophysical properties of the extracellular sensory domain (ESD) of DraK histidine kinase. ► The ESD of DraK showed a reversible pH-dependent conformational change in a wide pH range. ► The E83 is an important residue for the pH-dependent conformational change. -- Abstract: Recently, the DraR/DraK (Sco3063/Sco3062) two-component system (TCS) of Streptomycescoelicolor has been reported to be involved in the differential regulation of antibiotic biosynthesis. However, it has not been shown that under which conditions and how the DraR/DraK TCS is activated to initiate the signal transduction process. Therefore, to understand the sensing mechanism,more » structural study of the sensory domain of DraK is highly required. Here, we report the biochemical and biophysical properties of the extracellular sensory domain (ESD) of DraK. We observed a reversible pH-dependent conformational change of the ESD in a pH range of 2.5–10. Size-exclusion chromatography and AUC (analytical ultracentrifugation) data indicated that the ESD is predominantly monomeric in solution and exists in equilibrium between monomer and dimer states in acidic condition. Using NMR (nuclear magnetic resonance) and CD (circular dichroism) spectroscopy, our findings suggest that the structure of the ESD at low pH is more structured than that at high pH. In particular, the glutamate at position 83 is an important residue for the pH-dependent conformational change. These results suggest that this pH-dependent conformational change of ESD may be involved in signal transduction process of DraR/DraK TCS.« less

Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography

PubMed Central

Stagno, J. R.; Liu, Y.; Bhandari, Y. R.; Conrad, C. E.; Panja, S.; Swain, M.; Fan, L.; Nelson, G.; Li, C.; Wendel, D. R.; White, T. A.; Coe, J. D.; Wiedorn, M. O.; Knoska, J.; Oberthuer, D.; Tuckey, R. A.; Yu, P.; Dyba, M.; Tarasov, S. G.; Weierstall, U.; Grant, T. D.; Schwieters, C. D.; Zhang, J.; Ferré-D’Amaré, A. R.; Fromme, P.; Draper, D. E.; Liang, M.; Hunter, M. S.; Boutet, S.; Tan, K.; Zuo, X.; Ji, X.; Barty, A.; Zatsepin, N. A.; Chapman, H. N.; Spence, J. C. H.; Woodson, S. A.; Wang, Y.-X.

2017-01-01

Riboswitches are structural RNA elements that are generally located in the 5′ untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform1–3. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time4. Here we use femtosecond X-ray free electron laser (XFEL) pulses5,6 to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of ‘mix-and-inject’ time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes. PMID:27841871

[Requirements for CE-marking of apps and wearables].

PubMed

Berensmann, Michael; Gratzfeld, Markus

2018-03-01

Depending on the intended use, apps and wearables can be medical devices. In such cases, the manufacturer has to provide evidence that the requirements stated in directive 93/42/EWG are fulfilled. Depending on the classification of the medical device, several so-called conformity assessment procedures are possible. Once the conformity assessment procedure has been finished successfully, the manufacturer attaches the CE-marking to the product. This assures that all requirements of the directive have been fulfilled and the manufacturer is therefore authorized to put the product onto the market in all member states of the European union. In this article, the possible and practical conformity assessment procedures for apps and wearables are described and their implementation is outlined.For medical devices with sufficiently high-risk classification, the manufacturer has to involve a Notified Body. For the conformity assessment procedure according to annex II, the manufacturer implements a full quality management system and compiles technical documentation. These are supervised and evaluated by Notified Body audits. Especially for startups, it is important for the development of apps and wearables to implement a quality management system early and to fulfill the regulatory requirements, for example, related to the software life-cycle model. This also includes considering accompanying processes during development like risk management, usability engineering, and clinical evaluation.Additionally, it should be pointed out, that according to the new medical device regulation almost all apps will fall at least into class IIa. Thus, the involvement of a Notified Body in the related conformity assessment procedures would be required. Apps that have already been put onto the market as class I devices, and are now upgraded to a higher class, need the approval of a notified body starting from 26 May 2020.

THE ETIOLOGY OF ACUTE UPPER RESPIRATORY INFECTION (COMMON COLD)

PubMed Central

Long, Perrin H.; Doull, James A.; Bourn, Janet M.; McComb, Emily

1931-01-01

Experimental upper respiratory infections similar to "common colds" were transmitted singly and in series through two and four passages in nine out of fifteen persons, by intransal inoculations with bacteria-free filtrates of nasopharyngeal washings obtained from individuals ill with natural "colds." These observations conform with those reported by previous workers and lend further support to the view that the incitant of the "common cold" is a filtrable virus. PMID:19869857

Structural rationale for the cross-resistance of tumor cells bearing the A399V variant of elongation factor eEF1A1 to the structurally unrelated didemnin B, ternatin, nannocystin A and ansatrienin B

NASA Astrophysics Data System (ADS)

Sánchez-Murcia, Pedro A.; Cortés-Cabrera, Álvaro; Gago, Federico

2017-10-01

At least four classes of structurally distinct natural products with potent antiproliferative activities target the translation elongation factor eEF1A1, which is best known as the G-protein that delivers amino acyl transfer RNAs (aa-tRNAs) to ribosomes during mRNA translation. We present molecular models in atomic detail that provide a common structural basis for the high-affinity binding of didemnin B, ternatin, ansatrienin B and nannocystin A to eEF1A1, as well as a rationale based on molecular dynamics results that accounts for the deleterious effect of replacing alanine 399 with valine. The proposed binding site, at the interface between domains I and III, is eminently hydrophobic and exists only in the GTP-bound conformation. Drug binding at this site is expected to disrupt neither loading of aa-tRNAs nor GTP hydrolysis but would give rise to stabilization of this particular conformational state, in consonance with reported experimental findings. The experimental solution of the three-dimensional structure of mammalian eEF1A1 has proved elusive so far and the highly homologous eEF1A2 from rabbit muscle has been crystallized and solved only as a homodimer in a GDP-bound conformation. Interestingly, in this dimeric structure the large interdomain cavity where the drugs studied are proposed to bind is occupied by a mostly hydrophobic α-helix from domain I of the same monomer. Since binding of this α-helix and any of these drugs to domain III of eEF1A(1/2) is, therefore, mutually exclusive and involves two distinct protein conformations, one intriguing possibility that emerges from our study is that the potent antiproliferative effect of these natural products may arise not only from inhibition of protein synthesis, which is the current dogma, but also from interference with some other non-canonical functions. From this standpoint, this type of drugs could be considered antagonists of eEF1A1/2 oligomerization, a hypothesis that opens up novel areas of research.

AL Amyloidosis

PubMed Central

2012-01-01

Definition of the disease AL amyloidosis results from extra-cellular deposition of fibril-forming monoclonal immunoglobulin (Ig) light chains (LC) (most commonly of lambda isotype) usually secreted by a small plasma cell clone. Most patients have evidence of isolated monoclonal gammopathy or smoldering myeloma, and the occurrence of AL amyloidosis in patients with symptomatic multiple myeloma or other B-cell lymphoproliferative disorders is unusual. The key event in the development of AL amyloidosis is the change in the secondary or tertiary structure of an abnormal monoclonal LC, which results in instable conformation. This conformational change is responsible for abnormal folding of the LC, rich in β leaves, which assemble into monomers that stack together to form amyloid fibrils. Epidemiology AL amyloidosis is the most common type of systemic amyloidois in developed countries with an estimated incidence of 9 cases/million inhabitant/year. The average age of diagnosed patients is 65 years and less than 10% of patients are under 50. Clinical description The clinical presentation is protean, because of the wide number of tissues or organs that may be affected. The most common presenting symptoms are asthenia and dyspnoea, which are poorly specific and may account for delayed diagnosis. Renal manifestations are the most frequent, affecting two thirds of patients at presentation. They are characterized by heavy proteinuria, with nephrotic syndrome and impaired renal function in half of the patients. Heart involvement, which is present at diagnosis in more than 50% of patients, leading to restrictive cardiopathy, is the most serious complication and engages prognosis. Diagnostic methods The diagnosis relies on pathological examination of an involved site showing Congo red-positive amyloid deposits, with typical apple-green birefringence under polarized light, that stain positive with an anti-LC antibody by immunohistochemistry and/or immunofluorescence. Due to the systemic nature of the disease, non-invasive biopsies such as abdominal fat aspiration should be considered before taking biopsies from involved organs, in order to reduce the risk of bleeding complications. Differential diagnosis Systemic AL amyloidosis should be distinguished from other diseases related to deposition of monoclonal LC, and from other forms of systemic amyloidosis. When pathological studies have failed to identify the nature of amyloid deposits, genetic studies should be performed to diagnose hereditary amyloidosis. Management Treatment of AL amyloidosis is based on chemotherapy, aimed at controlling the underlying plasma clone that produces amyloidogenic LC. The hematological response should be carefully checked by serial measurements of serum free LC. The association of an alkylating agent with high-dose dexamethasone has proven to be effective in two thirds of patients and is considered as the current reference treatment. New agents used in the treatment of multiple myeloma are under investigation and appear to increase hematological response rates. Symptomatic measures and supportive care is necessary in patients with organ failure. Noticeably, usual treatments for cardiac failure (i.e. calcium inhibitors, β-blockers, angiotensin converting enzyme inhibitors) are inefficient or even dangerous in patients with amyloid heart disease, that should be managed using diuretics. Amiodarone and pace maker implantation should be considered in patients with rhythm or conduction abnormalities. In selected cases, heart and kidney transplantation may be associated with prolonged patient and graft survival. Prognosis Survival in AL amyloidosis depends on the spectrum of organ involvement (amyloid heart disease being the main prognosis factor), the severity of individual organs involved and haematological response to treatment. PMID:22909024

Exosites in the substrate specificity of blood coagulation reactions.

PubMed

Bock, P E; Panizzi, P; Verhamme, I M A

2007-07-01

The specificity of blood coagulation proteinases for substrate, inhibitor, and effector recognition is mediated by exosites on the surfaces of the catalytic domains, physically separated from the catalytic site. Some thrombin ligands bind specifically to either exosite I or II, while others engage both exosites. The involvement of different, overlapping constellations of exosite residues enables binding of structurally diverse ligands. The flexibility of the thrombin structure is central to the mechanism of complex formation and the specificity of exosite interactions. Encounter complex formation is driven by electrostatic ligand-exosite interactions, followed by conformational rearrangement to a stable complex. Exosites on some zymogens are in low affinity proexosite states and are expressed concomitant with catalytic site activation. The requirement for exosite expression controls the specificity of assembly of catalytic complexes on the coagulation pathway, such as the membrane-bound factor Xa*factor Va (prothrombinase) complex, and prevents premature assembly. Substrate recognition by prothrombinase involves a two-step mechanism with initial docking of prothrombin to exosites, followed by a conformational change to engage the FXa catalytic site. Prothrombin and its activation intermediates bind prothrombinase in two alternative conformations determined by the zymogen to proteinase transition that are hypothesized to involve prothrombin (pro)exosite I interactions with FVa, which underpin the sequential activation pathway. The role of exosites as the major source of substrate specificity has stimulated development of exosite-targeted anticoagulants for treatment of thrombosis.

Chemical substitutions in the selectivity filter of potassium channels do not rule out constricted-like conformations for C-type inactivation

PubMed Central

Li, Jing; Boulanger, Eliot; Rui, Huan; Perozo, Eduardo; Roux, Benoît

2017-01-01

In many K+ channels, prolonged activating stimuli lead to a time-dependent reduction in ion conduction, a phenomenon known as C-type inactivation. X-ray structures of the KcsA channel suggest that this inactivated state corresponds to a “constricted” conformation of the selectivity filter. However, the functional significance of the constricted conformation has become a matter of debate. Functional and structural studies based on chemically modified semisynthetic KcsA channels along the selectivity filter led to the conclusion that the constricted conformation does not correspond to the C-type inactivated state. The main results supporting this view include the observation that C-type inactivation is not suppressed by a substitution of D-alanine at Gly77, even though this modification is believed to lock the selectivity filter into its conductive conformation, whereas it is suppressed following amide-to-ester backbone substitutions at Gly77 and Tyr78, even though these structure-conserving modifications are not believed to prevent the selectivity filter from adopting the constricted conformation. However, several untested assumptions about the structural and functional impact of these chemical modifications underlie these arguments. To make progress, molecular dynamics simulations based on atomic models of the KcsA channel were performed. The computational results support the notion that the constricted conformation of the selectivity filter corresponds to the functional C-type inactivated state of the KcsA. Importantly, MD simulations reveal that the semisynthetic KcsAD-ala77 channel can adopt an asymmetrical constricted-like nonconductive conformation and that the amide-to-ester backbone substitutions at Gly77 and Tyr78 perturb the hydrogen bonding involving the buried water molecules stabilizing the constricted conformation. PMID:28973956

Conformational analysis of a covalently cross-linked Watson-Crick base pair model.

PubMed

Jensen, Erik A; Allen, Benjamin D; Kishi, Yoshito; O'Leary, Daniel J

2008-11-15

Low-temperature NMR experiments and molecular modeling have been used to characterize the conformational behavior of a covalently cross-linked DNA base pair model. The data suggest that Watson-Crick or reverse Watson-Crick hydrogen bonding geometries have similar energies and can interconvert at low temperatures. This low-temperature process involves rotation about the crosslink CH(2)C(5') (psi) carbon-carbon bond, which is energetically preferred over the alternate CH(2)N(3) (phi) carbon-nitrogen bond rotation.

Accurate calculation of conformational free energy differences in explicit water: the confinement-solvation free energy approach.

PubMed

Esque, Jeremy; Cecchini, Marco

2015-04-23

The calculation of the free energy of conformation is key to understanding the function of biomolecules and has attracted significant interest in recent years. Here, we present an improvement of the confinement method that was designed for use in the context of explicit solvent MD simulations. The development involves an additional step in which the solvation free energy of the harmonically restrained conformers is accurately determined by multistage free energy perturbation simulations. As a test-case application, the newly introduced confinement/solvation free energy (CSF) approach was used to compute differences in free energy between conformers of the alanine dipeptide in explicit water. The results are in excellent agreement with reference calculations based on both converged molecular dynamics and umbrella sampling. To illustrate the general applicability of the method, conformational equilibria of met-enkephalin (5 aa) and deca-alanine (10 aa) in solution were also analyzed. In both cases, smoothly converged free-energy results were obtained in agreement with equilibrium sampling or literature calculations. These results demonstrate that the CSF method may provide conformational free-energy differences of biomolecules with small statistical errors (below 0.5 kcal/mol) and at a moderate computational cost even with a full representation of the solvent.

LIM domain protein TES changes its conformational states in different cellular compartments.

PubMed

Zhong, Yingli; Zhu, Jiaolian; Wang, Yan; Zhou, Jianlin; Ren, Kaiqun; Ding, Xiaofeng; Zhang, Jian

2009-01-01

The human TESTIN (TES) is a putative tumor suppressor and localizes to the cytoplasm as a component of focal adhesions and cell contacts. TES contains a PET domain in the NH(2)-terminus and three tandem LIM domains in the COOH-terminus. It has been hypothesized that interactions between two termini of TES might lead to a "closed" conformational state of the protein. Here, we provide evidence for different conformational states of TES. We confirmed that the NH(2)-terminus of TES can interact with its third LIM domain in the COOH-terminus by GST pull-down assays. In addition, antisera against the full-length or two truncations of TES were prepared to examine the relationship between the conformation and cellular distribution of the protein. We found that these antisera recognize different regions of TES and showed that TES is co-localised with the marker protein B23 in nucleolus, in addition to its localization in endoplasmic reticulum (ER). Furthermore, our co-immunoprecipitation (co-IP) analysis of TES and B23 demonstrated their co-existence in the same complex. Taken together, our results suggest that TES has different conformational states in different cellular compartments, and a "closed" conformational state of TES may be involved in nucleolar localization.

Pharmacoperone drugs: targeting misfolded proteins causing lysosomal storage-, ion channels-, and G protein-coupled receptors-associated conformational disorders.

PubMed

Hou, Zhi-Shuai; Ulloa-Aguirre, Alfredo; Tao, Ya-Xiong

2018-06-01

Conformational diseases are caused by structurally abnormal proteins that cannot fold properly and achieve their native conformation. Misfolded proteins frequently originate from genetic mutations that may lead to loss-of-function diseases involving a variety of structurally diverse proteins including enzymes, ion channels, and membrane receptors. Pharmacoperones are small molecules that cross the cell surface plasma membrane and reach their target proteins within the cell, serving as molecular scaffolds to stabilize the native conformation of misfolded or well-folded but destabilized proteins, to prevent their degradation and promote correct trafficking to their functional site of action. Because of their high specificity toward the target protein, pharmacoperones are currently the focus of intense investigation as therapy for several conformational diseases. Areas covered: This review summarizes data on the mechanisms leading to protein misfolding and the use of pharmacoperone drugs as an experimental approach to rescue function of distinct misfolded/misrouted proteins associated with a variety of diseases, such as lysosomal storage diseases, channelopathies, and G protein-coupled receptor misfolding diseases. Expert commentary: The fact that many misfolded proteins may retain function, offers a unique therapeutic opportunity to cure disease by directly correcting misrouting through administering pharmacoperone drugs thereby rescuing function of disease-causing, conformationally abnormal proteins.

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

PubMed

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

2016-11-22

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

Single Molecule Cluster Analysis Identifies Signature Dynamic Conformations along the Splicing Pathway

PubMed Central

Blanco, Mario R.; Martin, Joshua S.; Kahlscheuer, Matthew L.; Krishnan, Ramya; Abelson, John; Laederach, Alain; Walter, Nils G.

2016-01-01

The spliceosome is the dynamic RNA-protein machine responsible for faithfully splicing introns from precursor messenger RNAs (pre-mRNAs). Many of the dynamic processes required for the proper assembly, catalytic activation, and disassembly of the spliceosome as it acts on its pre-mRNA substrate remain poorly understood, a challenge that persists for many biomolecular machines. Here, we developed a fluorescence-based Single Molecule Cluster Analysis (SiMCAn) tool to dissect the manifold conformational dynamics of a pre-mRNA through the splicing cycle. By clustering common dynamic behaviors derived from selectively blocked splicing reactions, SiMCAn was able to identify signature conformations and dynamic behaviors of multiple ATP-dependent intermediates. In addition, it identified a conformation adopted late in splicing by a 3′ splice site mutant, invoking a mechanism for substrate proofreading. SiMCAn presents a novel framework for interpreting complex single molecule behaviors that should prove widely useful for the comprehensive analysis of a plethora of dynamic cellular machines. PMID:26414013

A phenylalanine rotameric switch for signal-state control in bacterial chemoreceptors

NASA Astrophysics Data System (ADS)

Ortega, Davi R.; Yang, Chen; Ames, Peter; Baudry, Jerome; Parkinson, John S.; Zhulin, Igor B.

2013-12-01

Bacterial chemoreceptors are widely used as a model system for elucidating the molecular mechanisms of transmembrane signalling and have provided a detailed understanding of how ligand binding by the receptor modulates the activity of its associated kinase CheA. However, the mechanisms by which conformational signals move between signalling elements within a receptor dimer and how they control kinase activity remain unknown. Here, using long molecular dynamics simulations, we show that the kinase-activating cytoplasmic tip of the chemoreceptor fluctuates between two stable conformations in a signal-dependent manner. A highly conserved residue, Phe396, appears to serve as the conformational switch, because flipping of the stacked aromatic rings of an interacting F396-F396‧ pair in the receptor homodimer takes place concomitantly with the signal-related conformational changes. We suggest that interacting aromatic residues, which are common stabilizers of protein tertiary structure, might serve as rotameric molecular switches in other biological processes as well.

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

Prescribing pattern of psychotropic medications in child psychiatric practice in a mental referral hospital in Botswana

PubMed Central

Olashore, Anthony; Ayugi, James; Opondo, Philip

2017-01-01

Introduction There is a growing preference for psycho-pharmacological therapy over non-pharmacological care. The prescription pattern and the choice of psychotropic medications vary in different settings. Whilst newer agents and rational prescribing are favored in the more specialized settings, the pattern remains unclear in less specialized units, largely due to lack of data. The aims were to conduct a treatment audit in the only mental referral hospital in Botswana, which is a non-specialized child and adolescent care setting and see how it conforms to best practice. Methods A retrospective audit which involved the extraction of socio-demographic and clinical information from the records of patients who were ≤ 17 years and seen from January 1, 2012-July 31, 2016. Results A total of 238 files were used for this report. Mean age (SD) was 12.41 (4.1) years. Of the 120 (50.4%) patients who had pharmacological intervention, only 85(70.8%) had monotherapy. The most commonly prescribed psychotropic agents were antipsychotics (40%). Off-label use of antipsychotics and polypharmacy were 31.2% and 29.2% respectively. Conclusion The level of conformity to standard practice in terms of psychotropic prescribing in our setting is consistent with the reports from developed countries where more specialized care ostensibly exists. Further studies will be necessary to determine the scope of psychotropic use. PMID:28491214

THE EARTH SYSTEM PREDICTION SUITE: Toward a Coordinated U.S. Modeling Capability

PubMed Central

Theurich, Gerhard; DeLuca, C.; Campbell, T.; Liu, F.; Saint, K.; Vertenstein, M.; Chen, J.; Oehmke, R.; Doyle, J.; Whitcomb, T.; Wallcraft, A.; Iredell, M.; Black, T.; da Silva, AM; Clune, T.; Ferraro, R.; Li, P.; Kelley, M.; Aleinov, I.; Balaji, V.; Zadeh, N.; Jacob, R.; Kirtman, B.; Giraldo, F.; McCarren, D.; Sandgathe, S.; Peckham, S.; Dunlap, R.

2017-01-01

The Earth System Prediction Suite (ESPS) is a collection of flagship U.S. weather and climate models and model components that are being instrumented to conform to interoperability conventions, documented to follow metadata standards, and made available either under open source terms or to credentialed users. The ESPS represents a culmination of efforts to create a common Earth system model architecture, and the advent of increasingly coordinated model development activities in the U.S. ESPS component interfaces are based on the Earth System Modeling Framework (ESMF), community-developed software for building and coupling models, and the National Unified Operational Prediction Capability (NUOPC) Layer, a set of ESMF-based component templates and interoperability conventions. This shared infrastructure simplifies the process of model coupling by guaranteeing that components conform to a set of technical and semantic behaviors. The ESPS encourages distributed, multi-agency development of coupled modeling systems, controlled experimentation and testing, and exploration of novel model configurations, such as those motivated by research involving managed and interactive ensembles. ESPS codes include the Navy Global Environmental Model (NavGEM), HYbrid Coordinate Ocean Model (HYCOM), and Coupled Ocean Atmosphere Mesoscale Prediction System (COAMPS®); the NOAA Environmental Modeling System (NEMS) and the Modular Ocean Model (MOM); the Community Earth System Model (CESM); and the NASA ModelE climate model and GEOS-5 atmospheric general circulation model. PMID:29568125

THE EARTH SYSTEM PREDICTION SUITE: Toward a Coordinated U.S. Modeling Capability.

PubMed

Theurich, Gerhard; DeLuca, C; Campbell, T; Liu, F; Saint, K; Vertenstein, M; Chen, J; Oehmke, R; Doyle, J; Whitcomb, T; Wallcraft, A; Iredell, M; Black, T; da Silva, A M; Clune, T; Ferraro, R; Li, P; Kelley, M; Aleinov, I; Balaji, V; Zadeh, N; Jacob, R; Kirtman, B; Giraldo, F; McCarren, D; Sandgathe, S; Peckham, S; Dunlap, R

2016-07-01

The Earth System Prediction Suite (ESPS) is a collection of flagship U.S. weather and climate models and model components that are being instrumented to conform to interoperability conventions, documented to follow metadata standards, and made available either under open source terms or to credentialed users. The ESPS represents a culmination of efforts to create a common Earth system model architecture, and the advent of increasingly coordinated model development activities in the U.S. ESPS component interfaces are based on the Earth System Modeling Framework (ESMF), community-developed software for building and coupling models, and the National Unified Operational Prediction Capability (NUOPC) Layer, a set of ESMF-based component templates and interoperability conventions. This shared infrastructure simplifies the process of model coupling by guaranteeing that components conform to a set of technical and semantic behaviors. The ESPS encourages distributed, multi-agency development of coupled modeling systems, controlled experimentation and testing, and exploration of novel model configurations, such as those motivated by research involving managed and interactive ensembles. ESPS codes include the Navy Global Environmental Model (NavGEM), HYbrid Coordinate Ocean Model (HYCOM), and Coupled Ocean Atmosphere Mesoscale Prediction System (COAMPS ® ); the NOAA Environmental Modeling System (NEMS) and the Modular Ocean Model (MOM); the Community Earth System Model (CESM); and the NASA ModelE climate model and GEOS-5 atmospheric general circulation model.

The Earth System Prediction Suite: Toward a Coordinated U.S. Modeling Capability

NASA Technical Reports Server (NTRS)

Theurich, Gerhard; DeLuca, C.; Campbell, T.; Liu, F.; Saint, K.; Vertenstein, M.; Chen, J.; Oehmke, R.; Doyle, J.; Whitcomb, T.;

Mixture models for protein structure ensembles.

PubMed

Hirsch, Michael; Habeck, Michael

2008-10-01

Protein structure ensembles provide important insight into the dynamics and function of a protein and contain information that is not captured with a single static structure. However, it is not clear a priori to what extent the variability within an ensemble is caused by internal structural changes. Additional variability results from overall translations and rotations of the molecule. And most experimental data do not provide information to relate the structures to a common reference frame. To report meaningful values of intrinsic dynamics, structural precision, conformational entropy, etc., it is therefore important to disentangle local from global conformational heterogeneity. We consider the task of disentangling local from global heterogeneity as an inference problem. We use probabilistic methods to infer from the protein ensemble missing information on reference frames and stable conformational sub-states. To this end, we model a protein ensemble as a mixture of Gaussian probability distributions of either entire conformations or structural segments. We learn these models from a protein ensemble using the expectation-maximization algorithm. Our first model can be used to find multiple conformers in a structure ensemble. The second model partitions the protein chain into locally stable structural segments or core elements and less structured regions typically found in loops. Both models are simple to implement and contain only a single free parameter: the number of conformers or structural segments. Our models can be used to analyse experimental ensembles, molecular dynamics trajectories and conformational change in proteins. The Python source code for protein ensemble analysis is available from the authors upon request.

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.

PubChem3D: conformer ensemble accuracy

PubMed Central

2013-01-01

Background PubChem is a free and publicly available resource containing substance descriptions and their associated biological activity information. PubChem3D is an extension to PubChem containing computationally-derived three-dimensional (3-D) structures of small molecules. All the tools and services that are a part of PubChem3D rely upon the quality of the 3-D conformer models. Construction of the conformer models currently available in PubChem3D involves a clustering stage to sample the conformational space spanned by the molecule. While this stage allows one to downsize the conformer models to more manageable size, it may result in a loss of the ability to reproduce experimentally determined “bioactive” conformations, for example, found for PDB ligands. This study examines the extent of this accuracy loss and considers its effect on the 3-D similarity analysis of molecules. Results The conformer models consisting of up to 100,000 conformers per compound were generated for 47,123 small molecules whose structures were experimentally determined, and the conformers in each conformer model were clustered to reduce the size of the conformer model to a maximum of 500 conformers per molecule. The accuracy of the conformer models before and after clustering was evaluated using five different measures: root-mean-square distance (RMSD), shape-optimized shape-Tanimoto (STST-opt) and combo-Tanimoto (ComboTST-opt), and color-optimized color-Tanimoto (CTCT-opt) and combo-Tanimoto (ComboTCT-opt). On average, the effect of clustering decreased the conformer model accuracy, increasing the conformer ensemble’s RMSD to the bioactive conformer (by 0.18 ± 0.12 Å), and decreasing the STST-opt, ComboTST-opt, CTCT-opt, and ComboTCT-opt scores (by 0.04 ± 0.03, 0.16 ± 0.09, 0.09 ± 0.05, and 0.15 ± 0.09, respectively). Conclusion This study shows the RMSD accuracy performance of the PubChem3D conformer models is operating as designed. In addition, the effect of PubChem3D sampling on 3-D similarity measures shows that there is a linear degradation of average accuracy with respect to molecular size and flexibility. Generally speaking, one can likely expect the worst-case minimum accuracy of 90% or more of the PubChem3D ensembles to be 0.75, 1.09, 0.43, and 1.13, in terms of STST-opt, ComboTST-opt, CTCT-opt, and ComboTCT-opt, respectively. This expected accuracy improves linearly as the molecule becomes smaller or less flexible. PMID:23289532

Conformational dynamics of a short antigenic peptide in its free and antibody bound forms gives insight into the role of β-turns in peptide immunogenicity.

PubMed

Shukla, Rashmi Tambe; Sasidhar, Yellamraju U

2015-07-01

Earlier immunological experiments with a synthetic 36-residue peptide (75-110) from Influenza hemagglutinin have been shown to elicit anti-peptide antibodies (Ab) which could cross-react with the parent protein. In this article, we have studied the conformational features of a short antigenic (Ag) peptide ((98)YPYDVPDYASLRS(110)) from Influenza hemagglutinin in its free and antibody (Ab) bound forms with molecular dynamics simulations using GROMACS package and OPLS-AA/L all-atom force field at two different temperatures (293 K and 310 K). Multiple simulations for the free Ag peptide show sampling of ordered conformations and suggest different conformational preferences of the peptide at the two temperatures. The free Ag samples a conformation crucial for Ab binding (β-turn formed by "DYAS" sequence) with greater preference at 310 K while, it samples a native-like conformation with relatively greater propensity at 293 K. The sequence "DYAS" samples β-turn conformation with greater propensity at 310 K as part of the hemagglutinin protein also. The bound Ag too samples the β-turn involving "DYAS" sequence and in addition it also samples a β-turn formed by the sequence "YPYD" at its N-terminus, which seems to be induced upon binding to the Ab. Further, the bound Ag displays conformational flexibility at both 293 K and 310 K, particularly at terminal residues. The implications of these results for peptide immunogenicity and Ag-Ab recognition are discussed. © 2015 Wiley Periodicals, Inc.

The vicinal difluoro motif: The synthesis and conformation of erythro- and threo- diastereoisomers of 1,2-difluorodiphenylethanes, 2,3-difluorosuccinic acids and their derivatives

PubMed Central

O'Hagan, David; Rzepa, Henry S; Schüler, Martin; Slawin, Alexandra MZ

2006-01-01

Background It is well established that vicinal fluorines (RCHF-CHFR) prefer to adopt a gauche rather than an anti conformation when placed along aliphatic chains. This has been particularly recognised for 1,2-difluoroethane and extends to 2,3-difluorobutane and longer alkyl chains. It follows in these latter cases that if erythro and threo vicinal difluorinated stereoisomers are compared, they will adopt different overall conformations if the fluorines prefer to be gauche in each case. This concept is explored in this paper with erythro- and threo- diastereoisomers of 2,3-difluorosuccinates. Results A synthetic route to 2,3-difluorosuccinates has been developed through erythro- and threo- 1,2-difluoro-1,2-diphenylethane which involved the oxidation of the aryl rings to generate the corresponding 2,3-difluorosuccinic acids. Ester and amide derivatives of the erythro- and threo- 2,3-difluorosuccinic acids were then prepared. The solid and solution state conformation of these compounds was assessed by X-ray crystallography and NMR. Ab initio calculations were also carried out to model the conformation of erythro- and threo- 1,2-difluoro-1,2-diphenylethane as these differed from the 2,3-difluorosuccinates. Conclusion In general the overall chain conformations of the 2,3-difluorosuccinates diastereoisomers were found to be influenced by the fluorine gauche effect. The study highlights the prospects of utilising the vicinal difluorine motif (RCHF-CHFR) as a tool for influencing the conformation of performance organic molecules and particularly tuning conformation by selecting specific diastereoisomers (erythro or threo). PMID:17014729

The vicinal difluoro motif: The synthesis and conformation of erythro- and threo- diastereoisomers of 1,2-difluorodiphenylethanes, 2,3-difluorosuccinic acids and their derivatives.

PubMed

O'Hagan, David; Rzepa, Henry S; Schüler, Martin; Slawin, Alexandra M Z

2006-10-02

It is well established that vicinal fluorines (RCHF-CHFR) prefer to adopt a gauche rather than an anti conformation when placed along aliphatic chains. This has been particularly recognised for 1,2-difluoroethane and extends to 2,3-difluorobutane and longer alkyl chains. It follows in these latter cases that if erythro and threo vicinal difluorinated stereoisomers are compared, they will adopt different overall conformations if the fluorines prefer to be gauche in each case. This concept is explored in this paper with erythro- and threo- diastereoisomers of 2,3-difluorosuccinates. A synthetic route to 2,3-difluorosuccinates has been developed through erythro- and threo- 1,2-difluoro-1,2-diphenylethane which involved the oxidation of the aryl rings to generate the corresponding 2,3-difluorosuccinic acids. Ester and amide derivatives of the erythro- and threo- 2,3-difluorosuccinic acids were then prepared. The solid and solution state conformation of these compounds was assessed by X-ray crystallography and NMR. Ab initio calculations were also carried out to model the conformation of erythro- and threo- 1,2-difluoro-1,2-diphenylethane as these differed from the 2,3-difluorosuccinates. In general the overall chain conformations of the 2,3-difluorosuccinates diastereoisomers were found to be influenced by the fluorine gauche effect. The study highlights the prospects of utilising the vicinal difluorine motif (RCHF-CHFR) as a tool for influencing the conformation of performance organic molecules and particularly tuning conformation by selecting specific diastereoisomers (erythro or threo).

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

PubMed

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

2016-08-16

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

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

PubMed Central

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

2013-01-01

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

Conformational Order in Aggregates of Conjugated Polymers

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

Jackson, Nicholas E.; Kohlstedt, Kevin L.; Savoie, Brett M.

With the abundant variety and increasing chemical complexity of conjugated poly-friers proliferating the field of organic semiconductors, it has become increasingly important to correlate the polymer molecular structure with its mesoscale conformational and morphological attributes. For instance, it is unknown which combinations of chemical moieties and periodicities predictably produce mesoscale ordering. Interestingly) not all ordered morphologies result in efficient devices. In this work we have parametrized accurate classical force-fields and used these to compute the conformational and aggregation characteristics of single strands of common conjugated polymers. Molecular dynamics trajectories are shown to reproduce experimentally observed polymeric ordering, concluding that efficientmore » organic photovoltaic devices span a range of polymer conformational classes, and suggesting that the solution-phase morphologies have far-reaching effects. Encouragingly, these simulations indicate that despite the wide-range of conformational classes present in successful devices, local molecular ordering, and not long-range crystallinity, appears to be the necessary requirement for efficient devices. Finally, we examine what makes a "good" solvent for conjugated polymers, concluding that dispersive pi-electron solvent-polymer interactions, and not the electrostatic potential of the backbone interacting with the solvent, are what primarily determine a polymer's solubility in a particular solvent, and consequently its morphological characteristics.« less

Inherent structure versus geometric metric for state space discretization.

PubMed

Liu, Hanzhong; Li, Minghai; Fan, Jue; Huo, Shuanghong

2016-05-30

Inherent structure (IS) and geometry-based clustering methods are commonly used for analyzing molecular dynamics trajectories. ISs are obtained by minimizing the sampled conformations into local minima on potential/effective energy surface. The conformations that are minimized into the same energy basin belong to one cluster. We investigate the influence of the applications of these two methods of trajectory decomposition on our understanding of the thermodynamics and kinetics of alanine tetrapeptide. We find that at the microcluster level, the IS approach and root-mean-square deviation (RMSD)-based clustering method give totally different results. Depending on the local features of energy landscape, the conformations with close RMSDs can be minimized into different minima, while the conformations with large RMSDs could be minimized into the same basin. However, the relaxation timescales calculated based on the transition matrices built from the microclusters are similar. The discrepancy at the microcluster level leads to different macroclusters. Although the dynamic models established through both clustering methods are validated approximately Markovian, the IS approach seems to give a meaningful state space discretization at the macrocluster level in terms of conformational features and kinetics. © 2016 Wiley Periodicals, Inc.

On the effects of basis set truncation and electron correlation in conformers of 2-hydroxy-acetamide

NASA Astrophysics Data System (ADS)

Szarecka, A.; Day, G.; Grout, P. J.; Wilson, S.

Ab initio quantum chemical calculations have been used to study the differences in energy between two gas phase conformers of the 2-hydroxy-acetamide molecule that possess intramolecular hydrogen bonding. In particular, rotation around the central C-C bond has been considered as a factor determining the structure of the hydrogen bond and stabilization of the conformer. Energy calculations include full geometiy optimization using both the restricted matrix Hartree-Fock model and second-order many-body perturbation theory with a number of commonly used basis sets. The basis sets employed ranged from the minimal STO-3G set to [`]split-valence' sets up to 6-31 G. The effects of polarization functions were also studied. The results display a strong basis set dependence.

Chromosomal instability mediated by non-B DNA: cruciform conformation and not DNA sequence is responsible for recurrent translocation in humans.

PubMed

Inagaki, Hidehito; Ohye, Tamae; Kogo, Hiroshi; Kato, Takema; Bolor, Hasbaira; Taniguchi, Mariko; Shaikh, Tamim H; Emanuel, Beverly S; Kurahashi, Hiroki

2009-02-01

Chromosomal aberrations have been thought to be random events. However, recent findings introduce a new paradigm in which certain DNA segments have the potential to adopt unusual conformations that lead to genomic instability and nonrandom chromosomal rearrangement. One of the best-studied examples is the palindromic AT-rich repeat (PATRR), which induces recurrent constitutional translocations in humans. Here, we established a plasmid-based model that promotes frequent intermolecular rearrangements between two PATRRs in HEK293 cells. In this model system, the proportion of PATRR plasmid that extrudes a cruciform structure correlates to the levels of rearrangement. Our data suggest that PATRR-mediated translocations are attributable to unusual DNA conformations that confer a common pathway for chromosomal rearrangements in humans.

The Potential of Chitosan and Its Derivatives in Prevention and Treatment of Age-Related Diseases

PubMed Central

Kerch, Garry

2015-01-01

Age-related, diet-related and protein conformational diseases, such as atherosclerosis, diabetes mellitus, cancer, hypercholesterolemia, cardiovascular and neurodegenerative diseases are common in the elderly population. The potential of chitosan, chitooligosaccharides and their derivatives in prevention and treatment of age-related dysfunctions is reviewed and discussed in this paper. The influence of oxidative stress, low density lipoprotein oxidation, increase of tissue stiffness, protein conformational changes, aging-associated chronic inflammation and their pathobiological significance have been considered. The chitosan-based functional food also has been reviewed. PMID:25871293

The Relationship between Clients' Conformity to Masculine Norms and Their Perceptions of Helpful Therapist Actions

ERIC Educational Resources Information Center

Owen, Jesse; Wong, Y. Joel; Rodolfa, Emil

2010-01-01

T. J. G. Tracey et al.'s (2003) common factors model derived from therapists and psychotherapy researchers has provided a parsimonious structure to inform research and practice. Accordingly, the current authors used the 14 common factor categories identified in Tracey et al.'s model as a guide to code clients' perceptions of helpful therapist…

Unbiased, scalable sampling of protein loop conformations from probabilistic priors.

PubMed

Zhang, Yajia; Hauser, Kris

2013-01-01

Protein loops are flexible structures that are intimately tied to function, but understanding loop motion and generating loop conformation ensembles remain significant computational challenges. Discrete search techniques scale poorly to large loops, optimization and molecular dynamics techniques are prone to local minima, and inverse kinematics techniques can only incorporate structural preferences in adhoc fashion. This paper presents Sub-Loop Inverse Kinematics Monte Carlo (SLIKMC), a new Markov chain Monte Carlo algorithm for generating conformations of closed loops according to experimentally available, heterogeneous structural preferences. Our simulation experiments demonstrate that the method computes high-scoring conformations of large loops (>10 residues) orders of magnitude faster than standard Monte Carlo and discrete search techniques. Two new developments contribute to the scalability of the new method. First, structural preferences are specified via a probabilistic graphical model (PGM) that links conformation variables, spatial variables (e.g., atom positions), constraints and prior information in a unified framework. The method uses a sparse PGM that exploits locality of interactions between atoms and residues. Second, a novel method for sampling sub-loops is developed to generate statistically unbiased samples of probability densities restricted by loop-closure constraints. Numerical experiments confirm that SLIKMC generates conformation ensembles that are statistically consistent with specified structural preferences. Protein conformations with 100+ residues are sampled on standard PC hardware in seconds. Application to proteins involved in ion-binding demonstrate its potential as a tool for loop ensemble generation and missing structure completion.

Unbiased, scalable sampling of protein loop conformations from probabilistic priors

PubMed Central

2013-01-01

Background Protein loops are flexible structures that are intimately tied to function, but understanding loop motion and generating loop conformation ensembles remain significant computational challenges. Discrete search techniques scale poorly to large loops, optimization and molecular dynamics techniques are prone to local minima, and inverse kinematics techniques can only incorporate structural preferences in adhoc fashion. This paper presents Sub-Loop Inverse Kinematics Monte Carlo (SLIKMC), a new Markov chain Monte Carlo algorithm for generating conformations of closed loops according to experimentally available, heterogeneous structural preferences. Results Our simulation experiments demonstrate that the method computes high-scoring conformations of large loops (>10 residues) orders of magnitude faster than standard Monte Carlo and discrete search techniques. Two new developments contribute to the scalability of the new method. First, structural preferences are specified via a probabilistic graphical model (PGM) that links conformation variables, spatial variables (e.g., atom positions), constraints and prior information in a unified framework. The method uses a sparse PGM that exploits locality of interactions between atoms and residues. Second, a novel method for sampling sub-loops is developed to generate statistically unbiased samples of probability densities restricted by loop-closure constraints. Conclusion Numerical experiments confirm that SLIKMC generates conformation ensembles that are statistically consistent with specified structural preferences. Protein conformations with 100+ residues are sampled on standard PC hardware in seconds. Application to proteins involved in ion-binding demonstrate its potential as a tool for loop ensemble generation and missing structure completion. PMID:24565175

Diffusion maps, clustering and fuzzy Markov modeling in peptide folding transitions

NASA Astrophysics Data System (ADS)

Nedialkova, Lilia V.; Amat, Miguel A.; Kevrekidis, Ioannis G.; Hummer, Gerhard

2014-09-01

Using the helix-coil transitions of alanine pentapeptide as an illustrative example, we demonstrate the use of diffusion maps in the analysis of molecular dynamics simulation trajectories. Diffusion maps and other nonlinear data-mining techniques provide powerful tools to visualize the distribution of structures in conformation space. The resulting low-dimensional representations help in partitioning conformation space, and in constructing Markov state models that capture the conformational dynamics. In an initial step, we use diffusion maps to reduce the dimensionality of the conformational dynamics of Ala5. The resulting pretreated data are then used in a clustering step. The identified clusters show excellent overlap with clusters obtained previously by using the backbone dihedral angles as input, with small—but nontrivial—differences reflecting torsional degrees of freedom ignored in the earlier approach. We then construct a Markov state model describing the conformational dynamics in terms of a discrete-time random walk between the clusters. We show that by combining fuzzy C-means clustering with a transition-based assignment of states, we can construct robust Markov state models. This state-assignment procedure suppresses short-time memory effects that result from the non-Markovianity of the dynamics projected onto the space of clusters. In a comparison with previous work, we demonstrate how manifold learning techniques may complement and enhance informed intuition commonly used to construct reduced descriptions of the dynamics in molecular conformation space.

Diffusion maps, clustering and fuzzy Markov modeling in peptide folding transitions

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

Nedialkova, Lilia V.; Amat, Miguel A.; Kevrekidis, Ioannis G., E-mail: yannis@princeton.edu, E-mail: gerhard.hummer@biophys.mpg.de

Using the helix-coil transitions of alanine pentapeptide as an illustrative example, we demonstrate the use of diffusion maps in the analysis of molecular dynamics simulation trajectories. Diffusion maps and other nonlinear data-mining techniques provide powerful tools to visualize the distribution of structures in conformation space. The resulting low-dimensional representations help in partitioning conformation space, and in constructing Markov state models that capture the conformational dynamics. In an initial step, we use diffusion maps to reduce the dimensionality of the conformational dynamics of Ala5. The resulting pretreated data are then used in a clustering step. The identified clusters show excellent overlapmore » with clusters obtained previously by using the backbone dihedral angles as input, with small—but nontrivial—differences reflecting torsional degrees of freedom ignored in the earlier approach. We then construct a Markov state model describing the conformational dynamics in terms of a discrete-time random walk between the clusters. We show that by combining fuzzy C-means clustering with a transition-based assignment of states, we can construct robust Markov state models. This state-assignment procedure suppresses short-time memory effects that result from the non-Markovianity of the dynamics projected onto the space of clusters. In a comparison with previous work, we demonstrate how manifold learning techniques may complement and enhance informed intuition commonly used to construct reduced descriptions of the dynamics in molecular conformation space.« less

Properties of polyproline II, a secondary structure element implicated in protein-protein interactions.

PubMed

Cubellis, M V; Caillez, F; Blundell, T L; Lovell, S C

2005-03-01

The polyproline II (PPII) conformation of protein backbone is an important secondary structure type. It is unusual in that, due to steric constraints, its main-chain hydrogen-bond donors and acceptors cannot easily be satisfied. It is unable to make local hydrogen bonds, in a manner similar to that of alpha-helices, and it cannot easily satisfy the hydrogen-bonding potential of neighboring residues in polyproline conformation in a manner analogous to beta-strands. Here we describe an analysis of polyproline conformations using the HOMSTRAD database of structurally aligned proteins. This allows us not only to determine amino acid propensities from a much larger database than previously but also to investigate conservation of amino acids in polyproline conformations, and the conservation of the conformation itself. Although proline is common in polyproline helices, helices without proline represent 46% of the total. No other amino acid appears to be greatly preferred; glycine and aromatic amino acids have low propensities for PPII. Accordingly, the hydrogen-bonding potential of PPII main-chain is mainly satisfied by water molecules and by other parts of the main-chain. Side-chain to main-chain interactions are mostly nonlocal. Interestingly, the increased number of nonsatisfied H-bond donors and acceptors (as compared with alpha-helices and beta-strands) makes PPII conformers well suited to take part in protein-protein interactions. Copyright 2005 Wiley-Liss, Inc.

Diffusion maps, clustering and fuzzy Markov modeling in peptide folding transitions

PubMed Central

Nedialkova, Lilia V.; Amat, Miguel A.; Kevrekidis, Ioannis G.; Hummer, Gerhard

2014-01-01

Using the helix-coil transitions of alanine pentapeptide as an illustrative example, we demonstrate the use of diffusion maps in the analysis of molecular dynamics simulation trajectories. Diffusion maps and other nonlinear data-mining techniques provide powerful tools to visualize the distribution of structures in conformation space. The resulting low-dimensional representations help in partitioning conformation space, and in constructing Markov state models that capture the conformational dynamics. In an initial step, we use diffusion maps to reduce the dimensionality of the conformational dynamics of Ala5. The resulting pretreated data are then used in a clustering step. The identified clusters show excellent overlap with clusters obtained previously by using the backbone dihedral angles as input, with small—but nontrivial—differences reflecting torsional degrees of freedom ignored in the earlier approach. We then construct a Markov state model describing the conformational dynamics in terms of a discrete-time random walk between the clusters. We show that by combining fuzzy C-means clustering with a transition-based assignment of states, we can construct robust Markov state models. This state-assignment procedure suppresses short-time memory effects that result from the non-Markovianity of the dynamics projected onto the space of clusters. In a comparison with previous work, we demonstrate how manifold learning techniques may complement and enhance informed intuition commonly used to construct reduced descriptions of the dynamics in molecular conformation space. PMID:25240340

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

PubMed

Lindström, Ida; Dogan, Jakob

2018-05-18

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

Thermodynamic and conformational analysis of the interaction between antibody binding proteins and IgG.

PubMed

Tanwar, Neetu; Munde, Manoj

2018-06-01

Studying interaction of IgG with bacterial proteins such as proA (Protein A) and proG is essential for development in the areas of drug discovery and biotechnology. Some solution studies in the past have hinted at the possibility of variable binding ratios for IgG with proA and proG. Since earlier crystallographic studies focussed mostly on monomeric complexes, the knowledge about the binding interfaces and protein conformational changes involved in multimeric complexes is scarce. In this paper, we observed that single proA molecule was able to bind to three IgG molecules (1:3, proA:IgG) in ITC accentuating the presence of conformational flexibility in proA, corroborated also by CD results. By contrast, proG binds with 1:1 stoichiometry to IgG, which also involves key structural rearrangement within the binding interface of IgG-proG complex, confirmed by fluorescence KI quenching study. It is implicit from CD and fluorescence results that IgG does not undergo any significant conformational changes, which further suggests that proA and proG dictate the phenomenon of recognition in antibody complexes. ANS as a hydrophobic probe helped in revealing the distinctive antibody binding mechanism of proA and proG. Additionally, the binding competition experiments using ITC established that proA and proG cannot bind IgG concurrently. Copyright © 2018. Published by Elsevier B.V.

DNA-binding protects p53 from interactions with cofactors involved in transcription-independent functions.

PubMed

Lambrughi, Matteo; De Gioia, Luca; Gervasio, Francesco Luigi; Lindorff-Larsen, Kresten; Nussinov, Ruth; Urani, Chiara; Bruschi, Maurizio; Papaleo, Elena

2016-11-02

Binding-induced conformational changes of a protein at regions distant from the binding site may play crucial roles in protein function and regulation. The p53 tumour suppressor is an example of such an allosterically regulated protein. Little is known, however, about how DNA binding can affect distal sites for transcription factors. Furthermore, the molecular details of how a local perturbation is transmitted through a protein structure are generally elusive and occur on timescales hard to explore by simulations. Thus, we employed state-of-the-art enhanced sampling atomistic simulations to unveil DNA-induced effects on p53 structure and dynamics that modulate the recruitment of cofactors and the impact of phosphorylation at Ser215. We show that DNA interaction promotes a conformational change in a region 3 nm away from the DNA binding site. Specifically, binding to DNA increases the population of an occluded minor state at this distal site by more than 4-fold, whereas phosphorylation traps the protein in its major state. In the minor conformation, the interface of p53 that binds biological partners related to p53 transcription-independent functions is not accessible. Significantly, our study reveals a mechanism of DNA-mediated protection of p53 from interactions with partners involved in the p53 transcription-independent signalling. This also suggests that conformational dynamics is tightly related to p53 signalling. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Stalking Higher Energy Conformers on the Potential Energy Surface of Charged Species.

PubMed

Brites, Vincent; Cimas, Alvaro; Spezia, Riccardo; Sieffert, Nicolas; Lisy, James M; Gaigeot, Marie-Pierre

2015-03-10

Combined theoretical DFT-MD and RRKM methodologies and experimental spectroscopic infrared predissociation (IRPD) strategies to map potential energy surfaces (PES) of complex ionic clusters are presented, providing lowest and high energy conformers, thresholds to isomerization, and cluster formation pathways. We believe this association not only represents a significant advance in the field of mapping minima and transition states on the PES but also directly measures dynamical pathways for the formation of structural conformers and isomers. Pathways are unraveled over picosecond (DFT-MD) and microsecond (RRKM) time scales while changing the amount of internal energy is experimentally achieved by changing the loss channel for the IRPD measurements, thus directly probing different kinetic and isomerization pathways. Demonstration is provided for Li(+)(H2O)3,4 ionic clusters. Nonstatistical formation of these ionic clusters by both direct and cascade processes, involving isomerization processes that can lead to trapping of high energy conformers along the paths due to evaporative cooling, has been unraveled.

Amyloidogenesis of Natively Unfolded Proteins

PubMed Central

Uversky, Vladimir N.

2009-01-01

Aggregation and subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. The accumulated data support the model where protein fibrillogenesis proceeds via the formation of a relatively unfolded amyloidogenic conformation, which shares many structural properties with the pre-molten globule state, a partially folded intermediate first found during the equilibrium and kinetic (un)folding studies of several globular proteins and later described as one of the structural forms of natively unfolded proteins. The flexibility of this structural form is essential for the conformational rearrangements driving the formation of the core cross-beta structure of the amyloid fibril. Obviously, molecular mechanisms describing amyloidogenesis of ordered and natively unfolded proteins are different. For ordered protein to fibrillate, its unique and rigid structure has to be destabilized and partially unfolded. On the other hand, fibrillogenesis of a natively unfolded protein involves the formation of partially folded conformation; i.e., partial folding rather than unfolding. In this review recent findings are surveyed to illustrate some unique features of the natively unfolded proteins amyloidogenesis. PMID:18537543

Selective binding of proteins on functional nanoparticles via reverse charge parity model: an in vitro study

NASA Astrophysics Data System (ADS)

Ghosh, Goutam; Panicker, Lata; Barick, K. C.

2014-03-01

The conformation of proteins absorbed on nanoparticles surface plays a crucial role in applications of nanoparticles in biomedicine. The surface protein conformation depends on several factors, namely, nature of protein-nanoparticles interaction, chemical composition of the surface of nanoparticles etc. A model of the electrostatic binding of proteins on charged surface nanoparticles has been proposed earlier (Ghosh et al 2013 Colloids Surf. B 103 267). Also, the irreversible denaturation of the protein conformation due to binding of counterions was reported. In this paper, we have used this model, involving reverse charge parity, to show selective binding of proteins on charged surface iron oxide nanoparticles (IONPs). IONPs were surface functionalized with cetylpyridinium chloride (CPC), cetyl(trimethyl)ammonium bromide (CTAB) and cetylpyridinium iodide (CPI). The effect of counterions (Cl-, Br- and I-) on protein conformation has also been investigated. Several proteins such as α-lactalbumin (ALA), β-lactoglobulin (BLG), ovalbumin (OVA), bovin serum albumin (BSA) and HEWL were chosen for this investigation.

Differential Impact of the Monovalent Ions Li+, Na+, K+, and Rb+ on DNA Conformational Properties

PubMed Central

2015-01-01

The present report demonstrates that the conformational properties of DNA in solution are sensitive to the type of monovalent ion. Results are based on the ability of a polarizable force field using the classical Drude oscillator to reproduce experimental solution X-ray scattering data more accurately than two nonpolarizable DNA models, AMBER Parmbsc0 and CHARMM36. The polarizable model is then used to calculate scattering profiles of DNA in the presence of four different monovalent salts, LiCl, NaCl, KCl, and RbCl, showing the conformational properties of DNA to vary as a function of ion type, with that effect being sequence-dependent. The primary conformational mode associated with the variations is contraction of the DNA minor groove width with decreasing cation size. These results indicate that the Drude polarizable model provides a more realistic representation of ion–DNA interactions than additive models that may lead to a new level of understanding of the physical mechanisms driving salt-mediated biological processes involving nucleic acids. PMID:25580188

Utility of the advanced chronic kidney disease patient management tools: case studies.

PubMed

Patwardhan, Meenal B; Matchar, David B; Samsa, Gregory P; Haley, William E

2008-01-01

Appropriate management of advanced chronic kidney disease (CKD) delays or limits its progression. The Advanced CKD Patient Management Toolkit was developed using a process-improvement technique to assist patient management and address CKD-specific management issues. We pilot tested the toolkit in 2 community nephrology practices, assessed the utility of individual tools, and evaluated the impact on conformance to an advanced CKD guideline through patient chart abstraction. Tool use was distinct in the 2 sites and depended on the site champion's involvement, the extent of process reconfiguration demanded by a tool, and its perceived value. Baseline conformance varied across guideline recommendations (averaged 54%). Posttrial conformance increased in all clinical areas (averaged 59%). Valuable features of the toolkit in real-world settings were its ability to: facilitate tool selection, direct implementation efforts in response to a baseline performance audit, and allow selection of tool versions and customizing them. Our results suggest that systematically created, multifaceted, and customizable tools can promote guideline conformance.

Hsp90 dependence of a kinase is determined by its conformational landscape

PubMed Central

Luo, Qi; Boczek, Edgar E.; Wang, Qi; Buchner, Johannes; Kaila, Ville R. I.

2017-01-01

Heat shock protein 90 (Hsp90) is an abundant molecular chaperone, involved in the folding and activation of 60% of the human kinome. The oncogenic tyrosine kinase v-Src is one of the most stringent client proteins of Hsp90, whereas its almost identical homolog c-Src is only weakly affected by the chaperone. Here, we perform atomistic molecular simulations and in vitro kinase assays to explore the mechanistic differences in the activation of v-Src and c-Src. While activation in c-Src is strictly controlled by ATP-binding and phosphorylation, we find that activating conformational transitions are spontaneously sampled in Hsp90-dependent Src mutants. Phosphorylation results in an enrichment of the active conformation and in an increased affinity for Hsp90. Thus, the conformational landscape of the mutated kinase is reshaped by a broken “control switch”, resulting in perturbations of long-range electrostatics, higher activity and increased Hsp90-dependence. PMID:28290541

Structural basis for gating and activation of RyR1

PubMed Central

des Georges, Amédée; Clarke, Oliver B.; Zalk, Ran; Yuan, Qi; Condon, Kendall J.; Grassucci, Robert A.; Hendrickson, Wayne A.; Marks, Andrew R.; Frank, Joachim

2016-01-01

Summary The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca2+) release channel required for skeletal muscle contraction. Here we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca2+, ATP and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca2+ alone induce conformational changes in the cytoplasmic assembly (‘priming’), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement and deformation of the S4-S5 linker, and conformational changes in the pseudo-voltage-sensor domain. PMID:27662087

Methyl group conformation and hydrogen bonds in proteins determined by neutron protein crystallography

NASA Astrophysics Data System (ADS)

Yamaguchi, Atsushi; Shibata, Kouji; Tanaka, Ichiro; Niimura, Nobuo

2009-02-01

Using 'Hydrogen and Hydration in Proteins Data Base' (HHDB) that catalogs all H atom positions in biological macromolecules and in hydration water molecules that have been determined thus far by neutron macromolecular crystallography, methyl group conformation and hydrogen bonds (H.B.) in proteins are explored. It is found that most of the methyl groups belong to the stable staggered conformation but 11% of them seemed to be close to the eclipsed conformation. And geometrical consideration has been done for H.B. involved in α-helices. 125 H.B. were identified as donors for acceptor C dbnd O in the main chain α-helix. For these H.B., it is found that co-linear H.B. were rare, that hydrogen atoms seen from acceptors C dbnd O can localize upon certain arrangements, that H.B. are not parallel to the helix axis but rather inclined to C-terminal direction, and that hydrogen atoms except water are located inside, not outside of cylinders which the backbones of α-helices form.

Scale factor duality for conformal cyclic cosmologies

NASA Astrophysics Data System (ADS)

Camara da Silva, U.; Alves Lima, A. L.; Sotkov, G. M.

2016-11-01

The scale factor duality is a symmetry of dilaton gravity which is known to lead to pre-big-bang cosmologies. A conformal time version of the scale factor duality (SFD) was recently implemented as a UV/IR symmetry between decelerated and accelerated phases of the post-big-bang evolution within Einstein gravity coupled to a scalar field. The problem investigated in the present paper concerns the employment of the conformal time SFD methods to the construction of pre-big-bang and cyclic extensions of these models. We demonstrate that each big-bang model gives rise to two qualitatively different pre-big-bang evolutions: a contraction/expansion SFD model and Penrose's Conformal Cyclic Cosmology (CCC). A few examples of SFD symmetric cyclic universes involving certain gauged Kähler sigma models minimally coupled to Einstein gravity are studied. We also describe the specific SFD features of the thermodynamics and the conditions for validity of the generalized second law in the case of Gauss-Bonnet (GB) extension of these selected CCC models.

Loop propensity of the sequence YKGQP from staphylococcal nuclease: implications for the folding of nuclease.

PubMed

Patel, Sunita; Sasidhar, Yellamraju U

2007-10-01

Recently we performed molecular dynamics (MD) simulations on the folding of the hairpin peptide DTVKLMYKGQPMTFR from staphylococcal nuclease in explicit water. We found that the peptide folds into a hairpin conformation with native and nonnative hydrogen-bonding patterns. In all the folding events observed in the folding of the hairpin peptide, loop formation involving the region YKGQP was an important event. In order to trace the origins of the loop propensity of the sequence YKGQP, we performed MD simulations on the sequence starting from extended, polyproline II and native type I' turn conformations for a total simulation length of 300 ns, using the GROMOS96 force field under constant volume and temperature (NVT) conditions. The free-energy landscape of the peptide YKGQP shows minima corresponding to loop conformation with Tyr and Pro side-chain association, turn and extended conformational forms, with modest free-energy barriers separating the minima. To elucidate the role of Gly in facilitating loop formation, we also performed MD simulations of the mutated peptide YKAQP (Gly --> Ala mutation) under similar conditions starting from polyproline II conformation for 100 ns. Two minima corresponding to bend/turn and extended conformations were observed in the free-energy landscape for the peptide YKAQP. The free-energy barrier between the minima in the free-energy landscape of the peptide YKAQP was also modest. Loop conformation is largely sampled by the YKGQP peptide, while extended conformation is largely sampled by the YKAQP peptide. We also explain why the YKGQP sequence samples type II turn conformation in these simulations, whereas the sequence as part of the hairpin peptide DTVKLMYKGQPMTFR samples type I' turn conformation both in the X-ray crystal structure and in our earlier simulations on the folding of the hairpin peptide. We discuss the implications of our results to the folding of the staphylococcal nuclease. Copyright (c) 2007 European Peptide Society and John Wiley & Sons, Ltd.

Examining Masculinity Norms, Problem Drinking, and Athletic Involvement as Predictors of Sexual Aggression in College Men

ERIC Educational Resources Information Center

Locke, Benjamin D.; Mahalik, James R.

2005-01-01

Male sexual aggression toward women is a serious social problem, particularly on college campuses. In this study, college men's sexually aggressive behavior and rape myth acceptance were examined using conformity to 11 masculine norms and 2 variables previously linked to sexual aggression: problem drinking and athletic involvement. Results…

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.

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

PubMed

van der Vaart, Arjan

2015-05-01

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

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

Illuminating the Mechanistic Roles of Enzyme Conformational Dynamics

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

Hanson, Jeffrey A.; Dunderstadt, Karl; Watkins, Lucas P.

2007-11-13

Many enzymes mold their structures to enclose substrates in their active sites such that conformational remodeling may be required during each catalytic cycle. In adenylate kinase (AK), this involves a large-amplitude rearrangement of the enzyme’s lid domain. Using our method of high-resolution single-molecule FRET, we directly followed AK’s domain movements on its catalytic time scale. To quantitatively measure the enzyme’s entire conformational distribution, we have applied maximum entropy-based methods to remove photon-counting noise from single-molecule data. This analysis shows unambiguously that AK is capable of dynamically sampling two distinct states, which correlate well with those observed by x-ray crystallography. Unexpectedly,more » the equilibrium favors the closed, active-site-forming configurations even in the absence of substrates. Our experiments further showed that interaction with substrates, rather than locking the enzyme into a compact state, restricts the spatial extent of conformational fluctuations and shifts the enzyme’s conformational equilibrium toward the closed form by increasing the closing rate of the lid. Integrating these microscopic dynamics into macroscopic kinetics allows us to model lid opening-coupled product release as the enzyme’s rate-limiting step.« less

The comparison of automated clustering algorithms for resampling representative conformer ensembles with RMSD matrix.

PubMed

Kim, Hyoungrae; Jang, Cheongyun; Yadav, Dharmendra K; Kim, Mi-Hyun

2017-03-23

The accuracy of any 3D-QSAR, Pharmacophore and 3D-similarity based chemometric target fishing models are highly dependent on a reasonable sample of active conformations. Since a number of diverse conformational sampling algorithm exist, which exhaustively generate enough conformers, however model building methods relies on explicit number of common conformers. In this work, we have attempted to make clustering algorithms, which could find reasonable number of representative conformer ensembles automatically with asymmetric dissimilarity matrix generated from openeye tool kit. RMSD was the important descriptor (variable) of each column of the N × N matrix considered as N variables describing the relationship (network) between the conformer (in a row) and the other N conformers. This approach used to evaluate the performance of the well-known clustering algorithms by comparison in terms of generating representative conformer ensembles and test them over different matrix transformation functions considering the stability. In the network, the representative conformer group could be resampled for four kinds of algorithms with implicit parameters. The directed dissimilarity matrix becomes the only input to the clustering algorithms. Dunn index, Davies-Bouldin index, Eta-squared values and omega-squared values were used to evaluate the clustering algorithms with respect to the compactness and the explanatory power. The evaluation includes the reduction (abstraction) rate of the data, correlation between the sizes of the population and the samples, the computational complexity and the memory usage as well. Every algorithm could find representative conformers automatically without any user intervention, and they reduced the data to 14-19% of the original values within 1.13 s per sample at the most. The clustering methods are simple and practical as they are fast and do not ask for any explicit parameters. RCDTC presented the maximum Dunn and omega-squared values of the four algorithms in addition to consistent reduction rate between the population size and the sample size. The performance of the clustering algorithms was consistent over different transformation functions. Moreover, the clustering method can also be applied to molecular dynamics sampling simulation results.

HPLC study on the 'history' dependence of gramicidin A conformation in phospholipid model membranes.

PubMed

Bañó, M C; Braco, L; Abad, C

1989-06-19

A novel HPLC methodology for the study of gramicidin A reconstituted in model membranes has been tested in comparison with circular dichroism data. It is shown that this chromatographic technique not only corroborates most of the recent spectroscopic results but allows one to explain them in terms of mass fractions of different actual conformational species of GA in the phospholipid assemblies. In particular, the dependence of the inserted peptide configuration on the organic solvent and other parameters involved in the 'history' of the sample preparation and handling has been analyzed by HPLC in two phospholipid model systems: small unilamellar vesicles and micelles. Moreover, a slow conformational transition of GA towards a beta 6.3-helical configuration, accelerated by heat incubation, has been also chromatographically visualized and quantitatively interpreted.

Non-degradative Ubiquitination of Protein Kinases

PubMed Central

Ball, K. Aurelia; Johnson, Jeffrey R.; Lewinski, Mary K.; Guatelli, John; Verschueren, Erik; Krogan, Nevan J.; Jacobson, Matthew P.

2016-01-01

Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well. PMID:27253329

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.

The use of time-averaged 3JHH restrained molecular dynamics (tar-MD) simulations for the conformational analysis of five-membered ring systems: methodology and applications.

PubMed

Hendrickx, Pieter M S; Corzana, Francisco; Depraetere, Stefaan; Tourwé, Dirk A; Augustyns, Koen; Martins, José C

2010-02-01

Because of its presence in many molecules of biological relevance, the conformational analysis of five-membered rings using (3)J(HH) scalar coupling data from NMR is a topic of considerable interest. Typically, conformational analysis involves the use of a well-established mathematical procedure, originally developed by de Leeuw et al., that fits two rigid conformations to the available experimental data. This so-called pseudorotation analysis approach is not without problems, however, as chemically unrealistic conformations are sometimes generated from the data. Here, we present our investigations in the use of time-averaged restrained molecular dynamics simulations as a generic tool to determine the conformations that agree with experimental (3)J(HH) scalar coupling data. For this purpose, a set of six ribose-based molecules has been used as model compounds. The influence of several modeling parameters is assessed and optimized values are proposed. The results obtained with the tar-MD approach are compared to those obtained from the two conformer fitting procedure. Interpretation of the latter is facilitated by the introduction of a fitting error analysis that allows mapping the solution space of the fitting procedure. The relative merits of both methods and the advantages that result from the use of a force field and a time-averaged restraint potential for the experimental data are discussed. When combined, both techniques allow an enhanced understanding of the molecules' conformational behavior and prevent possible overinterpretation. In view of the very reasonable computational burden of a tar-MD simulation for the systems investigated here, the approach should be generally applicable. Copyright 2009 Wiley Periodicals, Inc.

Ligand induced change of β2 adrenergic receptor from active to inactive conformation and its implication for the closed/open state of the water channel: insight from molecular dynamics simulation, free energy calculation and Markov state model analysis.

PubMed

Bai, Qifeng; Pérez-Sánchez, Horacio; Zhang, Yang; Shao, Yonghua; Shi, Danfeng; Liu, Huanxiang; Yao, Xiaojun

2014-08-14

The reported crystal structures of β2 adrenergic receptor (β2AR) reveal that the open and closed states of the water channel are correlated with the inactive and active conformations of β2AR. However, more details about the process by which the water channel states are affected by the active to inactive conformational change of β2AR remain illusive. In this work, molecular dynamics simulations are performed to study the dynamical inactive and active conformational change of β2AR induced by inverse agonist ICI 118,551. Markov state model analysis and free energy calculation are employed to explore the open and close states of the water channel. The simulation results show that inverse agonist ICI 118,551 can induce water channel opening during the conformational transition of β2AR. Markov state model (MSM) analysis proves that the energy contour can be divided into seven states. States S1, S2 and S5, which represent the active conformation of β2AR, show that the water channel is in the closed state, while states S4 and S6, which correspond to the intermediate state conformation of β2AR, indicate the water channel opens gradually. State S7, which represents the inactive structure of β2AR, corresponds to the full open state of the water channel. The opening mechanism of the water channel is involved in the ligand-induced conformational change of β2AR. These results can provide useful information for understanding the opening mechanism of the water channel and will be useful for the rational design of potent inverse agonists of β2AR.

Molecular mechanics calculations on deaminooxytocin and on deamino-arginine-vasopressin and its analogues

NASA Astrophysics Data System (ADS)

Liwo, A.; Tempczyk, A.; Grzonka, Z.

1989-01-01

The backbone conformations of the cyclic moieties of 1-[ β-mercaptopropionic acid]-oxytocin ([Mpa1]-OT), [1- β-mercaptopropionic acid]-arginine-vasopressin ([Mpa1]-AVP), [1-( β'-mercapto- β,β-cyclopentamethylene)propionic acid]-arginine-vasopressin ([Cpp1]-AVP), and [1-thiosalicylic acid]-arginine-vasopressin ([Ths1]-AVP) have been analyzed by means of molecular mechanics. In these calculations, the side chains were simulated by pseudoatoms. For the three last compounds, the calculations were also performed on the whole molecules, in order to shed light on the differences in their biological activity. Their starting conformations were obtained by attaching the acyclic tail and side chains to the lowest energy conformations of the cyclic parts. In the case of [Ths1]-AVP, however, other starting conformations were also examined, which were obtained by attaching the planar benzene ring to the lowest energy conformations of [Mpa1]-AVP. In the calculations, all the degrees of freedom were relaxed and Weiner's force field was used, the parameters required for the benzene parts of [Ths1]-AVP being determined from the experimental data available, as well as from the results of molecular dynamics calculations on the model compounds. The lowest energy conformations of [Mpa1]-AVP and [Cpp1]-AVP are similar, while [Ths1]-AVP differs from them near the disulphide region, due to the presence of a planar benzene ring. Interactions involving the charged guanidine group of arginine make, in each case, an important contribution to the conformational energy. A model description of the shapes of the oxytocin and vasopressin ring has been proposed, which is based on the cyclohexane geometry. This description is in good correlation with the energetics of the conformations corresponding to different shapes.

SIMS: A Hybrid Method for Rapid Conformational Analysis

PubMed Central

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

2013-01-01

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

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.

HMG-D is an architecture-specific protein that preferentially binds to DNA containing the dinucleotide TG.

PubMed Central

Churchill, M E; Jones, D N; Glaser, T; Hefner, H; Searles, M A; Travers, A A

1995-01-01

The high mobility group (HMG) protein HMG-D from Drosophila melanogaster is a highly abundant chromosomal protein that is closely related to the vertebrate HMG domain proteins HMG1 and HMG2. In general, chromosomal HMG domain proteins lack sequence specificity. However, using both NMR spectroscopy and standard biochemical techniques we show that binding of HMG-D to a single DNA site is sequence selective. The preferred duplex DNA binding site comprises at least 5 bp and contains the deformable dinucleotide TG embedded in A/T-rich sequences. The TG motif constitutes a common core element in the binding sites of the well-characterized sequence-specific HMG domain proteins. We show that a conserved aromatic residue in helix 1 of the HMG domain may be involved in recognition of this core sequence. In common with other HMG domain proteins HMG-D binds preferentially to DNA sites that are stably bent and underwound, therefore HMG-D can be considered an architecture-specific protein. Finally, we show that HMG-D bends DNA and may confer a superhelical DNA conformation at a natural DNA binding site in the Drosophila fushi tarazu scaffold-associated region. Images PMID:7720717

The role of conformational selection in the molecular recognition of the wild type and mutants XPA67-80 peptides by ERCC1.

PubMed

Fadda, Elisa

2015-07-01

Molecular recognition is a fundamental step in the coordination of biomolecular pathways. Understanding how recognition and binding occur between highly flexible protein domains is a complex task. The conformational selection theory provides an elegant rationalization of the recognition mechanism, especially valid in cases when unstructured protein regions are involved. The recognition of a poorly structured peptide, namely XPA67-80 , by its target receptor ERCC1, falls in this challenging study category. The microsecond molecular dynamics (MD) simulations, discussed in this work, show that the conformational propensity of the wild type XPA67-80 peptide in solution supports conformational selection as the key mechanism driving its molecular recognition by ERCC1. Moreover, all the mutations of the XPA67-80 peptide studied here cause a significant increase of its conformational disorder, relative to the wild type. Comparison to experimental data suggests that the loss of the recognized structural motifs at the microscopic time scale can contribute to the critical decrease in binding observed for one of the mutants, further substantiating the key role of conformational selection in recognition. Ultimately, because of the high sequence identity and analogy in binding, it is conceivable that the conclusions of this study on the XPA67-80 peptide also apply to the ERCC1-binding domain of the XPA protein. © 2015 Wiley Periodicals, Inc.

Discriminating binding mechanisms of an intrinsically disordered protein via a multi-state coarse-grained model

NASA Astrophysics Data System (ADS)

Knott, Michael; Best, Robert B.

2014-05-01

Many proteins undergo a conformational transition upon binding to their cognate binding partner, with intrinsically disordered proteins (IDPs) providing an extreme example in which a folding transition occurs. However, it is often not clear whether this occurs via an "induced fit" or "conformational selection" mechanism, or via some intermediate scenario. In the first case, transient encounters with the binding partner favour transitions to the bound structure before the two proteins dissociate, while in the second the bound structure must be selected from a subset of unbound structures which are in the correct state for binding, because transient encounters of the incorrect conformation with the binding partner are most likely to result in dissociation. A particularly interesting situation involves those intrinsically disordered proteins which can bind to different binding partners in different conformations. We have devised a multi-state coarse-grained simulation model which is able to capture the binding of IDPs in alternate conformations, and by applying it to the binding of nuclear coactivator binding domain (NCBD) to either ACTR or IRF-3 we are able to determine the binding mechanism. By all measures, the binding of NCBD to either binding partner appears to occur via an induced fit mechanism. Nonetheless, we also show how a scenario closer to conformational selection could arise by choosing an alternative non-binding structure for NCBD.

Conformational trapping of mismatch recognition complex MSH2/MSH3 on repair-resistant DNA loops.

PubMed

Lang, Walter H; Coats, Julie E; Majka, Jerzy; Hura, Greg L; Lin, Yuyen; Rasnik, Ivan; McMurray, Cynthia T

2011-10-18

Insertion and deletion of small heteroduplex loops are common mutations in DNA, but why some loops are prone to mutation and others are efficiently repaired is unknown. Here we report that the mismatch recognition complex, MSH2/MSH3, discriminates between a repair-competent and a repair-resistant loop by sensing the conformational dynamics of their junctions. MSH2/MSH3 binds, bends, and dissociates from repair-competent loops to signal downstream repair. Repair-resistant Cytosine-Adenine-Guanine (CAG) loops adopt a unique DNA junction that traps nucleotide-bound MSH2/MSH3, and inhibits its dissociation from the DNA. We envision that junction dynamics is an active participant and a conformational regulator of repair signaling, and governs whether a loop is removed by MSH2/MSH3 or escapes to become a precursor for mutation.

Media effects in modulating the conformational equilibrium of a model compound for tumor necrosis factor converting enzyme inhibition

NASA Astrophysics Data System (ADS)

Banchelli, Martina; Guardiani, Carlo; Sandberg, Robert B.; Menichetti, Stefano; Procacci, Piero; Caminati, Gabriella

2015-07-01

Small-molecule inhibitors of Tumor Necrosis Factor α Converting Enzyme (TACE) are a promising therapeutic tool for Rheumatoid Arthritis, Multiple Sclerosis and other autoimmune diseases. Here we report on an extensive chemical-physical analysis of the media effects in modulating the conformational landscape of MBET306, the common scaffold and a synthetic precursor of a family of recently discovered tartrate-based TACE inhibitors. The structural features of this molecule with potential pharmaceutical applications have been disclosed by interpreting extensive photophysical measurements in various solvents with the aid of enhanced sampling molecular dynamics simulations and time dependent density functional calculations. Using a combination of experimental and computational techniques, the paper provides a general protocol for studying the structure in solution of molecular systems characterized by the existence of conformational metastable states.

Mechanism of autophosphorylation of mycobacterial PknB explored by molecular dynamics simulations.

PubMed

Damle, Nikhil P; Mohanty, Debasisa

2014-07-22

Mycobacterial Ser/Thr kinase, PknB, is essential for the growth of the pathogen. Unphosphorylated PknB is catalytically inactive, and its activation requires autophosphorylation of Thr residues on the activation loop. Autophosphorylation can in principle take place via two distinct mechanisms. Intermolecular trans autophosphorylation involves dimerization and phosphorylation of the activation loop of one chain in the catalytic pocket of the other chain. On the other hand, intramolecular cis autophosphorylation involves phosphorylation of the activation loop of the kinases in its own catalytic pocket within a monomer. On the basis of the crystal structure of PknB in the front-to-front dimeric form, it is currently believed that activation of PknB involves trans autophosphorylation. However, because of the lack of coordinates of the activation loop in the crystal structures, atomic details of the conformational changes associated with activation are yet to be deciphered. Therefore, to understand the conformational transitions associated with activation via autophosphorylation, a series of explicit solvent molecular dynamics simulations with a duration of 1 μs have been performed on each of the phosphorylated and nonphosphorylated forms of the PknB catalytic domain in monomeric and dimeric states. Simulations on phosphorylated PknB revealed a differential network of crucial electrostatic and hydrophobic residues that stabilize the phosphorylated form in the active conformation. Interestingly, in our simulations on nonphosphorylated monomers, the activation loop was observed to fold into its own active site, thereby opening the novel possibility of activation through intramolecular cis autophosphorylation. Thus, our simulations suggest that autophosphorylation of PknB might also involve cis initiation followed by trans amplification as reported for other eukaryotic kinases based on recent reaction kinetics studies.

Evidence that Membrane Insertion of the Cytosolic Domain of Bcl-xL is Governed by an Electrostatic Mechanism

PubMed Central

Thuduppathy, Guruvasuthevan R.; Craig, Jeffrey W.; Schon, Victoria Kholodenko Arne; Hill, R. Blake

2006-01-01

Signals from different cellular networks are integrated at the mitochondria in the regulation of apoptosis. This integration is controlled by the Bcl-2 proteins, many of which change localization fromthe cytosol to the mitochondrial outer membrane in this regulation. For Bcl-xL, this change in localization reflects the ability to undergo a conformational change from a solution to integral membrane conformation. To characterize this conformational change, structural and thermodynamic measurements were performed in the absence and presence of lipid vesicles with Bcl-xL. A pH-dependent model is proposed for the solution to membrane conformational change that consists of three stable conformations: a solution conformation, a conformation similar to the solution conformation but anchored to the membrane by its C-terminal transmembrane domain, and a membrane conformation that is fully associated with the membrane. This model predicts that the solution to membrane conformational change is independent of the C-terminal trans-membrane domain, which is experimentally demonstrated. The conformational change is associated with changes in secondary and, especially, tertiary structure of the protein, as measured by far and near-UV circular dichroism spectroscopy, respectively. Membrane insertion was distinguished from peripheral association with the membrane by quenching of intrinsic tryptophan fluorescence by acrylamide and brominated lipids. For the cytosolic domain, the free energy of insertion ( ΔGox) into lipid vesicles was determined to be −6.5 k cal mol−1 at pH4.9 by vesicle binding experiments. To test whether electrostatic interactions were significant to this process, the salt dependence of this conformational change was measured and analyzed in terms of Gouy–Chapman theory to estimate an electrostatic contribution of ΔGoel ~−2.5 kcal mol−1 and a non-electrostatic contribution of ΔGonel ~−4.0 kcal mol−1 to the free energy of insertion, ΔGox. Calcium, which blocks ion channel activity of Bcl-xL, did not affect the solution to membrane conformational change more than predicted by these electrostatic considerations. The lipid cardiolipin, that is enriched at mitochondrial contact sites and reported to be important for the localization of Bcl-2 proteins, did not affect the solution to membrane conformational change of the cytosolic domain, suggesting that this lipid is not involved in the localization of Bcl-xL in vivo. Collectively, these data suggest the solution to membrane conformational change is controlled by an electrostatic mechanism. Given the distinct biological activities of these conformations, the possibility that this conformational change might be a regulatory checkpoint for apoptosis is discussed. PMID:16650855

The polar T1 interface is linked to conformational changes that open the voltage-gated potassium channel.

PubMed

Minor, D L; Lin, Y F; Mobley, B C; Avelar, A; Jan, Y N; Jan, L Y; Berger, J M

2000-09-01

Kv voltage-gated potassium channels share a cytoplasmic assembly domain, T1. Recent mutagenesis of two T1 C-terminal loop residues implicates T1 in channel gating. However, structural alterations of these mutants leave open the question concerning direct involvement of T1 in gating. We find in mammalian Kv1.2 that gating depends critically on residues at complementary T1 surfaces in an unusually polar interface. An isosteric mutation in this interface causes surprisingly little structural alteration while stabilizing the closed channel and increasing the stability of T1 tetramers. Replacing T1 with a tetrameric coiled-coil destabilizes the closed channel. Together, these data suggest that structural changes involving the buried polar T1 surfaces play a key role in the conformational changes leading to channel opening.

Trend-Analysis of Solid-State Structures: Low-Energy Conformational 'Reactions' Involving Directed and Coupled Movements in Half-Sandwich Compounds [CpFe(CO){C(=O)R}PPh3].

PubMed

Brunner, Henri; Tsuno, Takashi

2018-05-01

Invited for this month's cover picture are Prof. Dr. Henri Brunner from the University of Regensburg (Germany) and Prof. Dr. Takashi Tsuno from Nihon University (Japan). The cover picture shows the conformational reaction of JIDLUD→FIHTUL. The order of sample points of solid-state structures reveals information concerning low-energy, directed, and coupled movements in molecules. Read the full text of their Communication at https://doi.org/10.1002/open.201800007.

Towards vast libraries of scaffold-diverse, conformationally constrained oligomers.

PubMed

Kodadek, Thomas; McEnaney, Patrick J

2016-05-04

There is great interest in the development of probe molecules and drug leads that would bind tightly and selectively to protein surfaces that are difficult to target with traditional molecules, such as those involved in protein-protein interactions. The currently available evidence suggests that this will require molecules that are larger and have quite different chemical properties than typical Lipinski-compliant molecules that target enzyme active sites. We describe here efforts to develop vast libraries of conformationally constrained oligomers as a potentially rich source of these molecules.

Algebraic classification of Weyl anomalies in arbitrary dimensions.

PubMed

Boulanger, Nicolas

2007-06-29

Conformally invariant systems involving only dimensionless parameters are known to describe particle physics at very high energy. In the presence of an external gravitational field, the conformal symmetry may generalize to the Weyl invariance of classical massless field systems in interaction with gravity. In the quantum theory, the latter symmetry no longer survives: A Weyl anomaly appears. Anomalies are a cornerstone of quantum field theory, and, for the first time, a general, purely algebraic understanding of the universal structure of the Weyl anomalies is obtained, in arbitrary dimensions and independently of any regularization scheme.

Towards Vast Libraries of Scaffold-Diverse, Conformationally Constrained Oligomers

PubMed Central

Kodadek, Thomas; McEnaney, Patrick

2016-01-01

There is great interest in the development of probe molecules and drug leads that would bind tightly and selectively to protein surfaces that are difficult to target with traditional molecules, such as those involved in protein-protein interactions. The currently available evidence suggests that this will require molecules that are larger and have quite different chemical properties than typical Lipinski-compliant molecules that target enzyme active sites. We describe here efforts to develop vast libraries of conformationally constrained oligomers as a potentially rich source of these molecules. PMID:26996593

Soil Carbon Data: long tail recovery

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

2017-07-25

The software is intended to be part of an open source effort regarding soils data. The software provides customized data ingestion scripts for soil carbon related data sets and scripts for output databases that conform to common templates.

Effect of Ca2+ on the promiscuous target-protein binding of calmodulin

PubMed Central

Westerlund, Annie M.

2018-01-01

Calmodulin (CaM) is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling pathways. CaM has the ability to bind more than 300 different target peptides in a Ca2+-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question. Here, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca2+-bound and Ca2+-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca2+ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity. This work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity. PMID:29614072

Effect of Ca2+ on the promiscuous target-protein binding of calmodulin.

PubMed

Westerlund, Annie M; Delemotte, Lucie

2018-04-01

Calmodulin (CaM) is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling pathways. CaM has the ability to bind more than 300 different target peptides in a Ca2+-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question. Here, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca2+-bound and Ca2+-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca2+ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity. This work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity.

Interplay of non-Markov and internal friction effects in the barrier crossing kinetics of biopolymers: insights from an analytically solvable model.

PubMed

Makarov, Dmitrii E

2013-01-07

Conformational rearrangements in biomolecules (such as protein folding or enzyme-ligand binding) are often interpreted in terms of low-dimensional models of barrier crossing such as Kramers' theory. Dimensionality reduction, however, entails memory effects; as a result, the effective frictional drag force along the reaction coordinate nontrivially depends on the time scale of the transition. Moreover, when both solvent and "internal" friction effects are important, their interplay results in a highly nonlinear dependence of the effective friction on solvent viscosity that is not captured by common phenomenological models of barrier crossing. Here, these effects are illustrated using an analytically solvable toy model of an unstructured polymer chain involved in an inter- or intramolecular transition. The transition rate is calculated using the Grote-Hynes and Langer theories, which--unlike Kramers' theory--account for memory. The resulting effective frictional force exerted by the polymer along the reaction coordinate can be rationalized in terms of the effective number of monomers engaged in the transition. Faster transitions (relative to the polymer reconfiguration time scale) involve fewer monomers and, correspondingly, lower friction forces, because the polymer chain does not have enough time to reconfigure in response to the transition.

Structure of the transcriptional regulator LmrR and its mechanism of multidrug recognition.

PubMed

Madoori, Pramod Kumar; Agustiandari, Herfita; Driessen, Arnold J M; Thunnissen, Andy-Mark W H

2009-01-21

LmrR is a PadR-related transcriptional repressor that regulates the production of LmrCD, a major multidrug ABC transporter in Lactococcus lactis. Transcriptional regulation is presumed to follow a drug-sensitive induction mechanism involving the direct binding of transporter ligands to LmrR. Here, we present crystal structures of LmrR in an apo state and in two drug-bound states complexed with Hoechst 33342 and daunomycin. LmrR shows a common topology containing a typical beta-winged helix-turn-helix domain with an additional C-terminal helix involved in dimerization. Its dimeric organization is highly unusual with a flat-shaped hydrophobic pore at the dimer centre serving as a multidrug-binding site. The drugs bind in a similar manner with their aromatic rings sandwiched in between the indole groups of two dimer-related tryptophan residues. Multidrug recognition is facilitated by conformational plasticity and the absence of drug-specific hydrogen bonds. Combined analyses using site-directed mutagenesis, fluorescence-based drug binding and protein-DNA gel shift assays reveal an allosteric coupling between the multidrug- and DNA-binding sites of LmrR that most likely has a function in the induction mechanism.

On the ambiguity of conformational states: A B&S-LEUS simulation study of the helical conformations of decaalanine in water

NASA Astrophysics Data System (ADS)

Bieler, Noah S.; Hünenberger, Philippe H.

2015-04-01

Estimating the relative stabilities of different conformational states of a (bio-)molecule using molecular dynamics simulations involves two challenging problems: the conceptual problem of how to define the states of interest and the technical problem of how to properly sample these states, along with achieving a sufficient number of interconversion transitions. In this study, the two issues are addressed in the context of a decaalanine peptide in water, by considering the 310-, α-, and π-helical states. The simulations rely on the ball-and-stick local-elevation umbrella-sampling (B&S-LEUS) method. In this scheme, the states are defined as hyperspheres (balls) in a (possibly high dimensional) collective-coordinate space and connected by hypercylinders (sticks) to ensure transitions. A new object, the pipe, is also introduced here to handle curvilinear pathways. Optimal sampling within the so-defined space is ensured by confinement and (one-dimensional) memory-based biasing potentials associated with the three different kinds of objects. The simulation results are then analysed in terms of free energies using reweighting, possibly relying on two distinct sets of collective coordinates for the state definition and analysis. The four possible choices considered for these sets are Cartesian coordinates, hydrogen-bond distances, backbone dihedral angles, or pairwise sums of successive backbone dihedral angles. The results concerning decaalanine underline that the concept of conformational state may be extremely ambiguous, and that its tentative absolute definition as a free-energy basin remains subordinated to the choice of a specific analysis space. For example, within the force-field employed and depending on the analysis coordinates selected, the 310-helical state may refer to weakly overlapping collections of conformations, differing by as much as 25 kJ mol-1 in terms of free energy. As another example, the π-helical state appears to correspond to a free-energy basin for three choices of analysis coordinates, but to be unstable with the fourth one. The problem of conformational-state definition may become even more intricate when comparison with experiment is involved, where the state definition relies on spectroscopic or functional observables.

On the ambiguity of conformational states: A B&S-LEUS simulation study of the helical conformations of decaalanine in water.

PubMed

Bieler, Noah S; Hünenberger, Philippe H

2015-04-28

Estimating the relative stabilities of different conformational states of a (bio-)molecule using molecular dynamics simulations involves two challenging problems: the conceptual problem of how to define the states of interest and the technical problem of how to properly sample these states, along with achieving a sufficient number of interconversion transitions. In this study, the two issues are addressed in the context of a decaalanine peptide in water, by considering the 310-, α-, and π-helical states. The simulations rely on the ball-and-stick local-elevation umbrella-sampling (B&S-LEUS) method. In this scheme, the states are defined as hyperspheres (balls) in a (possibly high dimensional) collective-coordinate space and connected by hypercylinders (sticks) to ensure transitions. A new object, the pipe, is also introduced here to handle curvilinear pathways. Optimal sampling within the so-defined space is ensured by confinement and (one-dimensional) memory-based biasing potentials associated with the three different kinds of objects. The simulation results are then analysed in terms of free energies using reweighting, possibly relying on two distinct sets of collective coordinates for the state definition and analysis. The four possible choices considered for these sets are Cartesian coordinates, hydrogen-bond distances, backbone dihedral angles, or pairwise sums of successive backbone dihedral angles. The results concerning decaalanine underline that the concept of conformational state may be extremely ambiguous, and that its tentative absolute definition as a free-energy basin remains subordinated to the choice of a specific analysis space. For example, within the force-field employed and depending on the analysis coordinates selected, the 310-helical state may refer to weakly overlapping collections of conformations, differing by as much as 25 kJ mol(-1) in terms of free energy. As another example, the π-helical state appears to correspond to a free-energy basin for three choices of analysis coordinates, but to be unstable with the fourth one. The problem of conformational-state definition may become even more intricate when comparison with experiment is involved, where the state definition relies on spectroscopic or functional observables.

Conformational Space and Stability of ETD Charge Reduction Products of Ubiquitin

NASA Astrophysics Data System (ADS)

Lermyte, Frederik; Łącki, Mateusz Krzysztof; Valkenborg, Dirk; Gambin, Anna; Sobott, Frank

2017-01-01

Owing to its versatility, electron transfer dissociation (ETD) has become one of the most commonly utilized fragmentation techniques in both native and non-native top-down mass spectrometry. However, several competing reactions—primarily different forms of charge reduction—occur under ETD conditions, as evidenced by the distorted isotope patterns usually observed. In this work, we analyze these isotope patterns to compare the stability of nondissociative electron transfer (ETnoD) products, specifically noncovalent c/ z fragment complexes, across a range of ubiquitin conformational states. Using ion mobility, we find that more extended states are more prone to fragment release. We obtain evidence that for a given charge state, populations of ubiquitin ions formed either directly by electrospray ionization or through collapse of more extended states upon charge reduction, span a similar range of collision cross-sections. Products of gas-phase collapse are, however, less stabilized towards unfolding than the native conformation, indicating that the ions retain a memory of previous conformational states. Furthermore, this collapse of charge-reduced ions is promoted if the ions are `preheated' using collisional activation, with possible implications for the kinetics of gas-phase compaction.

Inherent Structure versus Geometric Metric for State Space Discretization

PubMed Central

Liu, Hanzhong; Li, Minghai; Fan, Jue; Huo, Shuanghong

2016-01-01

Inherent structure (IS) and geometry-based clustering methods are commonly used for analyzing molecular dynamics trajectories. ISs are obtained by minimizing the sampled conformations into local minima on potential/effective energy surface. The conformations that are minimized into the same energy basin belong to one cluster. We investigate the influence of the applications of these two methods of trajectory decomposition on our understanding of the thermodynamics and kinetics of alanine tetrapeptide. We find that at the micro cluster level, the IS approach and root-mean-square deviation (RMSD) based clustering method give totally different results. Depending on the local features of energy landscape, the conformations with close RMSDs can be minimized into different minima, while the conformations with large RMSDs could be minimized into the same basin. However, the relaxation timescales calculated based on the transition matrices built from the micro clusters are similar. The discrepancy at the micro cluster level leads to different macro clusters. Although the dynamic models established through both clustering methods are validated approximately Markovian, the IS approach seems to give a meaningful state space discretization at the macro cluster level. PMID:26915811

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.

Controlling the Conformational Energy of a Phenyl Group by Tuning the Strength of a Nonclassical CH···O Hydrogen Bond: The Case of 5-Phenyl-1,3-dioxane.

PubMed

Bailey, William F; Lambert, Kyle M; Stempel, Zachary D; Wiberg, Kenneth B; Mercado, Brandon Q

2016-12-16

Anancomeric 5-phenyl-1,3-dioxanes provide a unique opportunity to study factors that control conformation. Whereas one might expect an axial phenyl group at C(5) of 1,3-dioxane to adopt a conformation similar to that in axial phenylcyclohexane, a series of studies including X-ray crystallography, NOE measurements, and DFT calculations demonstrate that the phenyl prefers to lie over the dioxane ring in order to position an ortho-hydrogen to participate in a stabilizing, nonclassical CH···O hydrogen bond with a ring oxygen of the dioxane. Acid-catalyzed equilibration of a series of anancomeric 2-tert-butyl-5-aryl-1,3-dioxane isomers demonstrates that remote substituents on the phenyl ring affect the conformational energy of a 5-aryl-1,3-dioxane: electron-withdrawing substituents decrease the conformational energy of the aryl group, while electron-donating substituents increase the conformational energy of the group. This effect is correlated in a very linear way to Hammett substituent parameters. In short, the strength of the CH···O hydrogen bond may be tuned in a predictable way in response to the electron-withdrawing or electron-donating ability of substituents positioned remotely on the aryl ring. This effect may be profound: a 3,5-bis-CF 3 phenyl group at C(5) in 1,3-dioxane displays a pronounced preference for the axial orientation. The results are relevant to broader conformational issues involving heterocyclic systems bearing aryl substituents.

49 CFR 599.303 - Agency disposition of dealer application for reimbursement.

Code of Federal Regulations, 2010 CFR

2010-10-01

... correct a non-conforming submission. (d) Electronic rejection. An application is automatically rejected... transaction, or identifies the vehicle identification number of a new or trade-in vehicle that was involved in...

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

Pathophysiological condition changes the conformation of a flexible FBG-related protein, switching it from pathogen-recognition to host-interaction.

PubMed

Zhang, Jing; Yang, Lifeng; Anand, Ganesh Srinivasan; Ho, Bow; Ding, Jeak Ling

2011-10-01

Although homeostatic disturbance of the blood pH and calcium in the vicinity of tissue injury/malignancy/local infection seems subtle, it can cause substantial pathophysiological consequences, a phenomenon which has remained largely unexplored. The fibrinogen-related proteins (FREPs) containing fibrinogen-like domain (FBG) represent a conserved protein family with a common calcium-binding region, implying the presence of elements responsive to physiological perturbation. Here, we studied the molecular interaction between a representative FREP, the M-ficolin, and an acute phase blood protein, the C-reactive protein (CRP), both of which are known to trigger and control seminal pathways in infection and injury. Using hydrogen-deuterium exchange mass spectrometry, we showed that the C-terminal region of M-ficolin FBG underwent dramatic conformational change upon pH and calcium perturbations. Biochemical and biophysical assays showed that under defined pathophysiological condition (pH 6.5, 2.0 mM calcium), the FBG:CRP interaction occurred more strongly compared to that under physiological condition (pH 7.4, 2.5 mM calcium). We identified the binding interface between CRP and FBG, locating it to the pH- and calcium-sensitive C-terminal region of FBG. By site-directed mutagenesis, we determined H284 in the N-acetylglucosamine (GlcNAc)-binding pocket of the FBG, to be the critical CRP-binding residue. This conformational switch involving H284, explains how the pathophysiologically-driven FBG:CRP interaction diverts the M-ficolin away from GlcNAc/pathogen-recognition to host protein-protein interaction, thus enabling the host to regain homeostatic control. Our elucidation of the binding interface at the flexible FBG domain provides insights into the bioactive centre of the M-ficolin, and possibly other FREPs, which might aid future development of immunomodulators. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Computational Modeling of Allosteric Regulation in the Hsp90 Chaperones: A Statistical Ensemble Analysis of Protein Structure Networks and Allosteric Communications

PubMed Central

Blacklock, Kristin; Verkhivker, Gennady M.

2014-01-01

A fundamental role of the Hsp90 chaperone in regulating functional activity of diverse protein clients is essential for the integrity of signaling networks. In this work we have combined biophysical simulations of the Hsp90 crystal structures with the protein structure network analysis to characterize the statistical ensemble of allosteric interaction networks and communication pathways in the Hsp90 chaperones. We have found that principal structurally stable communities could be preserved during dynamic changes in the conformational ensemble. The dominant contribution of the inter-domain rigidity to the interaction networks has emerged as a common factor responsible for the thermodynamic stability of the active chaperone form during the ATPase cycle. Structural stability analysis using force constant profiling of the inter-residue fluctuation distances has identified a network of conserved structurally rigid residues that could serve as global mediating sites of allosteric communication. Mapping of the conformational landscape with the network centrality parameters has demonstrated that stable communities and mediating residues may act concertedly with the shifts in the conformational equilibrium and could describe the majority of functionally significant chaperone residues. The network analysis has revealed a relationship between structural stability, global centrality and functional significance of hotspot residues involved in chaperone regulation. We have found that allosteric interactions in the Hsp90 chaperone may be mediated by modules of structurally stable residues that display high betweenness in the global interaction network. The results of this study have suggested that allosteric interactions in the Hsp90 chaperone may operate via a mechanism that combines rapid and efficient communication by a single optimal pathway of structurally rigid residues and more robust signal transmission using an ensemble of suboptimal multiple communication routes. This may be a universal requirement encoded in protein structures to balance the inherent tension between resilience and efficiency of the residue interaction networks. PMID:24922508

Computational modeling of allosteric regulation in the hsp90 chaperones: a statistical ensemble analysis of protein structure networks and allosteric communications.

PubMed

Blacklock, Kristin; Verkhivker, Gennady M

2014-06-01

A fundamental role of the Hsp90 chaperone in regulating functional activity of diverse protein clients is essential for the integrity of signaling networks. In this work we have combined biophysical simulations of the Hsp90 crystal structures with the protein structure network analysis to characterize the statistical ensemble of allosteric interaction networks and communication pathways in the Hsp90 chaperones. We have found that principal structurally stable communities could be preserved during dynamic changes in the conformational ensemble. The dominant contribution of the inter-domain rigidity to the interaction networks has emerged as a common factor responsible for the thermodynamic stability of the active chaperone form during the ATPase cycle. Structural stability analysis using force constant profiling of the inter-residue fluctuation distances has identified a network of conserved structurally rigid residues that could serve as global mediating sites of allosteric communication. Mapping of the conformational landscape with the network centrality parameters has demonstrated that stable communities and mediating residues may act concertedly with the shifts in the conformational equilibrium and could describe the majority of functionally significant chaperone residues. The network analysis has revealed a relationship between structural stability, global centrality and functional significance of hotspot residues involved in chaperone regulation. We have found that allosteric interactions in the Hsp90 chaperone may be mediated by modules of structurally stable residues that display high betweenness in the global interaction network. The results of this study have suggested that allosteric interactions in the Hsp90 chaperone may operate via a mechanism that combines rapid and efficient communication by a single optimal pathway of structurally rigid residues and more robust signal transmission using an ensemble of suboptimal multiple communication routes. This may be a universal requirement encoded in protein structures to balance the inherent tension between resilience and efficiency of the residue interaction networks.

Synthesis of Continuous Conductive PEDOT:PSS Nanofibers by Electrospinning: A Conformal Coating for Optoelectronics.

PubMed

Bessaire, Bastien; Mathieu, Maillard; Salles, Vincent; Yeghoyan, Taguhi; Celle, Caroline; Simonato, Jean-Pierre; Brioude, Arnaud

2017-01-11

A process to synthesize continuous conducting nanofibers were developed using PEDOT:PSS as a conducting polymer and an electrospinning method. Experimental parameters were carefully explored to achieve reproducible conductive nanofibers synthesis in large quantities. In particular, relative humidity during the electrospinning process was proven to be of critical importance, as well as doping post-treatment involving glycols and alcohols. The synthesized fibers were assembled as a mat on glass substrates, forming a conductive and transparent electrode and their optoelectronic have been fully characterized. This method produces a conformable conductive and transparent coating that is well-adapted to nonplanar surfaces, having very large aspect ratio features. A demonstration of this property was made using surfaces having deep trenches and high steps, where conventional transparent conductive materials fail because of a lack of conformability.

Modern Radiation Therapy for Hodgkin Lymphoma: Field and Dose Guidelines From the International Lymphoma Radiation Oncology Group (ILROG)

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

Specht, Lena, E-mail: lena.specht@regionh.dk; Yahalom, Joachim; Illidge, Tim

2014-07-15

Radiation therapy (RT) is the most effective single modality for local control of Hodgkin lymphoma (HL) and an important component of therapy for many patients. These guidelines have been developed to address the use of RT in HL in the modern era of combined modality treatment. The role of reduced volumes and doses is addressed, integrating modern imaging with 3-dimensional (3D) planning and advanced techniques of treatment delivery. The previously applied extended field (EF) and original involved field (IF) techniques, which treated larger volumes based on nodal stations, have now been replaced by the use of limited volumes, based solelymore » on detectable nodal (and extranodal extension) involvement at presentation, using contrast-enhanced computed tomography, positron emission tomography/computed tomography, magnetic resonance imaging, or a combination of these techniques. The International Commission on Radiation Units and Measurements concepts of gross tumor volume, clinical target volume, internal target volume, and planning target volume are used for defining the targeted volumes. Newer treatment techniques, including intensity modulated radiation therapy, breath-hold, image guided radiation therapy, and 4-dimensional imaging, should be implemented when their use is expected to decrease significantly the risk for normal tissue damage while still achieving the primary goal of local tumor control. The highly conformal involved node radiation therapy (INRT), recently introduced for patients for whom optimal imaging is available, is explained. A new concept, involved site radiation therapy (ISRT), is introduced as the standard conformal therapy for the scenario, commonly encountered, wherein optimal imaging is not available. There is increasing evidence that RT doses used in the past are higher than necessary for disease control in this era of combined modality therapy. The use of INRT and of lower doses in early-stage HL is supported by available data. Although the use of ISRT has not yet been validated in a formal study, it is more conservative than INRT, accounting for suboptimal information and appropriately designed for safe local disease control. The goal of modern smaller field radiation therapy is to reduce both treatment volume and treatment dose while maintaining efficacy and minimizing acute and late sequelae. This review is a consensus of the International Lymphoma Radiation Oncology Group (ILROG) Steering Committee regarding the modern approach to RT in the treatment of HL, outlining a new concept of ISRT in which reduced treatment volumes are planned for the effective control of involved sites of HL. Nodal and extranodal non-Hodgkin lymphomas (NHL) are covered separately by ILROG guidelines.« less

Modern radiation therapy for Hodgkin lymphoma: field and dose guidelines from the international lymphoma radiation oncology group (ILROG).

PubMed

Specht, Lena; Yahalom, Joachim; Illidge, Tim; Berthelsen, Anne Kiil; Constine, Louis S; Eich, Hans Theodor; Girinsky, Theodore; Hoppe, Richard T; Mauch, Peter; Mikhaeel, N George; Ng, Andrea

2014-07-15

Radiation therapy (RT) is the most effective single modality for local control of Hodgkin lymphoma (HL) and an important component of therapy for many patients. These guidelines have been developed to address the use of RT in HL in the modern era of combined modality treatment. The role of reduced volumes and doses is addressed, integrating modern imaging with 3-dimensional (3D) planning and advanced techniques of treatment delivery. The previously applied extended field (EF) and original involved field (IF) techniques, which treated larger volumes based on nodal stations, have now been replaced by the use of limited volumes, based solely on detectable nodal (and extranodal extension) involvement at presentation, using contrast-enhanced computed tomography, positron emission tomography/computed tomography, magnetic resonance imaging, or a combination of these techniques. The International Commission on Radiation Units and Measurements concepts of gross tumor volume, clinical target volume, internal target volume, and planning target volume are used for defining the targeted volumes. Newer treatment techniques, including intensity modulated radiation therapy, breath-hold, image guided radiation therapy, and 4-dimensional imaging, should be implemented when their use is expected to decrease significantly the risk for normal tissue damage while still achieving the primary goal of local tumor control. The highly conformal involved node radiation therapy (INRT), recently introduced for patients for whom optimal imaging is available, is explained. A new concept, involved site radiation therapy (ISRT), is introduced as the standard conformal therapy for the scenario, commonly encountered, wherein optimal imaging is not available. There is increasing evidence that RT doses used in the past are higher than necessary for disease control in this era of combined modality therapy. The use of INRT and of lower doses in early-stage HL is supported by available data. Although the use of ISRT has not yet been validated in a formal study, it is more conservative than INRT, accounting for suboptimal information and appropriately designed for safe local disease control. The goal of modern smaller field radiation therapy is to reduce both treatment volume and treatment dose while maintaining efficacy and minimizing acute and late sequelae. This review is a consensus of the International Lymphoma Radiation Oncology Group (ILROG) Steering Committee regarding the modern approach to RT in the treatment of HL, outlining a new concept of ISRT in which reduced treatment volumes are planned for the effective control of involved sites of HL. Nodal and extranodal non-Hodgkin lymphomas (NHL) are covered separately by ILROG guidelines. Copyright © 2014 Elsevier Inc. All rights reserved.

Exploring the structural insights on human laforin mutation K87A in Lafora disease--a molecular dynamics study.

PubMed

Srikumar, P S; Rohini, K

2013-10-01

Lafora disease (LD) is an autosomal recessive, progressive form of myoclonus epilepsy which affects worldwide. LD occurs mainly in countries like southern Europe, northern Africa, South India, and in the Middle East. LD occurs with its onset mainly in teenagers and leads to decline and death within 2 to 10 years. The genes EPM2A and EPM2B are commonly involved in 90 % of LD cases. EPM2A codes for protein laforin which contains an amino terminal carbohydrate binding module (CBM) belonging to the CBM20 family and a carboxy terminal dual specificity phosphatase domain. Mutations in laforin are found to abolish glycogen binding and have been reported in wet lab methods. In order to investigate on structural insights on laforin mutation K81A, we performed molecular dynamics (MD) simulation studies for native and mutant protein. MD simulation results showed loss of stability due to mutation K87A which confirmed the structural reason for conformational changes observed in laforin. The conformational change of mutant laforin was confirmed by analysis using root mean square deviation, root mean square fluctuation, solvent accessibility surface area, radius of gyration, hydrogen bond, and principle component analysis. Our results identified that the flexibility of K87A mutated laforin structure, with replacement of acidic amino acid to aliphatic amino acid in functional CBM domain, have more impact in abolishing glycogen binding that favors LD.

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

Dirix, Piet; Nuyts, Sandra; Geussens, Yasmyne

Purpose: To evaluate the long-term clinical outcome and toxicity of conventional and three-dimensional conformal radiotherapy for malignancies of the nasal cavity and paranasal sinuses. Methods and Materials: Between January 1976 and February 2003, 127 patients with histologically proven cancer of the paranasal sinuses (n = 119) or nasal cavity (n = 8) were treated with preoperative (n = 61), postoperative (n = 51), or primary (n = 15) radiotherapy, using conventional (n = 74) or three-dimensional conformal (n = 53) techniques. No elective neck irradiation of the cervical lymph nodes was performed in N0 patients. Results: Median follow-up was 5.6more » years (range, 3-307 months) for all patients, and 7.3 years (range, 47-307 months) for patients still alive at the close-out date. The actuarial 5-year local control, overall survival, and disease-free survival rates were 53%, 54%, and 37%, respectively. Only 6 (5%) of all 127 patients and 4 (3%) of 122 originally N0 patients developed a regional failure in the neck. Distant metastasis occurred in 20% of patients. Both primary tumor extent and lymph node involvement were the most important prognostic factors, together with squamous cell carcinoma histology. Conclusion: Local failure remains the dominant cause of poor outcome for patients with sinonasal cancer, despite aggressive local treatment with combined surgery and radiotherapy in operable patients. Distant metastasis and certainly regional relapse were much less common sites of failure. Overall survival remains poor, suggesting the need for more efficacious local and possibly systemic therapy.« less

The Earth System Prediction Suite: Toward a Coordinated U.S. Modeling Capability

DOE PAGES

Theurich, Gerhard; DeLuca, C.; Campbell, T.; ...

2016-08-22

The Earth System Prediction Suite (ESPS) is a collection of flagship U.S. weather and climate models and model components that are being instrumented to conform to interoperability conventions, documented to follow metadata standards, and made available either under open-source terms or to credentialed users. Furthermore, the ESPS represents a culmination of efforts to create a common Earth system model architecture, and the advent of increasingly coordinated model development activities in the United States. ESPS component interfaces are based on the Earth System Modeling Framework (ESMF), community-developed software for building and coupling models, and the National Unified Operational Prediction Capability (NUOPC)more » Layer, a set of ESMF-based component templates and interoperability conventions. Our shared infrastructure simplifies the process of model coupling by guaranteeing that components conform to a set of technical and semantic behaviors. The ESPS encourages distributed, multiagency development of coupled modeling systems; controlled experimentation and testing; and exploration of novel model configurations, such as those motivated by research involving managed and interactive ensembles. ESPS codes include the Navy Global Environmental Model (NAVGEM), the Hybrid Coordinate Ocean Model (HYCOM), and the Coupled Ocean–Atmosphere Mesoscale Prediction System (COAMPS); the NOAA Environmental Modeling System (NEMS) and the Modular Ocean Model (MOM); the Community Earth System Model (CESM); and the NASA ModelE climate model and the Goddard Earth Observing System Model, version 5 (GEOS-5), atmospheric general circulation model.« less

The Earth System Prediction Suite: Toward a Coordinated U.S. Modeling Capability

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

Theurich, Gerhard; DeLuca, C.; Campbell, T.

The Earth System Prediction Suite (ESPS) is a collection of flagship U.S. weather and climate models and model components that are being instrumented to conform to interoperability conventions, documented to follow metadata standards, and made available either under open-source terms or to credentialed users. Furthermore, the ESPS represents a culmination of efforts to create a common Earth system model architecture, and the advent of increasingly coordinated model development activities in the United States. ESPS component interfaces are based on the Earth System Modeling Framework (ESMF), community-developed software for building and coupling models, and the National Unified Operational Prediction Capability (NUOPC)more » Layer, a set of ESMF-based component templates and interoperability conventions. Our shared infrastructure simplifies the process of model coupling by guaranteeing that components conform to a set of technical and semantic behaviors. The ESPS encourages distributed, multiagency development of coupled modeling systems; controlled experimentation and testing; and exploration of novel model configurations, such as those motivated by research involving managed and interactive ensembles. ESPS codes include the Navy Global Environmental Model (NAVGEM), the Hybrid Coordinate Ocean Model (HYCOM), and the Coupled Ocean–Atmosphere Mesoscale Prediction System (COAMPS); the NOAA Environmental Modeling System (NEMS) and the Modular Ocean Model (MOM); the Community Earth System Model (CESM); and the NASA ModelE climate model and the Goddard Earth Observing System Model, version 5 (GEOS-5), atmospheric general circulation model.« less

Kinetics and thermodynamics of the interchange of the morpheein forms of human porphobilinogen synthase.

PubMed

Selwood, Trevor; Tang, Lei; Lawrence, Sarah H; Anokhina, Yana; Jaffe, Eileen K

2008-03-11

A morpheein is a homo-oligomeric protein that can adopt different nonadditive quaternary assemblies (morpheein forms) with different functionalities. The human porphobilinogen synthase (PBGS) morpheein forms are a high activity octamer, a low activity hexamer, and two structurally distinct dimer conformations. Conversion between hexamer and octamer involves dissociation to dimers, conformational change at the dimer level, followed by association to the alternate assembly. The current work promotes an alternative and novel view of the physiologically relevant dimeric structures, which are derived from the crystal structures, but are distinct from the asymmetric units of their crystal forms. Using a well characterized heteromeric system (WT+F12L; Tang, L. et al. (2005) J. Biol. Chem. 280, 15786-15793), extensive study of the human PBGS morpheein reequilibration process now reveals that the intervening dimers do not dissociate to monomers. The morpheein equilibria of wild type (WT) human PBGS are found to respond to changes in pH, PBGS concentration, and substrate turnover. Notably, the WT enzyme is predominantly an octamer at neutral pH, but increasing pH results in substantial conversion to lower order oligomers. Most significantly, the free energy of activation for the conversion of WT+F12L human PBGS heterohexamers to hetero-octamers is determined to be the same as that for the catalytic conversion of substrate to product by the octamer, remarkably suggesting a common rate-limiting step for both processes, which is postulated to be the opening/closing of the active site lid.

Effects of conformism on the cultural evolution of social behaviour.

PubMed

Molleman, Lucas; Pen, Ido; Weissing, Franz J

2013-01-01

Models of cultural evolution study how the distribution of cultural traits changes over time. The dynamics of cultural evolution strongly depends on the way these traits are transmitted between individuals by social learning. Two prominent forms of social learning are payoff-based learning (imitating others that have higher payoffs) and conformist learning (imitating locally common behaviours). How payoff-based and conformist learning affect the cultural evolution of cooperation is currently a matter of lively debate, but few studies systematically analyse the interplay of these forms of social learning. Here we perform such a study by investigating how the interaction of payoff-based and conformist learning affects the outcome of cultural evolution in three social contexts. First, we develop a simple argument that provides insights into how the outcome of cultural evolution will change when more and more conformist learning is added to payoff-based learning. In a social dilemma (e.g. a Prisoner's Dilemma), conformism can turn cooperation into a stable equilibrium; in an evasion game (e.g. a Hawk-Dove game or a Snowdrift game) conformism tends to destabilize the polymorphic equilibrium; and in a coordination game (e.g. a Stag Hunt game), conformism changes the basin of attraction of the two equilibria. Second, we analyse a stochastic event-based model, revealing that conformism increases the speed of cultural evolution towards pure equilibria. Individual-based simulations as well as the analysis of the diffusion approximation of the stochastic model by and large confirm our findings. Third, we investigate the effect of an increasing degree of conformism on cultural group selection in a group-structured population. We conclude that, in contrast to statements in the literature, conformism hinders rather than promotes the evolution of cooperation.

Positively selected FimH residues enhance virulence during urinary tract infection by altering FimH conformation.

PubMed

Schwartz, Drew J; Kalas, Vasilios; Pinkner, Jerome S; Chen, Swaine L; Spaulding, Caitlin N; Dodson, Karen W; Hultgren, Scott J

2013-09-24

Chaperone-usher pathway pili are a widespread family of extracellular, Gram-negative bacterial fibers with important roles in bacterial pathogenesis. Type 1 pili are important virulence factors in uropathogenic Escherichia coli (UPEC), which cause the majority of urinary tract infections (UTI). FimH, the type 1 adhesin, binds mannosylated glycoproteins on the surface of human and murine bladder cells, facilitating bacterial colonization, invasion, and formation of biofilm-like intracellular bacterial communities. The mannose-binding pocket of FimH is invariant among UPEC. We discovered that pathoadaptive alleles of FimH with variant residues outside the binding pocket affect FimH-mediated acute and chronic pathogenesis of two commonly studied UPEC strains, UTI89 and CFT073. In vitro binding studies revealed that, whereas all pathoadaptive variants tested displayed the same high affinity for mannose when bound by the chaperone FimC, affinities varied when FimH was incorporated into pilus tip-like, FimCGH complexes. Structural studies have shown that FimH adopts an elongated conformation when complexed with FimC, but, when incorporated into the pilus tip, FimH can adopt a compact conformation. We hypothesize that the propensity of FimH to adopt the elongated conformation in the tip corresponds to its mannose binding affinity. Interestingly, FimH variants, which maintain a high-affinity conformation in the FimCGH tip-like structure, were attenuated during chronic bladder infection, implying that FimH's ability to switch between conformations is important in pathogenesis. Our studies argue that positively selected residues modulate fitness during UTI by affecting FimH conformation and function, providing an example of evolutionary tuning of structural dynamics impacting in vivo survival.

The Conformational Landscape of L-Threonine Matrix Isolation Infrared and {AB-INITIO Studies

NASA Astrophysics Data System (ADS)

Dubey, Pankaj; Mukhopadhyay, Anamika; Viswanathan, K. S.

2017-06-01

Amino acids, containing hydroxy side chains such as L-threonine and tyrosine play an important role in molecular recognition, such as in the docking of propofol, which is a commonly used anaesthetic. A rich conformational landscape of these amino acids makes them interesting candidates in the study of intra and intermolecular interactions. In this work, the conformational landscape of L-threonine was studied, as it can be expected to serve as a basis for understanding structure and functions of polypeptides and other biomolecules. The matrix isolation technique (MI) coupled with a high temperature effusive molecular beam (EMB) nozzle was used to trap conformers of amino acid, which were then characterized using FTIR spectroscopy. The usefulness of MI-EMB-FTIR spectroscopy is that it can trap structures corresponding to the local minima along with the global minimum and hence allows for a better exploration of the potential energy surface. A major challenge in conformational analysis of amino acids using matrix isolation FTIR arises from its non-volatile nature. A home built heating system which was mounted close to the cryotip, was used to evaporate the non-volatile amino acids. Our infrared spectra show that three conformations were trapped in the matrix. Experimental results were supported by {ab-initio calculations performed using the CCSD(T), MP2 and M06-2X methods together with 6-311++G(d,p) and aug/cc-pVDZ basis sets. The side chains of the amino acids appeared to have an influence on the preferential stabilisation of a particular backbone structure of amino acids. Factors such as entropy, anomeric effect and intramolecular H-bonding were also found to play an important role in determining conformal preferences, which will be discussed.

A residue in helical conformation in the native state adopts a β-strand conformation in the folding transition state despite its high and canonical Φ-value.

PubMed

Zarrine-Afsar, Arash; Dahesh, Samira; Davidson, Alan R

2012-05-01

Delineating structures of the transition states in protein folding reactions has provided great insight into the mechanisms by which proteins fold. The most common method for obtaining this information is Φ-value analysis, which is carried out by measuring the changes in the folding and unfolding rates caused by single amino acid substitutions at various positions within a given protein. Canonical Φ-values range between 0 and 1, and residues displaying high values within this range are interpreted to be important in stabilizing the transition state structure, and to elicit this stabilization through native-like interactions. Although very successful in defining the general features of transition state structures, Φ-value analysis can be confounded when non-native interactions stabilize this state. In addition, direct information on backbone conformation within the transition state is not provided. In the work described here, we have investigated structure formation at a conserved β-bulge (with helical conformation) in the Fyn SH3 domain by characterizing the effects of substituting all natural amino acids at one position within this structural motif. By comparing the effects on folding rates of these substitutions with database-derived local structure propensity values, we have determined that this position adopts a non-native backbone conformation in the folding transition state. This result is surprising because this position displays a high and canonical Φ-value of 0.7. This work emphasizes the potential role of non-native conformations in folding pathways and demonstrates that even positions displaying high and canonical Φ-values may, nevertheless, adopt a non-native conformation in the transition state. Copyright © 2012 Wiley Periodicals, Inc.

Distance-Based Configurational Entropy of Proteins from Molecular Dynamics Simulations

PubMed Central

Fogolari, Federico; Corazza, Alessandra; Fortuna, Sara; Soler, Miguel Angel; VanSchouwen, Bryan; Brancolini, Giorgia; Corni, Stefano; Melacini, Giuseppe; Esposito, Gennaro

2015-01-01

Estimation of configurational entropy from molecular dynamics trajectories is a difficult task which is often performed using quasi-harmonic or histogram analysis. An entirely different approach, proposed recently, estimates local density distribution around each conformational sample by measuring the distance from its nearest neighbors. In this work we show this theoretically well grounded the method can be easily applied to estimate the entropy from conformational sampling. We consider a set of systems that are representative of important biomolecular processes. In particular: reference entropies for amino acids in unfolded proteins are obtained from a database of residues not participating in secondary structure elements;the conformational entropy of folding of β2-microglobulin is computed from molecular dynamics simulations using reference entropies for the unfolded state;backbone conformational entropy is computed from molecular dynamics simulations of four different states of the EPAC protein and compared with order parameters (often used as a measure of entropy);the conformational and rototranslational entropy of binding is computed from simulations of 20 tripeptides bound to the peptide binding protein OppA and of β2-microglobulin bound to a citrate coated gold surface. This work shows the potential of the method in the most representative biological processes involving proteins, and provides a valuable alternative, principally in the shown cases, where other approaches are problematic. PMID:26177039

Distance-Based Configurational Entropy of Proteins from Molecular Dynamics Simulations.

PubMed

Fogolari, Federico; Corazza, Alessandra; Fortuna, Sara; Soler, Miguel Angel; VanSchouwen, Bryan; Brancolini, Giorgia; Corni, Stefano; Melacini, Giuseppe; Esposito, Gennaro

2015-01-01

Estimation of configurational entropy from molecular dynamics trajectories is a difficult task which is often performed using quasi-harmonic or histogram analysis. An entirely different approach, proposed recently, estimates local density distribution around each conformational sample by measuring the distance from its nearest neighbors. In this work we show this theoretically well grounded the method can be easily applied to estimate the entropy from conformational sampling. We consider a set of systems that are representative of important biomolecular processes. In particular: reference entropies for amino acids in unfolded proteins are obtained from a database of residues not participating in secondary structure elements;the conformational entropy of folding of β2-microglobulin is computed from molecular dynamics simulations using reference entropies for the unfolded state;backbone conformational entropy is computed from molecular dynamics simulations of four different states of the EPAC protein and compared with order parameters (often used as a measure of entropy);the conformational and rototranslational entropy of binding is computed from simulations of 20 tripeptides bound to the peptide binding protein OppA and of β2-microglobulin bound to a citrate coated gold surface. This work shows the potential of the method in the most representative biological processes involving proteins, and provides a valuable alternative, principally in the shown cases, where other approaches are problematic.

Influence of Trp flipping on carbohydrate binding in lectins. An example on Aleuria aurantia lectin AAL.

PubMed

Houser, Josef; Kozmon, Stanislav; Mishra, Deepti; Mishra, Sushil K; Romano, Patrick R; Wimmerová, Michaela; Koča, Jaroslav

2017-01-01

Protein-carbohydrate interactions are very often mediated by the stacking CH-π interactions involving the side chains of aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr) or phenylalanine (Phe). Especially suitable for stacking is the Trp residue. Analysis of the PDB database shows Trp stacking for 265 carbohydrate or carbohydrate like ligands in 5 208 Trp containing motives. An appropriate model system to study such an interaction is the AAL lectin family where the stacking interactions play a crucial role and are thought to be a driving force for carbohydrate binding. In this study we present data showing a novel finding in the stacking interaction of the AAL Trp side chain with the carbohydrate. High resolution X-ray structure of the AAL lectin from Aleuria aurantia with α-methyl-l-fucoside ligand shows two possible Trp side chain conformations with the same occupation in electron density. The in silico data shows that the conformation of the Trp side chain does not influence the interaction energy despite the fact that each conformation creates interactions with different carbohydrate CH groups. Moreover, the PDB data search shows that the conformations are almost equally distributed across all Trp-carbohydrate complexes, which would suggest no substantial preference for one conformation over another.

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

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

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

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

Structure of the uncleaved ectodomain of the paramyxovirus (hPIV3) fusion protein

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

Yin, Hsien-Sheng; Paterson, Reay G.; Wen, Xiaolin

2010-03-08

Class I viral fusion proteins share common mechanistic and structural features but little sequence similarity. Structural insights into the protein conformational changes associated with membrane fusion are based largely on studies of the influenza virus hemagglutinin in pre- and postfusion conformations. Here, we present the crystal structure of the secreted, uncleaved ectodomain of the paramyxovirus, human parainfluenza virus 3 fusion (F) protein, a member of the class I viral fusion protein group. The secreted human parainfluenza virus 3 F forms a trimer with distinct head, neck, and stalk regions. Unexpectedly, the structure reveals a six-helix bundle associated with the postfusionmore » form of F, suggesting that the anchor-minus ectodomain adopts a conformation largely similar to the postfusion state. The transmembrane anchor domains of F may therefore profoundly influence the folding energetics that establish and maintain a metastable, prefusion state.« less

Analysis of vibrational, structural, and electronic properties of rivastigmine by density functional theory

NASA Astrophysics Data System (ADS)

Prasad, O.; Sinha, L.; Misra, N.; Narayan, V.; Kumar, N.; Kumar, A.

2010-09-01

The present work deals with the structural, electronic, and vibrational analysis of rivastigmine. Rivastigmine, an antidementia medicament, is credited with significant therapeutic effects on the cognitive, functional, and behavioural problems that are commonly associated with Alzheimer’s dementia. For rivastigmine, a number of minimum energy conformations are possible. The geometry of twelve possible conformers has been analyzed and the most stable conformer was further optimized at a higher basis set. The electronic properties and vibrational frequencies were then calculated using a density functional theory at the B3LYP level with the 6-311+G(d, p) basis set. The different molecular surfaces have also been drawn to understand the activity of the molecule. A narrower frontier orbital energy gap in rivastigmine makes it softer and more reactive than water and dimethylfuran. The calculated value of the dipole moment is 2.58 debye.

Electrowetting of Weak Polyelectrolyte-Coated Surfaces.

PubMed

Sénéchal, Vincent; Saadaoui, Hassan; Rodriguez-Hernandez, Juan; Drummond, Carlos

2017-05-23

Polymer coatings are commonly used to modify interfacial properties like wettability, lubrication, or biocompatibility. These properties are determined by the conformation of polymer molecules at the interface. Polyelectrolytes are convenient elementary bricks to build smart materials, given that polyion chain conformation is very sensitive to different environmental variables. Here we discuss the effect of an applied electric field on the properties of surfaces coated with poly(acrylic acid) brushes. By combining atomic force microscopy, quartz crystal microbalance, and contact angle experiments, we show that it is possible to precisely tune polyion chain conformation, surface adhesion, and surface wettability using very low applied voltages if the polymer grafting density and environmental conditions (pH and ionic strength) are properly formulated. Our results indicate that the effective ionization degree of the grafted weak polyacid can be finely controlled with the externally applied field, with important consequences for the macroscopic surface properties.

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

PubMed

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

2017-10-04

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

Conformations of the HIV-1 protease: A crystal structure data set analysis.

PubMed

Palese, Luigi Leonardo

2017-11-01

The HIV protease is an important drug target for HIV/AIDS therapy, and its structure and function have been extensively investigated. This enzyme performs an essential role in viral maturation by processing specific cleavage sites in the Gag and Gag-Pol precursor polyproteins so as to release their mature forms. This 99 amino acid aspartic protease works as a homodimer, with the active site localized in a central cavity capped by two flexible flap regions. The dimer presents closed or open conformations, which are involved in the substrate binding and release. Here the results of the analysis of a HIV-1 protease data set containing 552 dimer structures are reported. Different dimensionality reduction methods have been used in order to get information from this multidimensional database. Most of the structures in the data set belong to two conformational clusters. An interesting observation that comes from the analysis of these data is that some protease sequences are localized preferentially in specific areas of the conformational landscape of this protein. Copyright © 2017 Elsevier B.V. All rights reserved.

Antibacterial action mode of quaternized carboxymethyl chitosan/poly(amidoamine) dendrimer core-shell nanoparticles against Escherichia coli correlated with molecular chain conformation.

PubMed

Wen, Yan; Yao, Fanglian; Sun, Fang; Tan, Zhilei; Tian, Liang; Xie, Lei; Song, Qingchao

2015-03-01

The action mode of quaternized carboxymethyl chitosan/poly(amidoamine) dendrimer core-shell nanoparticles (CM-HTCC/PAMAM) against Escherichia coli (E. coli) was investigated via a combination of approaches including measurements of cell membrane integrity, outer membrane (OM) and inner membrane (IM) permeability, and scanning electron microscopy (SEM). CM-HTCC/PAMAM dendrimer nanoparticles likely acted in a sequent event-driven mechanism, beginning with the binding of positively charged groups from nanoparticle surface with negative cell surface, thereby causing the disorganization of cell membrane, and subsequent leakage of intracellular components which might ultimately lead to cell death. Moreover, the chain conformation of polymers was taken into account for a better understanding of the antibacterial action mode by means of viscosity and GPC measurements. High utilization ratio of positive charge and large specific surface area generated from a compacted conformation of CM-HTCC/PAMAM, significantly different from the extended conformation of HTCC, were proposed to be involved in the antibacterial action. Copyright © 2014 Elsevier B.V. All rights reserved.

Phi (Φ) and psi (Ψ) angles involved in malarial peptide bonds determine sterile protective immunity.

PubMed

Patarroyo, Manuel E; Moreno-Vranich, Armando; Bermúdez, Adriana

2012-12-07

Modified HABP (mHABP) regions interacting with HLA-DRβ1(∗) molecules have a more restricted conformation and/or sequence than other mHABPs which do not fit perfectly into their peptide binding regions (PBR) and do not induce an acceptable immune response due to the critical role of their Φ and Ψ torsion angles. These angle's critical role was determined in such highly immunogenic, protection-inducing response against experimental malaria using the conformers (mHABPs) obtained by (1)H-NMR and superimposed into HLA-DRβ1(∗)-like Aotus monkey molecules; their phi (Φ) and psi (Ψ) angles were measured and the H-bond formation between these molecules was evaluated. The aforementioned mHABP propensity to assume a regular conformation similar to a left-handed polyproline type II helix (PPII(L)) led to suggesting that favouring these conformations according to their amino acid sequence would lead to high antibody titre production and sterile protective immunity induction against malaria, thereby adding new principles or rules for vaccine development, malaria being one of them. Copyright © 2012 Elsevier Inc. All rights reserved.

Rotational Spectrum and Conformational Analysis of N-methyl-2-aminoethanol: Insights into the Shape of Adrenergic Neurotransmitters

NASA Astrophysics Data System (ADS)

Calabrese, Camilla; Maris, Assimo; Evangelisti, Luca; Piras, Anna; Parravicini, Valentina; Melandri, Sonia

2018-02-01

Abstract We describe an experimental and quantum chemical study for the accurate determination of the conformational space of small molecular systems governed by intramolecular non-covalent interactions. The model systems investigated belong to the biological relevant aminoalcohol’s family, and include 2-aminophenylethanol, 2-methylaminophenylethanol, noradrenaline, adrenaline 2-aminoethanol and N-methyl-2-aminoethanol. For the latter molecule, the rotational spectrum in the 6-18 and 59.6-74.4 GHz ranges was recorded in the isolated conditions of a free jet expansion. Based on the analysis of the rotational spectra, two different conformational species and 11 isotopologues were observed and their spectroscopic constants, including 14N-nuclear hyperfine coupling constants and methyl internal rotation barriers, were determined. From the experimental data a structural determination was obtained, which was also used to benchmark accurate quantum chemical calculations on the whole conformational space. Atom in molecules and non-covalent interactions theories allowed the characterization of the position of the intramolecular non-covalent interactions and the energies involved, highlighting the subtle balance responsible of the stabilization of all the molecular systems.

Can acyclic conformational control be achieved via a sulfur–fluorine gauche effect?† †Electronic supplementary information (ESI) available. CCDC 1048074–1048078. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5sc00871a

PubMed Central

Thiehoff, C.; Holland, M. C.; Daniliuc, C.

2015-01-01

The gauche conformation of the 1,2-difluoroethane motif is known to involve stabilising hyperconjugative interactions between donor (bonding, σC–H) and acceptor (antibonding, σ*C–F) orbitals. This model rationalises the generic conformational preference of F–Cβ–Cα–X systems (φFCCX ≈ 60°), where X is an electron deficient substituent containing a Period 2 atom. Little is known about the corresponding Period 3 systems, such as sulfur and phosphorus, where multiple oxidation states are possible. Conformational analyses of β-fluorosulfides, -sulfoxides and -sulfones are disclosed here, thus extending the scope of the fluorine gauche effect to the 3rd Period (F–C–C–S(O)n; φFCCS ≈ 60°). Synergy between experiment and computation has revealed that the gauche effect is only pronounced in structures bearing an electropositive vicinal sulfur atom (S+–O–, SO2). PMID:29511517

Matrix product state description of Halperin states

NASA Astrophysics Data System (ADS)

Crépel, V.; Estienne, B.; Bernevig, B. A.; Lecheminant, P.; Regnault, N.

2018-04-01

Many fractional quantum Hall states can be expressed as a correlator of a given conformal field theory used to describe their edge physics. As a consequence, these states admit an economical representation as an exact matrix product state (MPS) that was extensively studied for the systems without any spin or any other internal degrees of freedom. In that case, the correlators are built from a single electronic operator, which is primary with respect to the underlying conformal field theory. We generalize this construction to the archetype of Abelian multicomponent fractional quantum Hall wave functions, the Halperin states. These can be written as conformal blocks involving multiple electronic operators and we explicitly derive their exact MPS representation. In particular, we deal with the caveat of the full wave-function symmetry and show that any additional SU(2) symmetry is preserved by the natural MPS truncation scheme provided by the conformal dimension. We use our method to characterize the topological order of the Halperin states by extracting the topological entanglement entropy. We also evaluate their bulk correlation lengths, which are compared to plasma analogy arguments.

Conformational dependence of a protein kinase phosphate transfer reaction.

PubMed

Henkelman, Graeme; LaBute, Montiago X; Tung, Chang-Shung; Fenimore, P W; McMahon, Benjamin H

2005-10-25

Atomic motions and energetics for a phosphate transfer reaction catalyzed by the cAMP-dependent protein kinase are calculated by plane-wave density functional theory, starting from structures of proteins crystallized in both the reactant conformation (RC) and the transition-state conformation (TC). In TC, we calculate that the reactants and products are nearly isoenergetic with a 20-kJ/mol barrier, whereas phosphate transfer is unfavorable by 120 kJ/mol in the RC, with an even higher barrier. With the protein in TC, the motions involved in reaction are small, with only P(gamma) and the catalytic proton moving >0.5 A. Examination of the structures reveals that in the RC the active site cleft is not completely closed and there is insufficient space for the phosphorylated serine residue in the product state. Together, these observations imply that the phosphate transfer reaction occurs rapidly and reversibly in a particular conformation of the protein, and that the reaction can be gated by changes of a few tenths of an angstrom in the catalytic site.

Electrodes synthesized from carbon nanostructures coated with a smooth and conformal metal adlayer

DOEpatents

Adzic, Radoslav; Harris, Alexander

2014-04-15

High-surface-area carbon nanostructures coated with a smooth and conformal submonolayer-to-multilayer thin metal films and their method of manufacture are described. The preferred manufacturing process involves the initial oxidation of the carbon nanostructures followed by a surface preparation process involving immersion in a solution with the desired pH to create negative surface dipoles. The nanostructures are subsequently immersed in an alkaline solution containing a suitable quantity of non-noble metal ions which adsorb at surface reaction sites. The metal ions are then reduced via chemical or electrical means. The nanostructures are exposed to a solution containing a salt of one or more noble metals which replace adsorbed non-noble surface metal atoms by galvanic displacement. The process can be controlled and repeated to obtain a desired film coverage. The resulting coated nanostructures may be used, for example, as high-performance electrodes in supercapacitors, batteries, or other electric storage devices.

Sport-Related Identities and the "Toxic Jock"

PubMed

Miller, Kathleen E

2009-01-01

Little attention has been paid to the multidimensional nature of athletic involvement, which includes identity formation as well as participation in sports activities. Five hundred eighty-one sport-involved undergraduate students completed a questionnaire assessing their sport-related identities, goal orientations, primary sport ratings, and conformity to masculine norms. F-tested mean comparisons and hierarchical linear regressions were used to explore the characteristics associated with two distinct sport-related identities ("athletes" and "jocks"). Jock identity was associated with an ego-oriented approach to sports (men only), whereas athlete identity was associated with a task-oriented approach (both genders). Jock identity was positively associated with conformity to masculine norms, particularly for men, whereas athlete identity was positively associated with some masculine norms (i.e., Winning) and negatively associated with others (i.e., Playboy). These findings help to identify the correlates of a "toxic Jock" identity that may signal elevated risk for health-compromising behavior.

Sport-Related Identities and the “Toxic Jock”

PubMed Central

Miller, Kathleen E.

2010-01-01

Little attention has been paid to the multidimensional nature of athletic involvement, which includes identity formation as well as participation in sports activities. Five hundred eighty-one sport-involved undergraduate students completed a questionnaire assessing their sport-related identities, goal orientations, primary sport ratings, and conformity to masculine norms. F-tested mean comparisons and hierarchical linear regressions were used to explore the characteristics associated with two distinct sport-related identities (“athletes” and “jocks”). Jock identity was associated with an ego-oriented approach to sports (men only), whereas athlete identity was associated with a task-oriented approach (both genders). Jock identity was positively associated with conformity to masculine norms, particularly for men, whereas athlete identity was positively associated with some masculine norms (i.e., Winning) and negatively associated with others (i.e., Playboy). These findings help to identify the correlates of a “toxic Jock” identity that may signal elevated risk for health-compromising behavior. PMID:21643553

COREPA-M: NEW MULTI-DIMENSIONAL FUNCTIONALITY OF THE COREPA METHOD

EPA Science Inventory

The COmmon REactivity PAttern (COREPA) method is a recently developed pattern recognition technique accounting for conformational flexibility of chemicals in 3-D quantitative structure-activity relationships (QSARs). The method is based on the assumption that non-congeneric chemi...

Encapsulation of biomaterials in porous glass-like matrices prepared via an aqueous colloidal sol-gel process

DOEpatents

Liu, Dean-Mo; Chen, I-Wei

2001-01-01

The present invention provides a process for the encapsulation of biologically important proteins into transparent, porous silica matrices by an alcohol-free, aqueous, colloidal sol-gel process, and to the biological materials encapsulated thereby. The process is exemplified by studies involving encapsulated cytochrome c, catalase, myoglobin, and hemoglobin, although non-proteinaceous biomaterials, such as active DNA or RNA fragments, cells or even tissues, may also be encapsulated in accordance with the present methods. Conformation, and hence activity of the biomaterial, is successfully retained after encapsulation as demonstrated by optical characterization of the molecules, even after long-term storage. The retained conformation of the biomaterial is strongly correlated to both the rate of gelation and the subsequent drying speed of the encapsulatng matrix. Moreover, in accordance with this process, gelation is accelerated by the use of a higher colloidal solid concentration and a lower synthesis pH than conventional methods, thereby enhancing structural stability and retained conformation of the biomaterials. Thus, the invention also provides a remarkable improvement in retaining the biological activity of the encapsulated biomaterial, as compared with those involved in conventional alkoxide-based processes. It further provides new methods for the quantitative and qualitative detection of test substances that are reactive to, or catalyzed by, the active, encapsulated biological materials.

GalaxyGPCRloop: Template-Based and Ab Initio Structure Sampling of the Extracellular Loops of G-Protein-Coupled Receptors.

PubMed

Won, Jonghun; Lee, Gyu Rie; Park, Hahnbeom; Seok, Chaok

2018-06-07

The second extracellular loops (ECL2s) of G-protein-coupled receptors (GPCRs) are often involved in GPCR functions, and their structures have important implications in drug discovery. However, structure prediction of ECL2 is difficult because of its long length and the structural diversity among different GPCRs. In this study, a new ECL2 conformational sampling method involving both template-based and ab initio sampling was developed. Inspired by the observation of similar ECL2 structures of closely related GPCRs, a template-based sampling method employing loop structure templates selected from the structure database was developed. A new metric for evaluating similarity of the target loop to templates was introduced for template selection. An ab initio loop sampling method was also developed to treat cases without highly similar templates. The ab initio method is based on the previously developed fragment assembly and loop closure method. A new sampling component that takes advantage of secondary structure prediction was added. In addition, a conserved disulfide bridge restraining ECL2 conformation was predicted and analytically incorporated into sampling, reducing the effective dimension of the conformational search space. The sampling method was combined with an existing energy function for comparison with previously reported loop structure prediction methods, and the benchmark test demonstrated outstanding performance.

I222 crystal form of despentapeptide (B26-B30) insulin provides new insights into the properties of monomeric insulin.

PubMed

Whittingham, Jean L; Youshang, Zhang; Záková, Lenka; Dodson, Eleanor J; Turkenburg, Johan P; Brange, Jens; Dodson, G Guy

2006-05-01

Despentapeptide (des-B26-B30) insulin (DPI), an active modified insulin, has been crystallized in the presence of 20% acetic acid pH 2. A crystal structure analysis to 1.8 A spacing (space group I222) revealed that the DPI molecule, which is unable to make beta-strand interactions for physiological dimer formation and is apparently monomeric in solution, formed an alternative lattice-generated dimer. The formation of this dimer involved interactions between surfaces which included the B9-B19 alpha-helices (usually buried by the dimer-dimer contacts within the native hexamer). The two crystallographically independent molecules within the dimer were essentially identical and were similar in conformation to T-state insulin as seen in the T(6) insulin hexamer. An unusual feature of each molecule in the dimer was the presence of two independent conformations at the B-chain C-terminus (residues B20-B25). Both conformations were different from that of native insulin, involving a 3.5 A displacement of the B20-B23 beta-turn and a repositioning of residue PheB25 such that it made close van der Waals contact with the main body of the molecule, appearing to stabilize the B-chain C-terminus.

Monitoring Conformational Landscape of Ovine Prion Protein Monomer Using Ion Mobility Coupled to Mass Spectrometry

NASA Astrophysics Data System (ADS)

Van der Rest, Guillaume; Rezaei, Human; Halgand, Frédéric

2017-02-01

Prion protein is involved in deadly neurodegenerative diseases. Its pathogenicity is linked to its structural conversion (α-helix to β-strand transition). However, recent studies suggest that prion protein can follow a plurality of conversion pathways, which hints towards different conformers that might coexist in solution. To gain insights on the plasticity of the ovine prion protein (PrP) monomer, wild type (A136, R154, Q171), mutants and deletions of ARQ were studied by traveling wave ion mobility experiments coupled to mass spectrometry. In order to perform the analysis of a large body of data sets, we designed and evaluated the performance of a processing pipeline based on Driftscope peak detection and a homemade script for automated peak assignment, annotation, and quantification on specific multiply charged protein data. Using this approach, we showed that in the gas phase, PrPs are represented by at least three conformer families differing in both charge state distribution and collisional cross-section, in agreement with the work of Hilton et al. (2010). We also showed that this plasticity is borne both by the N- and C-terminal domains. Effect of protein concentration, pH and temperature were also assessed, showing that (1) pH does not affect conformer distributions, (2) protein concentration modifies the conformational landscape of one mutant (I208M) only, and (3) heating leads to other unfolded species and to a modification of the conformer intensity ratios.

Femtosecond Heterodyne Transient Grating Detection of Conformational Dynamics in the S0 (11Ag-) State of Carotenoids After Nonradiative Decay of the S2 (11Bu+) State

NASA Astrophysics Data System (ADS)

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

Transient grating spectroscopy was used to study the dynamics of nonradiative decay of the S1 (21Ag-) state in ß-carotene and peridinin after optical preparation of the S2) state. The kinetics of the recovery of the absorption and dispersion components of the third-order signal exhibit significantly different time constants. For β-carotene in benzonitrile, the absorption and dispersion recovery time constants are 11.6 and 10.2 ps. For peridinin in methanol, the time constants are 9.9 and 7.4 ps. These results indicate that the initial product of the decay of the S1 state is a conformationally displaced structure. The decay rate for the S1 state and the conformational relaxation rate are both slowed in peridinin as the polarity of the solvent decreases; in ethyl acetate, the conformational relaxation time constant is 45 ps, which rules out a dominant contribution from vibrational cooling. These results indicate that the S1 state develops intramolecular charge transfer character owing to distortions along torsional and out-of-plane coordinates, with a pyramidal structure favored as the most stable conformation. Recovery of the photoselected ground state conformation involves a reverse charge-transfer event followed by relaxation to a planar structure. Work supported by Photosynthetic Systems Program of the U.S. Department of Energy under Grant DE-SC0010847.

pKa values in proteins determined by electrostatics applied to molecular dynamics trajectories.

PubMed

Meyer, Tim; Knapp, Ernst-Walter

2015-06-09

For a benchmark set of 194 measured pKa values in 13 proteins, electrostatic energy computations are performed in which pKa values are computed by solving the Poisson-Boltzmann equation. In contrast to the previous approach of Karlsberg(+) (KB(+)) that essentially used protein crystal structures with variations in their side chain conformations, the present approach (KB2(+)MD) uses protein conformations from four molecular dynamics (MD) simulations of 10 ns each. These MD simulations are performed with different specific but fixed protonation patterns, selected to sample the conformational space for the different protonation patterns faithfully. The root-mean-square deviation between computed and measured pKa values (pKa RMSD) is shown to be reduced from 1.17 pH units using KB(+) to 0.96 pH units using KB2(+)MD. The pKa RMSD can be further reduced to 0.79 pH units, if each conformation is energy-minimized with a dielectric constant of εmin = 4 prior to calculating the electrostatic energy. The electrostatic energy expressions upon which the computations are based have been reformulated such that they do not involve terms that mix protein and solvent environment contributions and no thermodynamic cycle is needed. As a consequence, conformations of the titratable residues can be treated independently in the protein and solvent environments. In addition, the energy terms used here avoid the so-called intrinsic pKa and can therefore be interpreted without reference to arbitrary protonation states and conformations.

A transferable coarse-grained model for diphenylalanine: How to represent an environment driven conformational transition

NASA Astrophysics Data System (ADS)

Dalgicdir, Cahit; Sensoy, Ozge; Peter, Christine; Sayar, Mehmet

2013-12-01

One of the major challenges in the development of coarse grained (CG) simulation models that aim at biomolecular structure formation processes is the correct representation of an environment-driven conformational change, for example, a folding/unfolding event upon interaction with an interface or upon aggregation. In the present study, we investigate this transferability challenge for a CG model using the example of diphenylalanine. This dipeptide displays a transition from a trans-like to a cis-like conformation upon aggregation as well as upon transfer from bulk water to the cyclohexane/water interface. Here, we show that one can construct a single CG model that can reproduce both the bulk and interface conformational behavior and the segregation between hydrophobic/hydrophilic medium. While the general strategy to obtain nonbonded interactions in the present CG model is to reproduce solvation free energies of small molecules representing the CG beads in the respective solvents, the success of the model strongly depends on nontrivial decisions one has to make to capture the delicate balance between the bonded and nonbonded interactions. In particular, we found that the peptide's conformational behavior is qualitatively affected by the cyclohexane/water interaction potential, an interaction that does not directly involve the peptide at all but merely influences the properties of the hydrophobic/hydrophilic interface. Furthermore, we show that a small modification to improve the structural/conformational properties of the CG model could dramatically alter the thermodynamic properties.

Conformational heterogeneity in closed and open states of the KcsA potassium channel in lipid bicelles

PubMed Central

Kim, Dorothy M.; Dikiy, Igor; Upadhyay, Vikrant; Posson, David J.

2016-01-01

The process of ion channel gating—opening and closing—involves local and global structural changes in the channel in response to external stimuli. Conformational changes depend on the energetic landscape that underlies the transition between closed and open states, which plays a key role in ion channel gating. For the prokaryotic, pH-gated potassium channel KcsA, closed and open states have been extensively studied using structural and functional methods, but the dynamics within each of these functional states as well as the transition between them is not as well understood. In this study, we used solution nuclear magnetic resonance (NMR) spectroscopy to investigate the conformational transitions within specific functional states of KcsA. We incorporated KcsA channels into lipid bicelles and stabilized them into a closed state by using either phosphatidylcholine lipids, known to favor the closed channel, or mutations designed to trap the channel shut by disulfide cross-linking. A distinct state, consistent with an open channel, was uncovered by the addition of cardiolipin lipids. Using selective amino acid labeling at locations within the channel that are known to move during gating, we observed at least two different slowly interconverting conformational states for both closed and open channels. The pH dependence of these conformations and the predictable disruptions to this dependence observed in mutant channels with altered pH sensing highlight the importance of conformational heterogeneity for KcsA gating. PMID:27432996

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.

Refoldable Foldamers: Global Conformational Switching by Deletion or Insertion of a Single Hydrogen Bond

PubMed Central

Le Bailly, Bryden A. F.; Byrne, Liam

2016-01-01

Abstract Small changes in the structure of a foldamer may lead to gross changes in conformational preference. We show that the simple insertion or deletion of a single hydrogen bond by changes in pH or by photochemical deprotection is sufficient to refold a helical oligomer, interconverting M and P screw‐sense preference. As a consequence of the switch, information may be transmitted to a remote catalytic site, selectively directing the formation of either of two enantiomeric products by a reaction involving 1,22‐remote intermolecular asymmetric induction. PMID:26762559

Protein relaxation without a geminate phase in nanosecond photodissociated CO carp hemoglobin

NASA Astrophysics Data System (ADS)

Loupiac, Camille; Kruk, Nicolay; Valat, Pierre; Alpert, Bernard

1999-03-01

Transient heme-protein interactions upon passing from ligated to deligated carp hemoglobin were observed through time-resolved optical spectra following nanosecond CO photodissociation. The spectral evolution of the heme, in the nanosecond and microsecond time ranges, shows a protein conformational relaxation and the absence of a geminate CO recombination in carp hemoglobin. The comparison of the phenomena in carp and human hemoglobin implies that the physical basis of the geminate rebinding in human hemoglobin should involve an out-of-equilibrium protein conformation, close to a dissipative structure defined by the thermodynamics of Prigogine.

Conformational Transitions in Molecular Systems

NASA Astrophysics Data System (ADS)

Bachmann, M.; Janke, W.

2008-11-01

Proteins are the "work horses" in biological systems. In almost all functions specific proteins are involved. They control molecular transport processes, stabilize the cell structure, enzymatically catalyze chemical reactions; others act as molecular motors in the complex machinery of molecular synthetization processes. Due to their significance, misfolds and malfunctions of proteins typically entail disastrous diseases, such as Alzheimer's disease and bovine spongiform encephalopathy (BSE). Therefore, the understanding of the trinity of amino acid composition, geometric structure, and biological function is one of the most essential challenges for the natural sciences. Here, we glance at conformational transitions accompanying the structure formation in protein folding processes.

Conformal Lightweight Antenna Structures for Aeronautical Communication Technologies

NASA Technical Reports Server (NTRS)

Meador, Mary Ann

2017-01-01

This project is to develop antennas which enable beyond line of sight (BLOS) command and control for UAVs. We will take advantage of newly assigned provisional Ku-band spectrum for UAVs and use unique antenna designs to avoid interference with ground systems. This will involve designing antennas with high isotropic effective radiated power (EIRP) and ultra-low sidelobes. The antennas will be made with polymer aerogel as a substrate to both reduce weight and improve performance, as demonstrated in an Aero Seedling. In addition, designing the antennas to be conformal to the aircraft fuselage will reduce drag.

The Crystal Structure of a Cardiovirus RNA-Dependent RNA Polymerase Reveals an Unusual Conformation of the Polymerase Active Site

PubMed Central

Vives-Adrian, Laia; Lujan, Celia; Oliva, Baldo; van der Linden, Lonneke; Selisko, Barbara; Coutard, Bruno; Canard, Bruno; van Kuppeveld, Frank J. M.

2014-01-01

ABSTRACT Encephalomyocarditis virus (EMCV) is a member of the Cardiovirus genus within the large Picornaviridae family, which includes a number of important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for viral genome replication. In this study, we report the X-ray structures of two different crystal forms of the EMCV RdRp determined at 2.8- and 2.15-Å resolution. The in vitro elongation and VPg uridylylation activities of the purified enzyme have also been demonstrated. Although the overall structure of EMCV 3Dpol is shown to be similar to that of the known RdRps of other members of the Picornaviridae family, structural comparisons show a large reorganization of the active-site cavity in one of the crystal forms. The rearrangement affects mainly motif A, where the conserved residue Asp240, involved in ribonucleoside triphosphate (rNTP) selection, and its neighbor residue, Phe239, move about 10 Å from their expected positions within the ribose binding pocket toward the entrance of the rNTP tunnel. This altered conformation of motif A is stabilized by a cation-π interaction established between the aromatic ring of Phe239 and the side chain of Lys56 within the finger domain. Other contacts, involving Phe239 and different residues of motif F, are also observed. The movement of motif A is connected with important conformational changes in the finger region flanked by residues 54 to 63, harboring Lys56, and in the polymerase N terminus. The structures determined in this work provide essential information for studies on the cardiovirus RNA replication process and may have important implications for the development of new antivirals targeting the altered conformation of motif A. IMPORTANCE The Picornaviridae family is one of the largest virus families known, including many important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for picornavirus genome replication and a validated target for the development of antiviral therapies. Solving the X-ray structure of the first cardiovirus RdRp, EMCV 3Dpol, we captured an altered conformation of a conserved motif in the polymerase active site (motif A) containing the aspartic acid residue involved in rNTP selection and binding. This altered conformation of motif A, which interferes with the correct positioning of the rNTP substrate in the active site, is stabilized by a number of residues strictly conserved among picornaviruses. The rearrangements observed suggest that this motif A segment is a dynamic element that can be modulated by external effectors, either activating or inhibiting enzyme activity, and this type of modulation appears to be general to all picornaviruses. PMID:24600002

The crystal structure of a cardiovirus RNA-dependent RNA polymerase reveals an unusual conformation of the polymerase active site.

PubMed

Vives-Adrian, Laia; Lujan, Celia; Oliva, Baldo; van der Linden, Lonneke; Selisko, Barbara; Coutard, Bruno; Canard, Bruno; van Kuppeveld, Frank J M; Ferrer-Orta, Cristina; Verdaguer, Núria

2014-05-01

Encephalomyocarditis virus (EMCV) is a member of the Cardiovirus genus within the large Picornaviridae family, which includes a number of important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for viral genome replication. In this study, we report the X-ray structures of two different crystal forms of the EMCV RdRp determined at 2.8- and 2.15-Å resolution. The in vitro elongation and VPg uridylylation activities of the purified enzyme have also been demonstrated. Although the overall structure of EMCV 3Dpol is shown to be similar to that of the known RdRps of other members of the Picornaviridae family, structural comparisons show a large reorganization of the active-site cavity in one of the crystal forms. The rearrangement affects mainly motif A, where the conserved residue Asp240, involved in ribonucleoside triphosphate (rNTP) selection, and its neighbor residue, Phe239, move about 10 Å from their expected positions within the ribose binding pocket toward the entrance of the rNTP tunnel. This altered conformation of motif A is stabilized by a cation-π interaction established between the aromatic ring of Phe239 and the side chain of Lys56 within the finger domain. Other contacts, involving Phe239 and different residues of motif F, are also observed. The movement of motif A is connected with important conformational changes in the finger region flanked by residues 54 to 63, harboring Lys56, and in the polymerase N terminus. The structures determined in this work provide essential information for studies on the cardiovirus RNA replication process and may have important implications for the development of new antivirals targeting the altered conformation of motif A. The Picornaviridae family is one of the largest virus families known, including many important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for picornavirus genome replication and a validated target for the development of antiviral therapies. Solving the X-ray structure of the first cardiovirus RdRp, EMCV 3Dpol, we captured an altered conformation of a conserved motif in the polymerase active site (motif A) containing the aspartic acid residue involved in rNTP selection and binding. This altered conformation of motif A, which interferes with the correct positioning of the rNTP substrate in the active site, is stabilized by a number of residues strictly conserved among picornaviruses. The rearrangements observed suggest that this motif A segment is a dynamic element that can be modulated by external effectors, either activating or inhibiting enzyme activity, and this type of modulation appears to be general to all picornaviruses.

Pathological conformations involving the amino terminus of tau occur early in Alzheimer’s disease and are differentially detected by monoclonal antibodies

PubMed Central

Combs, Benjamin; Hamel, Chelsey; Kanaan, Nicholas M.

2016-01-01

Conformational changes involving the amino terminus of the tau protein are among the earliest alterations associated with tau pathology in Alzheimer’s disease and other tauopathies. This region of tau contains a phosphatase-activating domain (PAD) that is aberrantly exposed in pathological forms of the protein, an event that is associated with disruptions in anterograde fast axonal transport. We utilized four antibodies that recognize the amino terminus of tau, TNT1, TNT2 (a novel antibody), Tau12, and Tau13, to further study this important region. Using scanning alanine mutations in recombinant tau proteins, we refined the epitopes of each antibody. We examined the antibodies’ relative abilities to specifically label pathological tau in non-denaturing and denaturing assays to gain insight into some of the mechanistic details of PAD exposure. We then determined the pattern of tau pathology labeled by each antibody in human hippocampal sections at various disease stages in order to characterize PAD exposure in the context of disease progression. The characteristics of reactivity for the antibodies fell into two groups. TNT1 and TNT2 recognized epitopes within amino acids 7–12 and specifically identified recombinant tau aggregates and pathological tau from Alzheimer’s disease brains in a conformation-dependent manner. These antibodies labeled early pre-tangle pathology from neurons in early Braak stages and colocalized with thiazine red, a marker of fibrillar pathology, in classic neurofibrillary tangles. However, late tangles were negative for TNT1 and TNT2 indicating a loss of the epitope in later stages of tangle evolution. In contrast, Tau12 and Tau13 both identified discontinuous epitopes in the amino terminus and were unable to differentiate between normal and pathological tau in biochemical and tissue immunohistological assays. Despite the close proximity of these epitopes, the antibodies demonstrated remarkably different abilities to identify pathological changes in tau indicating that detection of conformational alterations involving PAD exposure is not achieved by all N-terminal tau antibodies and that a relatively discrete region of the N-terminus (i.e., amino acids 7–12, the TNT1 and TNT2 epitope) is central to the differences between normal and pathological tau. The appearance of PAD in early tau pathology and its disappearance in late-stage tangles suggest that toxic forms of tau are associated with the earliest forms of tau deposits. Collectively, these findings demonstrate that the TNT antibodies are useful markers for early conformational display of PAD and provide information regarding conformational changes that have potential implications in the toxic mechanisms of tau pathology. PMID:27260838

Tuning the conformational properties of the prion peptide.

PubMed

Ho, Chai-Chi; Lee, Lily Y-L; Huang, Kuo-Ting; Lin, Chun-Cheng; Ku, Mei-Yun; Yang, Chien-Chih; Chan, Sunney I; Hsu, Ruei-Lin; Chen, Rita P-Y

2009-07-01

Previously, we disclosed that O-linked glycosylation of Ser-132 or Ser-135 could dramatically change the amyloidogenic property of the hamster prion peptide (sequence 108-144). This peptide, which corresponds to the flexible loop and the first beta-strand in the structure of the prion protein, is a random coil when it is initially dissolved in buffer, but amyloid fibrils are formed with time. Thus, it offers a convenient model system to observe and compare how different chemical modifications and sequence mutations alter the amyloidogenic property of the peptide within a reasonable experimental time frame. In our earlier study, aside from uncovering a site-specificity of the glycosylation on the fibrillogenesis, different effects of alpha-GalNAc and beta-GlcNAc were observed. In this work, we explore further how different sugar configurations affect the conformational property of the polypeptide chain. We compare the effects of O-linked glycosylation by the common sugars alpha-GalNAc, beta-GlcNAc with their non-native analogs beta-GalNAc, alpha-GlcNAc in an effort to uncover the origin of the sugar-specificity on the fibril formation. We find that the anomeric configuration of the sugar is the most important factor affecting the fibrillogenesis. Sugars with the glycosidic bond in the alpha-configuration at Ser-135 have a dramatic inhibitory effect on the structural conversion of the glycosylated peptide. Because O-glycosylation of Ser-135 with alpha-linked sugars also promote the formation of three slowly converting conformations at the site of glycosylation, we surmise that the amyloidogenic property of the peptide is related to its conformational flexibility, and the proclivity of this region of the peptide to undergo the structural conversion from the random coil to form the beta-structure. Upon O-glycosylation with an alpha-linked sugar, this conversion is inhibited and the nucleation of fibril formation is largely retarded. Consistent with this scenario, Arg-136 is the residue most affected in the TOCSY NMR spectra of the glycosylated peptides, other than the serine site modified. In addition, when Arg-136 is substituted by Gly, a mutation that should provide higher structural flexibility in this part of the peptide, the amyloidogenic property of the peptide is greatly enhanced, and the inhibition effect of glycosylation is largely diminished. These results are consistent with Ser-135 and Arg-136 being part of the kink region involved in the structural conversion.

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

PubMed Central

McGowan, Lauren C.; Hamelberg, Donald

2013-01-01

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

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

PubMed

McGowan, Lauren C; Hamelberg, Donald

2013-01-08

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

Thermodynamics and docking of agonists to the β(2)-adrenoceptor determined using [(3)H](R,R')-4-methoxyfenoterol as the marker ligand.

PubMed

Toll, Lawrence; Pajak, Karolina; Plazinska, Anita; Jozwiak, Krzysztof; Jimenez, Lucita; Kozocas, Joseph A; Tanga, Mary J; Bupp, James E; Wainer, Irving W

2012-06-01

G protein-coupled receptors (GPCRs) are integral membrane proteins that change conformation after ligand binding so that they can transduce signals from an extracellular ligand to a variety of intracellular components. The detailed interaction of a molecule with a G protein-coupled receptor is a complicated process that is influenced by the receptor conformation, thermodynamics, and ligand conformation and stereoisomeric configuration. To better understand the molecular interactions of fenoterol analogs with the β(2)-adrenergic receptor, we developed a new agonist radioligand for binding assays. [(3)H](R,R')-methoxyfenoterol was used to probe the binding affinity for a series of fenoterol stereoisomers and derivatives. The results suggest that the radioligand binds with high affinity to an agonist conformation of the receptor, which represents approximately 25% of the total β(2)-adrenoceptor (AR) population as determined with the antagonist [(3)H]CGP-12177. The β(2)-AR agonists tested in this study have considerably higher affinity for the agonist conformation of the receptor, and K(i) values determined for fenoterol analogs model much better the cAMP activity of the β(2)-AR elicited by these ligands. The thermodynamics of binding are also different when interacting with an agonist conformation, being purely entropy-driven for each fenoterol isomer, rather than a mixture of entropy and enthalpy when the fenoterol isomers binding was determined using [(3)H]CGP-12177. Finally, computational modeling identified the molecular interactions involved in agonist binding and allow for the prediction of additional novel β(2)-AR agonists. The study underlines the possibility of using defined radioligand structure to probe a specific conformation of such shape-shifting system as the β(2)-adrenoceptor.

Thermodynamics and Docking of Agonists to the β2-Adrenoceptor Determined Using [3H](R,R′)-4-Methoxyfenoterol as the Marker Ligand

PubMed Central

Pajak, Karolina; Plazinska, Anita; Jozwiak, Krzysztof; Jimenez, Lucita; Kozocas, Joseph A.; Tanga, Mary J.; Bupp, James E.; Wainer, Irving W.

2012-01-01

G protein-coupled receptors (GPCRs) are integral membrane proteins that change conformation after ligand binding so that they can transduce signals from an extracellular ligand to a variety of intracellular components. The detailed interaction of a molecule with a G protein-coupled receptor is a complicated process that is influenced by the receptor conformation, thermodynamics, and ligand conformation and stereoisomeric configuration. To better understand the molecular interactions of fenoterol analogs with the β2-adrenergic receptor, we developed a new agonist radioligand for binding assays. [3H](R,R′)-methoxyfenoterol was used to probe the binding affinity for a series of fenoterol stereoisomers and derivatives. The results suggest that the radioligand binds with high affinity to an agonist conformation of the receptor, which represents approximately 25% of the total β2-adrenoceptor (AR) population as determined with the antagonist [3H]CGP-12177. The β2-AR agonists tested in this study have considerably higher affinity for the agonist conformation of the receptor, and Ki values determined for fenoterol analogs model much better the cAMP activity of the β2-AR elicited by these ligands. The thermodynamics of binding are also different when interacting with an agonist conformation, being purely entropy-driven for each fenoterol isomer, rather than a mixture of entropy and enthalpy when the fenoterol isomers binding was determined using [3H]CGP-12177. Finally, computational modeling identified the molecular interactions involved in agonist binding and allow for the prediction of additional novel β2-AR agonists. The study underlines the possibility of using defined radioligand structure to probe a specific conformation of such shape-shifting system as the β2-adrenoceptor. PMID:22434858

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

Secondary Structures in Phe-Containing Isolated Dipeptide Chains: Laser Spectroscopy vs Quantum Chemistry.

PubMed

Loquais, Yohan; Gloaguen, Eric; Habka, Sana; Vaquero-Vara, Vanesa; Brenner, Valérie; Tardivel, Benjamin; Mons, Michel

2015-06-11

The intrinsic conformational landscape of two phenylalanine-containing protein chain models (-Gly-Phe- and -Ala-Phe- sequences) has been investigated theoretically and experimentally in the gas phase. The near UV spectroscopy (first ππ* transition of the Phe ring) is obtained experimentally under jet conditions where the conformational features can be resolved. Single-conformation IR spectroscopy in the NH stretch region is then obtained by IR/UV double resonance in the ground state, leading to resolved vibrational spectra that are assigned in terms of conformation and H-bonding content from comparison with quantum chemistry calculations. For the main conformer, whose UV spectrum exhibits a significant Franck-Condon activity in low frequency modes involving peptide backbone motions relative to the Phe chromophore, excited state IR spectroscopy has also been recorded in a UV/IR/UV experiment. The NH stretch spectral changes observed in such a ππ* labeling experiment enable us to determine those NH bonds that are coupled to the phenyl ring; they are compared to CC2 excited state calculations to quantify the geometry change upon ππ* excitation. The complete and consistent series of data obtained enable us to propose an unambiguous assignment for the gallery of conformers observed and to demonstrate that, in these two sequences, three conceptually important local structural motifs of proteins (β-strands, 27 ribbons, and β-turns) are represented. The satisfactory agreement between the experimental conformational distribution and the predicted landscape anticipated from the DFT-D approach demonstrates the capabilities of a theoretical method that accounts for dispersive interactions. It also shows that the flaws, inherent to a resonant two-photon ionization detection scheme, often evoked for aromatic chromophores, do not seem to be significant in the case of Phe.

PREDICTING ER BINDING AFFINITY FOR EDC RANKING AND PRIORITIZATION: MODEL I

EPA Science Inventory

A Common Reactivity Pattern (COREPA) model, based on consideration of multiple energetically reasonable conformations of flexible chemicals was developed using a training set of 232 rat estrogen receptor (rER) relative binding affinity (RBA) measurements. The training set include...

Information Operations Team Training & Information Operations Training Aid, Information Warfare Effectiveness (IWE) Program, Delivery Order 8

DTIC Science & Technology

2010-03-01

submenus and toolbar with icon buttons 4. The IFOTA shall conform to Defense Information Infrastructure Common Operating Environment ( DII COE) and...him my business card , but it might come in the package we request via AFRL). PSYOP Instructor IWST is now called IWT (??) SME MD MD Instructor...Engineering and Software Engineering CTA Cognitive Task Analysis DII COE Defense Information Infrastructure Common Operating Environment EJB Enterprise Java

Conformational energy calculations on polypeptides and proteins: use of a statistical mechanical procedure for evaluating structure and properties.

PubMed

Scheraga, H A; Paine, G H

1986-01-01

We are using a variety of theoretical and computational techniques to study protein structure, protein folding, and higher-order structures. Our earlier work involved treatments of liquid water and aqueous solutions of nonpolar and polar solutes, computations of the stabilities of the fundamental structures of proteins and their packing arrangements, conformations of small cyclic and open-chain peptides, structures of fibrous proteins (collagen), structures of homologous globular proteins, introduction of special procedures as constraints during energy minimization of globular proteins, and structures of enzyme-substrate complexes. Recently, we presented a new methodology for predicting polypeptide structure (described here); the method is based on the calculation of the probable and average conformation of a polypeptide chain by the application of equilibrium statistical mechanics in conjunction with an adaptive, importance sampling Monte Carlo algorithm. As a test, it was applied to Met-enkephalin.

Dynamic Disorder in Quasi-Equilibrium Enzymatic Systems

PubMed Central

Chaudhury, Srabanti; Igoshin, Oleg A.

2010-01-01

Conformations and catalytic rates of enzymes fluctuate over a wide range of timescales. Despite these fluctuations, there exist some limiting cases in which the enzymatic catalytic rate follows the macroscopic rate equation such as the Michaelis-Menten law. In this paper we investigate the applicability of macroscopic rate laws for fluctuating enzyme systems in which catalytic transitions are slower than ligand binding-dissociation reactions. In this quasi-equilibrium limit, for an arbitrary reaction scheme we show that the catalytic rate has the same dependence on ligand concentrations as obtained from mass-action kinetics even in the presence of slow conformational fluctuations. These results indicate that the timescale of conformational dynamics – no matter how slow – will not affect the enzymatic rate in quasi-equilibrium limit. Our numerical results for two enzyme-catalyzed reaction schemes involving multiple substrates and inhibitors further support our general theory. PMID:20808776

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

Battles, Michael B.; Langedijk, Johannes P.; Furmanova-Hollenstein, Polina

Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in young children and the elderly. Therapeutic small molecules have been developed that bind the RSV F glycoprotein and inhibit membrane fusion, yet their binding sites and molecular mechanisms of action remain largely unknown. In this paper, we show that these inhibitors bind to a three-fold-symmetric pocket within the central cavity of the metastable prefusion conformation of RSV F. Inhibitor binding stabilizes this conformation by tethering two regions that must undergo a structural rearrangement to facilitate membrane fusion. Inhibitor-escape mutations occur in residues that directly contact the inhibitorsmore » or are involved in the conformational rearrangements required to accommodate inhibitor binding. Resistant viruses do not propagate as well as wild-type RSV in vitro, indicating a fitness cost for inhibitor escape. Finally and collectively, these findings provide new insight into class I viral fusion proteins and should facilitate development of optimal RSV fusion inhibitors.« less

Visualization of ligand-induced transmembrane signaling in the full-length human insulin receptor

PubMed Central

2018-01-01

Insulin receptor (IR) signaling plays a critical role in the regulation of metabolism and growth in multicellular organisms. IRs are unique among receptor tyrosine kinases in that they exist exclusively as covalent (αβ)2 homodimers at the cell surface. Transmembrane signaling by the IR can therefore not be based on ligand-induced dimerization as such but must involve structural changes within the existing receptor dimer. In this study, using glycosylated full-length human IR reconstituted into lipid nanodiscs, we show by single-particle electron microscopy that insulin binding to the dimeric receptor converts its ectodomain from an inverted U-shaped conformation to a T-shaped conformation. This structural rearrangement of the ectodomain propagates to the transmembrane domains, which are well separated in the inactive conformation but come close together upon insulin binding, facilitating autophosphorylation of the cytoplasmic kinase domains. PMID:29453311

Quantitative determination of the conformational properties of partially folded and intrinsically disordered proteins using NMR dipolar couplings.

PubMed

Jensen, Malene Ringkjøbing; Markwick, Phineus R L; Meier, Sebastian; Griesinger, Christian; Zweckstetter, Markus; Grzesiek, Stephan; Bernadó, Pau; Blackledge, Martin

2009-09-09

Intrinsically disordered proteins (IDPs) inhabit a conformational landscape that is too complex to be described by classical structural biology, posing an entirely new set of questions concerning the molecular understanding of functional biology. The characterization of the conformational properties of IDPs, and the elucidation of the role they play in molecular function, is therefore one of the major challenges remaining for modern structural biology. NMR is the technique of choice for studying this class of proteins, providing information about structure, flexibility, and interactions at atomic resolution even in completely disordered states. In particular, residual dipolar couplings (RDCs) have been shown to be uniquely sensitive and powerful tools for characterizing local and long-range structural behavior in disordered proteins. In this review we describe recent applications of RDCs to quantitatively describe the level of local structure and transient long-range order in IDPs involved in viral replication, neurodegenerative disease, and cancer.

Hot topic: Bovine milk samples yielding negative or nonspecific results in bacterial culturing--the possible role of PCR-single strand conformation polymorphism in mastitis diagnosis.

PubMed

Schwaiger, K; Wimmer, M; Huber-Schlenstedt, R; Fehlings, K; Hölzel, C S; Bauer, J

2012-01-01

A large proportion of mastitis milk samples yield negative or nonspecific results (i.e., no mastitis pathogen can be identified) in bacterial culturing. Therefore, the culture-independent PCR-single strand conformation polymorphism method was applied to the investigation of bovine mastitis milk samples. In addition to the known mastitis pathogens, the method was suitable for the detection of fastidious bacteria such as Mycoplasma spp., which are often missed by conventional culturing methods. The detection of Helcococcus ovis in 4 samples might indicate an involvement of this species in pathogenesis of bovine mastitis. In conclusion, PCR-single-strand conformation polymorphism is a promising tool for gaining new insights into the bacteriological etiology of mastitis. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Design of a novel freeform lens for LED uniform illumination and conformal phosphor coating.

PubMed

Hu, Run; Luo, Xiaobing; Zheng, Huai; Qin, Zong; Gan, Zhiqiang; Wu, Bulong; Liu, Sheng

2012-06-18

A conformal phosphor coating can realize a phosphor layer with uniform thickness, which could enhance the angular color uniformity (ACU) of light-emitting diode (LED) packaging. In this study, a novel freeform lens was designed for simultaneous realization of LED uniform illumination and conformal phosphor coating. The detailed algorithm of the design method, which involves an extended light source and double refractions, was presented. The packaging configuration of the LED modules and the modeling of the light-conversion process were also presented. Monte Carlo ray-tracing simulations were conducted to validate the design method by comparisons with a conventional freeform lens. It is demonstrated that for the LED module with the present freeform lens, the illumination uniformity and ACU was 0.89 and 0.9283, respectively. The present freeform lens can realize equivalent illumination uniformity, but the angular color uniformity can be enhanced by 282.3% when compared with the conventional freeform lens.

Molecular mechanism of respiratory syncytial virus fusion inhibitors

PubMed Central

Battles, Michael B; Langedijk, Johannes P; Furmanova-Hollenstein, Polina; Chaiwatpongsakorn, Supranee; Costello, Heather M; Kwanten, Leen; Vranckx, Luc; Vink, Paul; Jaensch, Steffen; Jonckers, Tim H M; Koul, Anil; Arnoult, Eric; Peeples, Mark E; Roymans, Dirk; McLellan, Jason S

2016-01-01

Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in young children and the elderly. Therapeutic small molecules have been developed that bind the RSV F glycoprotein and inhibit membrane fusion, yet their binding sites and molecular mechanisms of action remain largely unknown. Here we show that these inhibitors bind to a three-fold-symmetric pocket within the central cavity of the metastable prefusion conformation of RSV F. Inhibitor binding stabilizes this conformation by tethering two regions that must undergo a structural rearrangement to facilitate membrane fusion. Inhibitor-escape mutations occur in residues that directly contact the inhibitors or are involved in the conformational rearrangements required to accommodate inhibitor binding. Resistant viruses do not propagate as well as wild-type RSV in vitro, indicating a fitness cost for inhibitor escape. Collectively, these findings provide new insight into class I viral fusion proteins and should facilitate development of optimal RSV fusion inhibitors. PMID:26641933

Structural basis of lipid-driven conformational transitions in the KvAP voltage-sensing domain.

PubMed

Li, Qufei; Wanderling, Sherry; Sompornpisut, Pornthep; Perozo, Eduardo

2014-02-01

Voltage-gated ion channels respond to transmembrane electric fields through reorientations of the positively charged S4 helix within the voltage-sensing domain (VSD). Despite a wealth of structural and functional data, the details of this conformational change remain controversial. Recent electrophysiological evidence showed that equilibrium between the resting ('down') and activated ('up') conformations of the KvAP VSD from Aeropyrum pernix can be biased through reconstitution in lipids with or without phosphate groups. We investigated the structural transition between these functional states, using site-directed spin-labeling and EPR spectroscopic methods. Solvent accessibility and interhelical distance determinations suggest that KvAP gates through S4 movements involving an ∼3-Å upward tilt and simultaneous ∼2-Å axial shift. This motion leads to large accessibly changes in the intracellular water-filled crevice and supports a new model of gating that combines structural rearrangements and electric-field remodeling.

Structure of the parainfluenza virus 5 F protein in its metastable, prefusion conformation.

PubMed

Yin, Hsien-Sheng; Wen, Xiaolin; Paterson, Reay G; Lamb, Robert A; Jardetzky, Theodore S

2006-01-05

Enveloped viruses have evolved complex glycoprotein machinery that drives the fusion of viral and cellular membranes, permitting entry of the viral genome into the cell. For the paramyxoviruses, the fusion (F) protein catalyses this membrane merger and entry step, and it has been postulated that the F protein undergoes complex refolding during this process. Here we report the crystal structure of the parainfluenza virus 5 F protein in its prefusion conformation, stabilized by the addition of a carboxy-terminal trimerization domain. The structure of the F protein shows that there are profound conformational differences between the pre- and postfusion states, involving transformations in secondary and tertiary structure. The positions and structural transitions of key parts of the fusion machinery, including the hydrophobic fusion peptide and two helical heptad repeat regions, clarify the mechanism of membrane fusion mediated by the F protein.

Functional Advantages of Conserved Intrinsic Disorder in RNA-Binding Proteins.

PubMed

Varadi, Mihaly; Zsolyomi, Fruzsina; Guharoy, Mainak; Tompa, Peter

2015-01-01

Proteins form large macromolecular assemblies with RNA that govern essential molecular processes. RNA-binding proteins have often been associated with conformational flexibility, yet the extent and functional implications of their intrinsic disorder have never been fully assessed. Here, through large-scale analysis of comprehensive protein sequence and structure datasets we demonstrate the prevalence of intrinsic structural disorder in RNA-binding proteins and domains. We addressed their functionality through a quantitative description of the evolutionary conservation of disordered segments involved in binding, and investigated the structural implications of flexibility in terms of conformational stability and interface formation. We conclude that the functional role of intrinsically disordered protein segments in RNA-binding is two-fold: first, these regions establish extended, conserved electrostatic interfaces with RNAs via induced fit. Second, conformational flexibility enables them to target different RNA partners, providing multi-functionality, while also ensuring specificity. These findings emphasize the functional importance of intrinsically disordered regions in RNA-binding proteins.

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

PubMed Central

Peng, Enxi; Todorova, Nevena

2017-01-01

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

"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.

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

PubMed

Peng, Enxi; Todorova, Nevena; Yarovsky, Irene

2017-01-01

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

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

PubMed

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

2017-06-13

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

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

PubMed Central

2017-01-01

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

Conformational analysis of the anti-HIV Nikavir prodrug: comparisons with AZT and Thymidine, and establishment of structure-activity relationships/tendencies in other 6'-derivatives.

PubMed

El-Sayed, Ahmed A; Tamara Molina, A; Álvarez-Ros, M C; Alcolea Palafox, M

2015-01-01

A comprehensive theoretical conformational analysis of the anti-HIV Nikavir prodrug was carried out; this prodrug has noticeable advantage over the approved drug AZT. The whole conformational parameters (χ, α, β, γ, δ, ϕ, P and νmax) were analysed as well as the NBO natural atomic charges. The calculations were carried out by means of DFT/B3LYP and ab initio MP2 methods with full relaxation of all geometrical parameters. The search located at least 67 stable structures, 4 of which were within a 1 kcal/mol electronic energy range of the global minimum. By MP2 it corresponds to the calculated values of the exocyclic torsional angles χ=-108.0°, β=14.5°, γ=76.7° and ε=71.5°. The results obtained are in accordance to those found in related anti-HIV nucleoside analogues. Comparisons of the conformers with those determined in the common anti-HIV drug AZT were carried out. A detailed analysis of the lowest vibrations (<200 cm(-1)) in the best conformer of Nikavir was carried out. The most stable hydrated cluster of this conformer with 20 explicit water molecules was determined. Calculations in five of its 6'-derivatives were performed to identify structural trends that might correlate with the anti-HIV activity of these compounds. Ten structure-activity relationships/tendencies were established that can help for the design of new drugs. Several recommendations for this design were expressed.

Cholic acid derivatives containing both 2-naphthylcarbamate and 3,5-dinitrophenylcarbamate groups: a combined circular dichroism-molecular mechanics approach to the definition of their molecular conformation.

PubMed

Alagona, Giuliano; Ghio, Caterina; Iuliano, Anna; Monti, Susanna; Pieraccini, Ilaria; Salvadori, Piero

2003-04-18

CD spectra of the chiral auxiliaries for enantioselective HPLC N-allyl-N'-methyl-3,12-bis(2-naphthyl)carbamoyloxy-7-(3,5-dinitrophenyl)carbamoyloxycholan-24-amide (1), N-allyl-N'-methyl-3-(3,5-dinitrophenyl)carbamoyloxy-7,12-bis(2-naphthyl)carbamoyloxycholan-24-amide (2), N-allyl-N'-methyl-3,7-bis(2-naphthyl)carbamoyloxy-12-(3,5-dinitrophenyl)carbamoyloxycholan-24-amide (3), and N-allyl-N'-methyl-3,7,12-tris(2-naphthyl)carbamoyloxycholan-24-amide (4) are presented. To determine the preferred conformations of those chiral auxiliaries, a random search based on the aromatic side-chain conformational degrees of freedom was performed and the energy was minimized using two different molecular mechanics force fields. The low energy structures presenting common features were arranged in groups and selected exploiting appropriate filters. The calculation of theoretical CD spectra according to the De Voe model has allowed a further discrimination among the conformations, specifying which of them gave calculated CD spectra in acceptable agreement with the experimental ones. Finally, taking into account the additivity of the contributions of each 2-naphthylcarbamate chromophore to the CD spectrum of the cholic acid derivatives, and, hence, choosing, for derivatives 1-3, those conformations in which the 2-naphthylcarbamate groups take a similar disposition as in 4, the preferentially assumed conformation of each compound was obtained. A molecular dynamics simulation in the presence of acetonitrile allowed the fluctuations of one of the structures, used as a test case, depending on environmental effects, to be examined.

Exploring the conformational and binding properties of unphosphorylated/phosphorylated monomeric and trimeric Bcl-2 through docking and molecular dynamics simulations.

PubMed

Zacarías-Lara, Oscar J; Correa-Basurto, José; Bello, Martiniano

2016-07-01

B-cell lymphoma (Bcl-2) is commonly associated with the progression and preservation of cancer and certain lymphomas; therefore, it is considered as a biological target against cancer. Nevertheless, evidence of all its structural binding sites has been hidden because of the lack of a complete Bcl-2 model, given the presence of a flexible loop domain (FLD), which is responsible for its complex behavior. FLD region has been implicated in phosphorylation, homotrimerization, and heterodimerization associated with Bcl-2 antiapoptotic function. In this contribution, homology modeling, molecular dynamics (MD) simulations in the microsecond (µs) time-scale and docking calculations were combined to explore the conformational complexity of unphosphorylated/phosphorylated monomeric and trimeric Bcl-2 systems. Conformational ensembles generated through MD simulations allowed for identifying the most populated unphosphorylated/phosphorylated monomeric conformations, which were used as starting models to obtain trimeric complexes through protein-protein docking calculations, also submitted to µs MD simulations. Principal component analysis showed that FLD represents the main contributor to total Bcl-2 mobility, and is affected by phosphorylation and oligomerization. Subsequently, based on the most representative unphosphorylated/phosphorylated monomeric and trimeric Bcl-2 conformations, docking studies were initiated to identify the ligand binding site of several known Bcl-2 inhibitors to explain their influence in homo-complex formation and phosphorylation. Docking studies showed that the different conformational states experienced by FLD, such as phosphorylation and oligomerization, play an essential role in the ability to make homo and hetero-complexes. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 393-413, 2016. © 2016 Wiley Periodicals, Inc.

Synthesis and pharmacological evaluation of conformationally constrained glutamic acid higher homologues.

PubMed

Tamborini, Lucia; Cullia, Gregorio; Nielsen, Birgitte; De Micheli, Carlo; Conti, Paola; Pinto, Andrea

2016-11-15

Homologation of glutamic acid chain together with conformational constraint is a commonly used strategy to achieve selectivity towards different types of glutamate receptors. In the present work, starting from two potent and selective unnatural amino acids previously developed by us, we investigated the effects on the activity/selectivity profile produced by a further increase in the distance between the amino acidic moiety and the distal carboxylate group. Interestingly, the insertion of an aromatic ring as a spacer produced a low micromolar affinity NMDA ligand that might represent a lead for the development of a new class of NMDA antagonists. Copyright © 2016 Elsevier Ltd. All rights reserved.

Incorporating physically-based microstructures in materials modeling: Bridging phase field and crystal plasticity frameworks

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

Lim, Hojun; Abdeljawad, Fadi; Owen, Steven J.

Here, the mechanical properties of materials systems are highly influenced by various features at the microstructural level. The ability to capture these heterogeneities and incorporate them into continuum-scale frameworks of the deformation behavior is considered a key step in the development of complex non-local models of failure. In this study, we present a modeling framework that incorporates physically-based realizations of polycrystalline aggregates from a phase field (PF) model into a crystal plasticity finite element (CP-FE) framework. Simulated annealing via the PF model yields ensembles of materials microstructures with various grain sizes and shapes. With the aid of a novel FEmore » meshing technique, FE discretizations of these microstructures are generated, where several key features, such as conformity to interfaces, and triple junction angles, are preserved. The discretizations are then used in the CP-FE framework to simulate the mechanical response of polycrystalline α-iron. It is shown that the conformal discretization across interfaces reduces artificial stress localization commonly observed in non-conformal FE discretizations. The work presented herein is a first step towards incorporating physically-based microstructures in lieu of the overly simplified representations that are commonly used. In broader terms, the proposed framework provides future avenues to explore bridging models of materials processes, e.g. additive manufacturing and microstructure evolution of multi-phase multi-component systems, into continuum-scale frameworks of the mechanical properties.« less

Incorporating physically-based microstructures in materials modeling: Bridging phase field and crystal plasticity frameworks

DOE PAGES

Lim, Hojun; Abdeljawad, Fadi; Owen, Steven J.; ...

2016-04-25

Here, the mechanical properties of materials systems are highly influenced by various features at the microstructural level. The ability to capture these heterogeneities and incorporate them into continuum-scale frameworks of the deformation behavior is considered a key step in the development of complex non-local models of failure. In this study, we present a modeling framework that incorporates physically-based realizations of polycrystalline aggregates from a phase field (PF) model into a crystal plasticity finite element (CP-FE) framework. Simulated annealing via the PF model yields ensembles of materials microstructures with various grain sizes and shapes. With the aid of a novel FEmore » meshing technique, FE discretizations of these microstructures are generated, where several key features, such as conformity to interfaces, and triple junction angles, are preserved. The discretizations are then used in the CP-FE framework to simulate the mechanical response of polycrystalline α-iron. It is shown that the conformal discretization across interfaces reduces artificial stress localization commonly observed in non-conformal FE discretizations. The work presented herein is a first step towards incorporating physically-based microstructures in lieu of the overly simplified representations that are commonly used. In broader terms, the proposed framework provides future avenues to explore bridging models of materials processes, e.g. additive manufacturing and microstructure evolution of multi-phase multi-component systems, into continuum-scale frameworks of the mechanical properties.« less

Conformational transition in signal transduction: metastable states and transition pathways in the activation of a signaling protein.

PubMed

Banerjee, Rahul; Yan, Honggao; Cukier, Robert I

2015-06-04

Signal transduction is of vital importance to the growth and adaptation of living organisms. The key to understand mechanisms of biological signal transduction is elucidation of the conformational dynamics of its signaling proteins, as the activation of a signaling protein is fundamentally a process of conformational transition from an inactive to an active state. A predominant form of signal transduction for bacterial sensing of environmental changes in the wild or inside their hosts is a variety of two-component systems, in which the conformational transition of a response regulator (RR) from an inactive to an active state initiates responses to the environmental changes. Here, RR activation has been investigated using RR468 as a model system by extensive unbiased all-atom molecular dynamics (MD) simulations in explicit solvent, starting from snapshots along a targeted MD trajectory that covers the conformational transition. Markov state modeling, transition path theory, and geometric analyses of the wealth of the MD data have provided a comprehensive description of the RR activation. It involves a network of metastable states, with one metastable state essentially the same as the inactive state and another very similar to the active state that are connected via a small set of intermediates. Five major pathways account for >75% of the fluxes of the conformational transition from the inactive to the active-like state. The thermodynamic stability of the states and the activation barriers between states are found, to identify rate-limiting steps. The conformal transition is initiated predominantly by movements of the β3α3 loop, followed by movements of the β4α4-loop and neighboring α4 helix region, and capped by additional movements of the β3α3 loop. A number of transient hydrophobic and hydrogen bond interactions are revealed, and they may be important for the conformational transition.

Nucleophilic modification of human complement protein C3: correlation of conformational changes with acquisition of C3b-like functional properties.

PubMed

Isenman, D E; Kells, D I; Cooper, N R; Müller-Eberhard, H J; Pangburn, M K

1981-07-21

Inactivation of C3 by enzymatic cleavage, nucleophilic addition, or slow freezing and thawing resulted in the acquisition of similar end-state conformations as judged by near-UV circular dichroism. Although inactivation by the two nonenzymatic processes involves no peptide bond scission, the inactivated C3 resembled C3b in that it possessed a free sulfhydryl group not present in the native protein and an increased surface hydrophobicity as evidenced by enhanced binding of the fluorophore 8-anilino-1-naphthalensulfonate (ANS). The C3b-like functional properties of modified C3 [Pangburn, M. K., & Müller-Eberhard, H. J. (1980) J. Exp. Med. 152, 1102-1114] may thus be understood in terms of the similarity of its conformation to that of C3b. The rate of the conformational change following proteolytic cleavage was fast and appeared to be limited by the rate of the enzymatic reaction. In contrast, the rate of conformational change following addition of methylamine was slow and rate limited by the conformational rearrangement itself, not by the chemical modification. A kinetic analysis of the changes in circular dichroism and ANS fluorescence enhancement suggested that the nucleophilic addition was spectroscopically undetectable and was followed by a minimally biphasic, spectroscopically demonstrable conformational rearrangement. The appearance of C3b-like functional activity in nucleophile-modified C3 largely parallels the time course of the spectroscopically detectable conformational change but is distinctly slower than the rate at which hemolytic activity is lost. While fully transconformed methylamine-inactivated C3 can bind factor B and is susceptible to cleavage by C3b inactivator and its cofactor beta 1H, this cleavage occurs at a substantially slower rate than the equivalent process in C3b. The implications of these findings in terms of the mechanism through which the alterative pathway of complement is initiated are discussed.

Computational ligand-based rational design: Role of conformational sampling and force fields in model development.

PubMed

Shim, Jihyun; Mackerell, Alexander D

2011-05-01

A significant number of drug discovery efforts are based on natural products or high throughput screens from which compounds showing potential therapeutic effects are identified without knowledge of the target molecule or its 3D structure. In such cases computational ligand-based drug design (LBDD) can accelerate the drug discovery processes. LBDD is a general approach to elucidate the relationship of a compound's structure and physicochemical attributes to its biological activity. The resulting structure-activity relationship (SAR) may then act as the basis for the prediction of compounds with improved biological attributes. LBDD methods range from pharmacophore models identifying essential features of ligands responsible for their activity, quantitative structure-activity relationships (QSAR) yielding quantitative estimates of activities based on physiochemical properties, and to similarity searching, which explores compounds with similar properties as well as various combinations of the above. A number of recent LBDD approaches involve the use of multiple conformations of the ligands being studied. One of the basic components to generate multiple conformations in LBDD is molecular mechanics (MM), which apply an empirical energy function to relate conformation to energies and forces. The collection of conformations for ligands is then combined with functional data using methods ranging from regression analysis to neural networks, from which the SAR is determined. Accordingly, for effective application of LBDD for SAR determinations it is important that the compounds be accurately modelled such that the appropriate range of conformations accessible to the ligands is identified. Such accurate modelling is largely based on use of the appropriate empirical force field for the molecules being investigated and the approaches used to generate the conformations. The present chapter includes a brief overview of currently used SAR methods in LBDD followed by a more detailed presentation of issues and limitations associated with empirical energy functions and conformational sampling methods.

Intramolecular BSSE and dispersion affect the structure of a dipeptide conformer

NASA Astrophysics Data System (ADS)

Hameed, Rabia; Khan, Afsar; van Mourik, Tanja

2018-05-01

B3LYP and MP2 calculations with the commonly-used 6-31+G(d) basis set predict qualitatively different structures for the Tyr-Gly conformer book1, which is the most stable conformer identified in a previous study. The structures differ mainly in the ψtyr Ramachandran angle (138° in the B3LYP structure and 120° in the MP2 structure). The causes for the discrepant structures are attributed to missing dispersion in the B3LYP calculations and large intramolecular BSSE in the MP2 calculations. The correct ψtyr value is estimated to be 130°. The MP2/6-31+G(d) profile identified an additional conformer, not present on the B3LYP surface, with a ψtyr value of 96° and a more folded structure. This minimum is, however, likely an artefact of large intramolecular BSSE values. We recommend the use of basis sets of at least quadruple-zeta quality in density functional theory (DFT), DFTaugmented with an empirical dispersion term (DFT-D) and second-order Møller-Plesset perturbation theory (MP2 ) calculations in cases where intramolecular BSSE is expected to be large.

Conformational Explosion: Understanding the Complexity of the Para-Dialkylbenzene Potential Energy Surfaces

NASA Astrophysics Data System (ADS)

Mishra, Piyush; Hewett, Daniel M.; Zwier, Timothy S.

2017-06-01

This talk focuses on the single-conformation spectroscopy of small-chain para-dialkylbenzenes. This work builds on previous studies from our group on long-chain n-alkylbenzenes that identified the first folded structure in octylbenzene. The dialkylbenzenes are representative of a class of molecules that are common components of coal and aviation fuel and are known to be present in vehicle exhaust. We bring the molecules para-diethylbenzene, para-dipropylbenzene and para-dibutylbenzene into the gas phase and cool the molecules in a supersonic expansion. The jet-cooled molecules are then interrogated using laser-induced fluorescence excitation, fluorescence dip IR spectroscopy (FDIRS) and dispersed fluorescence. The LIF spectra in the S_{0}-S_{1} origin region show dramatic increases in the number of resolved transitions with increasing length of alkyl chains, reflecting an explosion in the number of unique low-energy conformations formed when two independent alkyl chains are present. Since the barriers to isomerization of the alkyl chain are similar in size, this results in an 'egg carton' shape to the potential energy surface. We use a combination of electronic frequency shift and alkyl CH stretch infrared spectra to generate a consistent set of conformational assignments.

Avalanche for shape and feature-based virtual screening with 3D alignment

NASA Astrophysics Data System (ADS)

Diller, David J.; Connell, Nancy D.; Welsh, William J.

2015-11-01

This report introduces a new ligand-based virtual screening tool called Avalanche that incorporates both shape- and feature-based comparison with three-dimensional (3D) alignment between the query molecule and test compounds residing in a chemical database. Avalanche proceeds in two steps. The first step is an extremely rapid shape/feature based comparison which is used to narrow the focus from potentially millions or billions of candidate molecules and conformations to a more manageable number that are then passed to the second step. The second step is a detailed yet still rapid 3D alignment of the remaining candidate conformations to the query conformation. Using the 3D alignment, these remaining candidate conformations are scored, re-ranked and presented to the user as the top hits for further visualization and evaluation. To provide further insight into the method, the results from two prospective virtual screens are presented which show the ability of Avalanche to identify hits from chemical databases that would likely be missed by common substructure-based or fingerprint-based search methods. The Avalanche method is extended to enable patent landscaping, i.e., structural refinements to improve the patentability of hits for deployment in drug discovery campaigns.

Using THz Spectroscopy, Evolutionary Network Analysis Methods, and MD Simulation to Map the Evolution of Allosteric Communication Pathways in c-Type Lysozymes.

PubMed

Woods, Kristina N; Pfeffer, Juergen

2016-01-01

It is now widely accepted that protein function is intimately tied with the navigation of energy landscapes. In this framework, a protein sequence is not described by a distinct structure but rather by an ensemble of conformations. And it is through this ensemble that evolution is able to modify a protein's function by altering its landscape. Hence, the evolution of protein functions involves selective pressures that adjust the sampling of the conformational states. In this work, we focus on elucidating the evolutionary pathway that shaped the function of individual proteins that make-up the mammalian c-type lysozyme subfamily. Using both experimental and computational methods, we map out specific intermolecular interactions that direct the sampling of conformational states and accordingly, also underlie shifts in the landscape that are directly connected with the formation of novel protein functions. By contrasting three representative proteins in the family we identify molecular mechanisms that are associated with the selectivity of enhanced antimicrobial properties and consequently, divergent protein function. Namely, we link the extent of localized fluctuations involving the loop separating helices A and B with shifts in the equilibrium of the ensemble of conformational states that mediate interdomain coupling and concurrently moderate substrate binding affinity. This work reveals unique insights into the molecular level mechanisms that promote the progression of interactions that connect the immune response to infection with the nutritional properties of lactation, while also providing a deeper understanding about how evolving energy landscapes may define present-day protein function. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The distal C-terminal region of the KcsA potassium channel is a pH-dependent tetramerization domain.

PubMed

Kamnesky, Guy; Shaked, Hadassa; Chill, Jordan H

2012-05-04

The intracellular C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is a 40-residue-long segment that natively adopts a helical bundle conformation with 4-fold symmetry. A hallmark of KcsA behavior is pH-induced conformational change, which leads to the opening of the channel at acidic pH. Previous studies have reached conflicting conclusions as to the role of the CTD in this transition. Here, we investigate the involvement of this domain in pH-mediated channel opening by NMR using a soluble peptide corresponding to residues 128-160 of the CTD (CTD34). At neutral pH, CTD34 exhibits concentration-dependent spectral changes consistent with oligomer formation. We prove this slowly tumbling species to be a tetramer with a dissociation constant of (2.0±0.5)×10(-)(11) M(3) by NMR and sedimentation equilibrium experiments. Whereas monomeric CTD34 is only mildly helical, secondary chemical shifts prove that the tetrameric species adopts a tight native-like helical bundle conformation. The tetrameric species undergoes pH-dependent dissociation, and CTD34 is fully monomeric below pH 5.0. The structural basis for this phenomenon is the destabilization of the tetrameric CTD34 by protonation of residue H145 in the monomeric form of the peptide. We conclude that (i) the CTD34 peptide is independently capable of forming a tetrameric helical bundle, and (ii) this structurally significant conformational shift is modulated by the effects of solution pH on residue H145. Therefore, the involvement of this domain in the pH gating of the channel is strongly suggested. Copyright © 2012 Elsevier Ltd. All rights reserved.

Unbiased Simulations Reveal the Inward-Facing Conformation of the Human Serotonin Transporter and Na+ Ion Release

PubMed Central

Koldsø, Heidi; Noer, Pernille; Grouleff, Julie; Autzen, Henriette Elisabeth; Sinning, Steffen; Schiøtt, Birgit

2011-01-01

Monoamine transporters are responsible for termination of synaptic signaling and are involved in depression, control of appetite, and anxiety amongst other neurological processes. Despite extensive efforts, the structures of the monoamine transporters and the transport mechanism of ions and substrates are still largely unknown. Structural knowledge of the human serotonin transporter (hSERT) is much awaited for understanding the mechanistic details of substrate translocation and binding of antidepressants and drugs of abuse. The publication of the crystal structure of the homologous leucine transporter has resulted in homology models of the monoamine transporters. Here we present extended molecular dynamics simulations of an experimentally supported homology model of hSERT with and without the natural substrate yielding a total of more than 1.5 µs of simulation of the protein dimer. The simulations reveal a transition of hSERT from an outward-facing occluded conformation to an inward-facing conformation in a one-substrate-bound state. Simulations with a second substrate in the proposed symport effector site did not lead to conformational changes associated with translocation. The central substrate binding site becomes fully exposed to the cytoplasm leaving both the Na+-ion in the Na2-site and the substrate in direct contact with the cytoplasm through water interactions. The simulations reveal how sodium is released and show indications of early events of substrate transport. The notion that ion dissociation from the Na2-site drives translocation is supported by experimental studies of a Na2-site mutant. Transmembrane helices (TMs) 1 and 6 are identified as the helices involved in the largest movements during transport. PMID:22046120

Proton relays in anomalous carbocations dictate spectroscopy, stability, and mechanisms: case studies on C2H5+ and C3H3.

PubMed

Sager, LeeAnn M; Iyengar, Srinivasan S

2017-10-18

We present a detailed analysis of the anomalous carbocations: C 2 H 5 + and C 3 H 3 + . This work involves (a) probing electronic structural properties, (b) ab initio dynamics simulations over a range of internal energies, (c) analysis of reduced dimensional potential surfaces directed along selected conformational transition pathways, (d) dynamically averaged vibrational spectra computed from ab initio dynamics trajectories, and (e) two-dimensional time-frequency analysis to probe conformational dynamics. Key findings are as follows: (i) as noted in our previous study on C 2 H 3 + , it appears that these non-classical carbocations are stabilized by delocalized nuclear frameworks and "proton shuttles". We analyze this nuclear delocalization and find critical parallels between conformational changes in C 2 H 3 + , C 2 H 5 + , and C 3 H 3 + . (ii) The vibrational signatures of C 2 H 5 + are dominated by the "bridge-proton" conformation, but also show critical contributions from the "classical" configuration, which is a transition state at almost all levels of theory. This result is further substantiated through two-dimensional time-frequency analysis and is at odds with earlier explanations of the experimental spectra, where frequencies close to the classical region were thought to arise from an impurity. While this is still possible, our results here indicate an additional (perhaps more likely) explanation that involves the "classical" isomer. (iii) Finally, in the case of C 3 H 3 + our explanation of the experimental result includes the presence of multiple, namely, "cyclic", "straight", and propargyl, configurations. Proton shuttles and nuclear delocalization, reminiscent of those seen in the case of C 2 H 3 + , were seen all through and have a critical role in all our observations.

Structural Characterization of Early Michaelis Complexes in the Reaction Catalyzed by (+)-Limonene Synthase from Citrus sinensis Using Fluorinated Substrate Analogues.

PubMed

Kumar, Ramasamy P; Morehouse, Benjamin R; Matos, Jason O; Malik, Karan; Lin, Hongkun; Krauss, Isaac J; Oprian, Daniel D

2017-03-28

The stereochemical course of monoterpene synthase reactions is thought to be determined early in the reaction sequence by selective binding of distinct conformations of the geranyl diphosphate (GPP) substrate. We explore here formation of early Michaelis complexes of the (+)-limonene synthase [(+)-LS] from Citrus sinensis using monofluorinated substrate analogues 2-fluoro-GPP (FGPP) and 2-fluoroneryl diphosphate (FNPP). Both are competitive inhibitors for (+)-LS with K I values of 2.4 ± 0.5 and 39.5 ± 5.2 μM, respectively. The K I values are similar to the K M for the respective nonfluorinated substrates, indicating that fluorine does not significantly perturb binding of the ligand to the enzyme. FGPP and FNPP are also substrates, but with dramatically reduced rates (k cat values of 0.00054 ± 0.00005 and 0.00024 ± 0.00002 s -1 , respectively). These data are consistent with a stepwise mechanism for (+)-LS involving ionization of the allylic GPP substrate to generate a resonance-stabilized carbenium ion in the rate-limiting step. Crystals of apo-(+)-LS were soaked with FGPP and FNPP to obtain X-ray structures at 2.4 and 2.2 Å resolution, respectively. The fluorinated analogues are found anchored in the active site through extensive interactions involving the diphosphate, three metal ions, and three active-site Asp residues. Electron density for the carbon chains extends deep into a hydrophobic pocket, while the enzyme remains mostly in the open conformation observed for the apoprotein. While FNPP was found in multiple conformations, FGPP, importantly, was in a single, relatively well-defined, left-handed screw conformation, consistent with predictions for the mechanism of stereoselectivity in the monoterpene synthases.

Receptor signaling: when dimerization is not enough.

PubMed

Jiang, G; Hunter, T

Activation of receptors that signal via tyrosine kinase domains has been thought to involve receptor dimerization and transphosphorylation of juxtaposed catalytic domains. Recent results suggest things might be more complex - specific intersubunit conformational changes within a dimer can also be important.

Predicting folding-unfolding transitions in proteins without a priori knowledge of the folded state

NASA Astrophysics Data System (ADS)

Okan, Osman; Turgut, Deniz; Garcia, Angel; Ozisik, Rahmi

2013-03-01

The common computational method of studying folding transitions in proteins is to compare simulated conformations against the folded structure, but this method obviously requires the folded structure to be known beforehand. In the current study, we show that the use of bond orientational order parameter (BOOP) Ql [Steinhardt PJ, Nelson DR, Ronchetti M, Phys. Rev. B 1983, 28, 784] is a viable alternative to the commonly adopted root mean squared distance (RMSD) measure in probing conformational transitions. Replica exchange molecular dynamics simulations of the trp-cage protein (with 20 residues) in TIP-3P water were used to compare BOOP against RMSD. The results indicate that the correspondence between BOOP and RMSD time series become stronger with increasing l. We finally show that robust linear models that incorporate different Ql can be parameterized from a given replica run and can be used to study other replica trajectories. This work is partially supported by NSF DUE-1003574.

Gonadotropin-Releasing Hormone (GnRH) Receptor Structure and GnRH Binding

PubMed Central

Flanagan, Colleen A.; Manilall, Ashmeetha

2017-01-01

Gonadotropin-releasing hormone (GnRH) regulates reproduction. The human GnRH receptor lacks a cytoplasmic carboxy-terminal tail but has amino acid sequence motifs characteristic of rhodopsin-like, class A, G protein-coupled receptors (GPCRs). This review will consider how recent descriptions of X-ray crystallographic structures of GPCRs in inactive and active conformations may contribute to understanding GnRH receptor structure, mechanism of activation and ligand binding. The structures confirmed that ligands bind to variable extracellular surfaces, whereas the seven membrane-spanning α-helices convey the activation signal to the cytoplasmic receptor surface, which binds and activates heterotrimeric G proteins. Forty non-covalent interactions that bridge topologically equivalent residues in different transmembrane (TM) helices are conserved in class A GPCR structures, regardless of activation state. Conformation-independent interhelical contacts account for a conserved receptor protein structure and their importance in the GnRH receptor structure is supported by decreased expression of receptors with mutations of residues in the network. Many of the GnRH receptor mutations associated with congenital hypogonadotropic hypogonadism, including the Glu2.53(90) Lys mutation, involve amino acids that constitute the conserved network. Half of the ~250 intramolecular interactions in GPCRs differ between inactive and active structures. Conformation-specific interhelical contacts depend on amino acids changing partners during activation. Conserved inactive conformation-specific contacts prevent receptor activation by stabilizing proximity of TM helices 3 and 6 and a closed G protein-binding site. Mutations of GnRH receptor residues involved in these interactions, such as Arg3.50(139) of the DRY/S motif or Tyr7.53(323) of the N/DPxxY motif, increase or decrease receptor expression and efficiency of receptor coupling to G protein signaling, consistent with the native residues stabilizing the inactive GnRH receptor structure. Active conformation-specific interhelical contacts stabilize an open G protein-binding site. Progress in defining the GnRH-binding site has recently slowed, with evidence that Tyr6.58(290) contacts Tyr5 of GnRH, whereas other residues affect recognition of Trp3 and Gly10NH2. The surprisingly consistent observations that GnRH receptor mutations that disrupt GnRH binding have less effect on “conformationally constrained” GnRH peptides may now be explained by crystal structures of agonist-bound peptide receptors. Analysis of GPCR structures provides insight into GnRH receptor function. PMID:29123501

Modeling competitive substitution in a polyelectrolyte complex

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

Peng, B.; Muthukumar, M., E-mail: muthu@polysci.umass.edu

2015-12-28

We have simulated the invasion of a polyelectrolyte complex made of a polycation chain and a polyanion chain, by another longer polyanion chain, using the coarse-grained united atom model for the chains and the Langevin dynamics methodology. Our simulations reveal many intricate details of the substitution reaction in terms of conformational changes of the chains and competition between the invading chain and the chain being displaced for the common complementary chain. We show that the invading chain is required to be sufficiently longer than the chain being displaced for effecting the substitution. Yet, having the invading chain to be longermore » than a certain threshold value does not reduce the substitution time much further. While most of the simulations were carried out in salt-free conditions, we show that presence of salt facilitates the substitution reaction and reduces the substitution time. Analysis of our data shows that the dominant driving force for the substitution process involving polyelectrolytes lies in the release of counterions during the substitution.« less

AgHalo: A Facile Fluorogenic Sensor to Detect Drug-Induced Proteome Stress.

PubMed

Liu, Yu; Fares, Matthew; Dunham, Noah P; Gao, Zi; Miao, Kun; Jiang, Xueyuan; Bollinger, Samuel S; Boal, Amie K; Zhang, Xin

2017-07-17

Drug-induced proteome stress that involves protein aggregation may cause adverse effects and undermine the safety profile of a drug. Safety of drugs is regularly evaluated using cytotoxicity assays that measure cell death. However, these assays provide limited insights into the presence of proteome stress in live cells. A fluorogenic protein sensor is reported to detect drug-induced proteome stress prior to cell death. An aggregation prone Halo-tag mutant (AgHalo) was evolved to sense proteome stress through its aggregation. Detection of such conformational changes was enabled by a fluorogenic ligand that fluoresces upon AgHalo forming soluble aggregates. Using 5 common anticancer drugs, we exemplified detection of differential proteome stress before any cell death was observed. Thus, this sensor can be used to evaluate drug safety in a regime that the current cytotoxicity assays cannot cover and be generally applied to detect proteome stress induced by other toxins. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Green synthesis of Pd NPs from Pimpinella tirupatiensis plant extract and their application in photocatalytic activity dye degradation

NASA Astrophysics Data System (ADS)

Narasaiah, Palajonna; Mandal, Badal Kumar; Sarada, N. C.

2017-11-01

The present report the synthesis of palladium nanoparticles through the green method route offers few advantages over the common chemical and physical procedures, as it is an easy and fast, eco-friendly and does not involve any costly chemicals as well as hazardous chemicals. In this study, we reported synthesis of Pd NPs by using the Pimpinella tirupatiensis plant Extract (PTPE). The synthesized Pd NPs was characterization using different technique such as UV-Visible for the formation of Pd NPs. FT-IR spectroscopy was performed to detect the bio-active molecules liable for reduction and capping of biogenic Pd NPs. Crystallinity of Pd NPs conformed by powder - XRD. In the present study performed photo catalytic activity of synthesized Pd NPs using organic dye such as Congo red (CR). Hence, this study concludes the PTPE aqueous extract produced Pd NPs can be act as promising material for the degradation of organic pollutants.

Systematic screening for mutations in the human serotonin 1F receptor gene in patients with bipolar affective disorder and schizophrenia

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

Shimron-Abarbanell, D.; Harms, H.; Erdmann, J.

1996-04-09

Using single strand conformational analysis we screened the complete coding sequence of the serotonin 1F (5-HT{sub 1F}) receptor gene for the presence of DNA sequence variation in a sample of 137 unrelated individuals including 45 schizophrenic patients, 46 bipolar patients, as well as 46 healthy controls. We detected only three rare sequence variants which are characterized by single base pair substitutions, namely a silent T{r_arrow}A transversion in the third position of codon 261 (encoding isoleucine), a silent C{r_arrow}T transition in the third position of codon 176 (encoding histidine), and a C{r_arrow}T transition in position -78 upstream from the start codon.more » The lack of significant mutations in patients suffering from schizophrenia and bipolar affective disorder indicates that the 5-HT{sub 1F} receptor is not commonly involved in the etiology of these diseases. 12 refs., 1 fig., 2 tabs.« less

Standardization of quantum key distribution and the ETSI standardization initiative ISG-QKD

NASA Astrophysics Data System (ADS)

Länger, Thomas; Lenhart, Gaby

2009-05-01

In recent years, quantum key distribution (QKD) has been the object of intensive research activities and of rapid progress, and it is now developing into a competitive industry with commercial products. Once QKD systems are transferred from the controlled environment of physical laboratories into a real-world environment for practical use, a number of practical security, compatibility and connectivity issues need to be resolved. In particular, comprehensive security evaluation and watertight security proofs need to be addressed to increase trust in QKD. System interoperability with existing infrastructures and applications as well as conformance with specific user requirements have to be assured. Finding common solutions to these problems involving all actors can provide an advantage for the commercialization of QKD as well as for further technological development. The ETSI industry specification group for QKD (ISG-QKD) offers a forum for creating such universally accepted standards and will promote significant leverage effects on coordination, cooperation and convergence in research, technical development and business application of QKD.

Insights from the structure of a smallpox virus topoisomerase-DNA transition state mimic

PubMed Central

Perry, Kay; Hwang, Young; Bushman, Frederic D.; Van Duyne, Gregory D.

2010-01-01

Summary Poxviruses encode their own type IB topoisomerases (TopIBs) which release superhelical tension generated by replication and transcription of their genomes. To investigate the reaction catalyzed viral TopIBs, we have determined the structure of a variola virus topoisomerase-DNA complex trapped as a vanadate transition state mimic. The structure reveals how the viral TopIB enzymes are likely to position the DNA duplex for ligation following relaxation of supercoils and identifies the sources of friction observed in single molecule experiments that argue against free rotation. The structure also identifies a conformational change in the leaving group sugar that must occur prior to cleavage and reveals a mechanism for promoting ligation following relaxation of supercoils that involves a novel Asp-minor groove interaction. Overall, the new structural data support a common catalytic mechanism for the TopIB superfamily but indicate distinct methods for controlling duplex rotation in the small vs. large enzyme subfamilies. PMID:20152159

Crystal structure of bovine Cu,Zn superoxide dismutase at 3 A resolution: chain tracing and metal ligands.

PubMed Central

Richardson, J; Thomas, K A; Rubin, B H; Richardson, D C

1975-01-01

An electron density map at 3 angstrom resolution has been calculated for Cu2+, Zn2+ superoxide dismutase from bovine erythrocytes, and the course of the main chain has been traced. The dominant structural feature is an 8-stranded barrel of antiparallel beta-pleated sheet. There is one very short helical section and two long loops of non-repetitive structure. The Cu and Zn are bound between the loops and one side of the beta barrel and are about 6 Angstrom apart, with a common histidine ligand. The Cu has four histidine ligands in a somewhat distorted square plane, and the Zn has three histidines and an aspartate in approximately tetrahedral arrangement. The two coppers of a dimer are about 34 Angstrom apart. The two subunits have essentially the same conformation and have an extensive contact area that mainly involves hydrophobic side chain interactions. The overall folding pattern of the polypeptide chain is very similar to that of an immunoglobulin domain. Images PMID:1055410

SU-F-T-520: Dosimetric Comparison of Radiation Treatment Plans for Whole Breast Irradiation Between 3D Conformal in Prone and Supine Positions Vs. VMAT and IMRT in Supine Positions

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

Bejarano Buele, A; Parsai, E

Purpose: The target volume for Whole Breast Irradiation (WBI) is dictated by location of tumor mass, breast tissue distribution, and involvement of lymph nodes. Dose coverage and Organs at Risk (OARs) sparing can be difficult to achieve in patients with unfavorable thoracic geometries. For these cases, inverse-planned and 3D-conformal prone treatments can be alternatives to traditional supine 3D-conformal plans. A dosimetric comparison can determine which of these techniques achieve optimal target coverage while sparing OARs. Methods: This study included simulation datasets for 8 patients, 5 of whom were simulated in both supine and prone positions. Positioning devices included breast boardsmore » and Vaclok bags for the supine position, and prone breast boards for the prone position. WBI 3-D conformal plans were created for patients simulated in both positions. Additional VMAT and IMRT WBI plans were made for all patients in the supine position. Results: Prone and supine 3D conformal plans had comparable PTV coverage. Prone 3D conformal plans received a significant 50% decrease to V20, V10, V5 and V30% for the ipsilateral lung in contrast to the supine plans. The heart also experienced a 10% decrease in maximum dose in the prone position, and V20, V10, V5 and V2 had significantly lower values than the supine plan. Supine IMRT and VMAT breast plans obtained comparable PTV coverage. The heart experienced a 10% decrease in maximum dose with inverse modulated plans when compared to the supine 3D conformal plan, while V20, V10, V5 and V2 showed higher values with inverse modulated plans than with supine 3D conformal plans. Conclusion: Prone 3D-conformal, and supine inverse planned treatments were generally superior in sparing OARs to supine plans with comparable PTV coverage. IMRT and VMAT plans offer sparing of OARs from high dose regions with an increase of irradiated volume in the low dose regions.« less

Squaraine rotaxanes with boat conformation macrocycles.

PubMed

Fu, Na; Baumes, Jeffrey M; Arunkumar, Easwaran; Noll, Bruce C; Smith, Bradley D

2009-09-04

Mechanical encapsulation of fluorescent, deep-red bis(anilino)squaraine dyes inside Leigh-type tetralactam macrocycles produces interlocked squaraine rotaxanes. The surrounding macrocycles are flexible and undergo rapid exchange of chair and boat conformations in solution. A series of X-ray crystal structures show how the rotaxane co-conformational exchange process involves simultaneous lateral oscillation of the macrocycle about the center of the encapsulated squaraine thread. Rotaxane macrocycles with 1,4-phenylene sidewalls and 2,6-pyridine dicarboxamide bridging units are more likely to adopt boat conformations in the solid state than analogous squaraine rotaxane systems with isophthalamide-containing macrocycles. A truncated squaraine dye, with a secondary amine attached directly to the central C(4)O(2) core, is less electrophilic than the extended bis(anilino)squaraine analogue, but it is still susceptible to chemical and photochemical bleaching. Its stability is greatly enhanced when it is encapsulated as an interlocked squaraine rotaxane. An X-ray crystal structure of this truncated squaraine rotaxane shows the macrocycle in a boat conformation, and NMR studies indicate that the boat is maintained in solution. Encapsulation as a rotaxane increases the dye's brightness by a factor of 6. The encapsulation process appears to constrain the dye and reduce deformation of the chromophore from planarity. This study shows how mechanical encapsulation as a rotaxane can be used as a rational design parameter to fine-tune the chemical and photochemical properties of squaraine dyes.

Squaraine Rotaxanes with Boat Conformation Macrocycles

PubMed Central

Fu, Na; Baumes, Jeffrey M.; Arunkumar, Easwaran; Noll, Bruce C.; Smith, Bradley D.

2010-01-01

Mechanical encapsulation of fluorescent, deep-red bis(anilino)squaraine dyes inside Leigh-type tetralactam macrocycles produces interlocked squaraine rotaxanes. The surrounding macrocycles are flexible and undergo rapid exchange of chair and boat conformations in solution. A series of X-ray crystal structures show how the rotaxane co-conformational exchange process involves simultaneous lateral oscillation of the macrocycle about the center of the encapsulated squaraine thread. Rotaxane macrocycles with 1,4-phenylene-sidewalls and 2,6-pyridine dicarboxamide bridging units are more likely to adopt boat conformations in the solid-state than analogous squaraine rotaxane systems with isophthalamide-containing macrocycles. A truncated squaraine dye, with a secondary amine attached directly to the central C4O2 core, is less electrophilic than the extended bis(anilino)squaraine analogue, but it is still susceptible to chemical and photochemical bleaching. Its stability is greatly enhanced when it is encapsulated as an interlocked squaraine rotaxane. An X-ray crystal structure of this truncated squaraine rotaxane shows the macrocycle in a boat conformation, and NMR studies indicate that the boat is maintained in solution. Encapsulation as a rotaxane increases the dye’s brightness by a factor of six. The encapsulation process appears to constrain the dye and reduce deformation of the chromophore from planarity. This study shows how mechanical encapsulation as a rotaxane can be used as a rational design parameter to fine-tune the chemical and photochemical properties of squaraine dyes. PMID:19639940

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

PubMed

Pandini, Alessandro; Fraccalvieri, Domenico; Bonati, Laura

2013-01-01

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

BamA POTRA Domain Interacts with a Native Lipid Membrane Surface.

PubMed

Fleming, Patrick J; Patel, Dhilon S; Wu, Emilia L; Qi, Yifei; Yeom, Min Sun; Sousa, Marcelo Carlos; Fleming, Karen G; Im, Wonpil

2016-06-21

The outer membrane of Gram-negative bacteria is an asymmetric membrane with lipopolysaccharides on the external leaflet and phospholipids on the periplasmic leaflet. This outer membrane contains mainly β-barrel transmembrane proteins and lipidated periplasmic proteins (lipoproteins). The multisubunit protein β-barrel assembly machine (BAM) catalyzes the insertion and folding of the β-barrel proteins into this membrane. In Escherichia coli, the BAM complex consists of five subunits, a core transmembrane β-barrel with a long periplasmic domain (BamA) and four lipoproteins (BamB/C/D/E). The BamA periplasmic domain is composed of five globular subdomains in tandem called POTRA motifs that are key to BAM complex formation and interaction with the substrate β-barrel proteins. The BAM complex is believed to undergo conformational cycling while facilitating insertion of client proteins into the outer membrane. Reports describing variable conformations and dynamics of the periplasmic POTRA domain have been published. Therefore, elucidation of the conformational dynamics of the POTRA domain in full-length BamA is important to understand the function of this molecular complex. Using molecular dynamics simulations, we present evidence that the conformational flexibility of the POTRA domain is modulated by binding to the periplasmic surface of a native lipid membrane. Furthermore, membrane binding of the POTRA domain is compatible with both BamB and BamD binding, suggesting that conformational selection of different POTRA domain conformations may be involved in the mechanism of BAM-facilitated insertion of outer membrane β-barrel proteins. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Traversing the folding pathway of proteins using temperature-aided cascade molecular dynamics with conformation-dependent charges.

PubMed

Jani, Vinod; Sonavane, Uddhavesh; Joshi, Rajendra

2016-07-01

Protein folding is a multi-micro second time scale event and involves many conformational transitions. Crucial conformational transitions responsible for biological functions of biomolecules are difficult to capture using current state-of-the-art molecular dynamics (MD) simulations. Protein folding, being a stochastic process, witnesses these transitions as rare events. Many new methodologies have been proposed for observing these rare events. In this work, a temperature-aided cascade MD is proposed as a technique for studying the conformational transitions. Folding studies for Engrailed homeodomain and Immunoglobulin domain B of protein A have been carried out. Using this methodology, the unfolded structures with RMSD of 20 Å were folded to a structure with RMSD of 2 Å. Three sets of cascade MD runs were carried out using implicit solvation, explicit solvation, and charge updation scheme. In the charge updation scheme, charges based on the conformation obtained are calculated and are updated in the topology file. In all the simulations, the structure of 2 Å was reached within a few nanoseconds using these methods. Umbrella sampling has been performed using snapshots from the temperature-aided cascade MD simulation trajectory to build an entire conformational transition pathway. The advantage of the method is that the possible pathways for a particular reaction can be explored within a short duration of simulation time and the disadvantage is that the knowledge of the start and end state is required. The charge updation scheme adds the polarization effects in the force fields. This improves the electrostatic interaction among the atoms, which may help the protein to fold faster.

Structural, kinetic and computational investigation of Vitis vinifera DHDPS reveals new insight into the mechanism of lysine-mediated allosteric inhibition.

PubMed

Atkinson, Sarah C; Dogovski, Con; Downton, Matthew T; Czabotar, Peter E; Dobson, Renwick C J; Gerrard, Juliet A; Wagner, John; Perugini, Matthew A

2013-03-01

Lysine is one of the most limiting amino acids in plants and its biosynthesis is carefully regulated through inhibition of the first committed step in the pathway catalyzed by dihydrodipicolinate synthase (DHDPS). This is mediated via a feedback mechanism involving the binding of lysine to the allosteric cleft of DHDPS. However, the precise allosteric mechanism is yet to be defined. We present a thorough enzyme kinetic and thermodynamic analysis of lysine inhibition of DHDPS from the common grapevine, Vitis vinifera (Vv). Our studies demonstrate that lysine binding is both tight (relative to bacterial DHDPS orthologs) and cooperative. The crystal structure of the enzyme bound to lysine (2.4 Å) identifies the allosteric binding site and clearly shows a conformational change of several residues within the allosteric and active sites. Molecular dynamics simulations comparing the lysine-bound (PDB ID 4HNN) and lysine free (PDB ID 3TUU) structures show that Tyr132, a key catalytic site residue, undergoes significant rotational motion upon lysine binding. This suggests proton relay through the catalytic triad is attenuated in the presence of lysine. Our study reveals for the first time the structural mechanism for allosteric inhibition of DHDPS from the common grapevine.

Cyclophilin A catalyzes proline isomerization by an electrostatic handle mechanism

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

Camilloni, Carlo; Sahakyan, Aleksander B.; Holliday, Michael

2014-07-15

Proline isomerization is a ubiquitous process that plays a key role in the folding of proteins and in the regulation of their functions1-3. Different families of enzymes, known as peptidyl-prolyl isomerases (PPIases), catalyse this reaction, which involves the interconversion between the cis and trans isomers of the Nterminal amide bond of the amino acid proline2,3. A complete description of the mechanisms by which these enzymes function, however, has remained elusive. Here, we show that cyclophilin A, one of the most common PPIases4, provides a catalytic environment that acts on the substrate through an electrostatic lever mechanism. In this mechanism, themore » electrostatic field in the catalytic site turns the electric dipole associated with the carboxylic group of the amino acid preceding the proline in the substrate, thus causing the rotation of the peptide bond between the two residues. This mechanism resulted from the analysis of an ensemble of conformations populated by cyclophilin A during the enzymatic reaction using a combination of NMR measurements, molecular dynamics simulations and density functional theory calculations. We anticipate that this approach will be helpful in elucidating whether the electrostatic lever mechanism that we describe is common to other PPIases, and more generally to characterise other enzymatic processes.« less

Cyclophilin A catalyzes proline isomerization by an electrostatic handle mechanism

PubMed Central

Camilloni, Carlo; Sahakyan, Aleksandr B.; Holliday, Michael J.; Isern, Nancy G.; Zhang, Fengli; Eisenmesser, Elan Z.; Vendruscolo, Michele

2014-01-01

Proline isomerization is a ubiquitous process that plays a key role in the folding of proteins and in the regulation of their functions. Different families of enzymes, known as “peptidyl-prolyl isomerases” (PPIases), catalyze this reaction, which involves the interconversion between the cis and trans isomers of the N-terminal amide bond of the amino acid proline. However, complete descriptions of the mechanisms by which these enzymes function have remained elusive. We show here that cyclophilin A, one of the most common PPIases, provides a catalytic environment that acts on the substrate through an electrostatic handle mechanism. In this mechanism, the electrostatic field in the catalytic site turns the electric dipole associated with the carbonyl group of the amino acid preceding the proline in the substrate, thus causing the rotation of the peptide bond between the two residues. We identified this mechanism using a combination of NMR measurements, molecular dynamics simulations, and density functional theory calculations to simultaneously determine the cis-bound and trans-bound conformations of cyclophilin A and its substrate as the enzymatic reaction takes place. We anticipate that this approach will be helpful in elucidating whether the electrostatic handle mechanism that we describe here is common to other PPIases and, more generally, in characterizing other enzymatic processes. PMID:24982184

Cyclophilin A catalyzes proline isomerization by an electrostatic handle mechanism.

PubMed

Camilloni, Carlo; Sahakyan, Aleksandr B; Holliday, Michael J; Isern, Nancy G; Zhang, Fengli; Eisenmesser, Elan Z; Vendruscolo, Michele

2014-07-15

Proline isomerization is a ubiquitous process that plays a key role in the folding of proteins and in the regulation of their functions. Different families of enzymes, known as "peptidyl-prolyl isomerases" (PPIases), catalyze this reaction, which involves the interconversion between the cis and trans isomers of the N-terminal amide bond of the amino acid proline. However, complete descriptions of the mechanisms by which these enzymes function have remained elusive. We show here that cyclophilin A, one of the most common PPIases, provides a catalytic environment that acts on the substrate through an electrostatic handle mechanism. In this mechanism, the electrostatic field in the catalytic site turns the electric dipole associated with the carbonyl group of the amino acid preceding the proline in the substrate, thus causing the rotation of the peptide bond between the two residues. We identified this mechanism using a combination of NMR measurements, molecular dynamics simulations, and density functional theory calculations to simultaneously determine the cis-bound and trans-bound conformations of cyclophilin A and its substrate as the enzymatic reaction takes place. We anticipate that this approach will be helpful in elucidating whether the electrostatic handle mechanism that we describe here is common to other PPIases and, more generally, in characterizing other enzymatic processes.

Effect of Urea on G-Quadruplex Stability.

PubMed

Aslanyan, Lusine; Ko, Jordan; Kim, Byul G; Vardanyan, Ishkhan; Dalyan, Yeva B; Chalikian, Tigran V

2017-07-13

G-quadruplexes represent a class of noncanonical nucleic acid structures implicated in transcriptional regulation, cellular function, and disease. An understanding of the forces involved in stabilization and destabilization of the G-quadruplex conformation relative to the duplex or single-stranded conformation is a key to elucidating the biological role of G-quadruplex-based genomic switches and the quest for therapeutic means for controlled induction or suppression of a G-quadruplex at selected genomic loci. Solute-solvent interactions provide a ubiquitous and, in many cases, the determining thermodynamic force in maintaining and modulating the stability of nucleic acids. These interactions involve water as well as water-soluble cosolvents that may be present in the solution or in the crowded environment in the cell. We present here the first quantitative investigation of the effect of urea, a destabilizing cosolvent, on the conformational preferences of a G-quadruplex formed by the telomeric d[A(G 3 T 2 A) 3 G 3 ] sequence (Tel22). At 20 mM NaCl and room temperature, Tel22 undergoes a two-state urea-induced unfolding transition. An increase in salt mitigates the deleterious effect of urea on Tel22. The urea m-value of Tel22 normalized per change in solvent-accessible surface area, ΔS A , is similar to those for other DNA and RNA structures while being several-fold larger than that of proteins. Our results suggest that urea can be employed as an analytical tool in thermodynamic characterizations of G-quadruplexes in a manner similar to the use of urea in protein studies. We emphasize the need for further studies involving a larger selection of G-quadruplexes varying in sequence, topology (parallel, antiparallel, hybrid), and molecularity (monomolecular, bimolecular, tetramolecular) to outline the advantages and the limits of the use of urea in G-quadruplex studies. A deeper understanding of the effect of solvent and cosolvents on the differential stability of the G-quadruplex and duplex conformations is a step toward elucidation of the modulating influence of different types of cosolvents on duplex-G-quadruplex molecular switches triggering genomic events.

NMR studies of conformational states of proteins involved in biosynthesis of iron-sulfur clusters

NASA Astrophysics Data System (ADS)

Dai, Ziqi

Iron-sulfur (Fe-S) clusters are the most ancient and ubiquitous cofactors that exist throughout evolution. The most important biosynthetic system of the cluster in both prokaryotes and eukaryotes is the ISC system. Defects in this system can be lethal and have been associated with a number of human diseases. Previous works show that a number of proteins are involved in the [Fe-S] biosynthetic processes and the structural flexibility may play an important role. For example, it was shown that apo-IscU, the scaffold protein, from Escherichia coli populates two functionally important conformational states, one dynamically disordered (D-state) and the other more structured (S-state) (Kim et al., 2009; Kim et al., 2012c). To further investigate the characteristics and transition of the conformational states of proteins involved in this system, I performed extensive NMR studies. Here, I present the findings based on my studies of two important players of the ISC system, IscU and HscB. In this research, I find that a peptidyl-prolyl cis/trans isomerization might account for the slow step in the S-D interconversion of IscU. More specifically, P14 and P101 are trans in the S-state, but become cis in the D-state. In addition, I discover that IscU is very responsive to pH changes, and I postulate that this response is correlated to conserved histidine residues, H10 and H105. Moreover, my thermodynamic analyses reveal that the S-D equilibrium of IscU is also very sensitive to change in temperature, pressure, and amino acid sequence compared to other proteins. In the study, I also discovered a novel state of IscU, the unfolded U-state. I suspect that this state may serve as an intermediate of interconversion between IscU S-/D-states. Finally, I extended the effort to HscB, and find that it may possess more conformational flexibility than expected earlier. I postulate that this flexibility may be the cause of the line-broadening observed during interaction of HscB with IscU (Fuzery et al., 2008; Kim et al., 2009) and HscA.

Helical tomotherapy with dynamic running-start-stop delivery compared to conventional tomotherapy delivery

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

Rong, Yi, E-mail: yi.rong@osumc.edu; Chen, Yu; Lu, Weiguo

Purpose: Despite superior target dose uniformity, helical tomotherapy{sup ®} (HT) may involve a trade-off between longitudinal dose conformity and beam-on time (BOT), due to the limitation of only three available jaw sizes with the conventional HT (1.0, 2.5, and 5.0 cm). The recently introduced dynamic running-start-stop (RSS) delivery allows smaller jaw opening at the superior and inferior ends of the target when a sharp penumbra is needed. This study compared the dosimetric performance of RSS delivery with the fixed jaw HT delivery. Methods: Twenty patient cases were selected and deidentified prior to treatment planning, including 16 common clinical cases (brain,more » head and neck (HN), lung, and prostate) and four special cases of whole brain with hippocampus avoidance (WBHA) that require a high degree of dose modulation. HT plans were generated for common clinical cases using the fixed 2.5 cm jaw width (HT2.5) and WBHA cases using 1.0 cm (HT1.0). The jaw widths for RSS were preset with a larger size (RSS5.0 vs HT2.5 and RSS2.5 vs HT1.0). Both delivery techniques were planned based on identical contours, prescriptions, and planning objectives. Dose indices for targets and critical organs were compared using dose-volume histograms, BOT, and monitor units. Results: The average BOT was reduced from 4.8 min with HT2.5 to 2.5 min with RSS5.0. Target dose homogeneity with RSS5.0 was shown comparable to HT2.5 for common clinical sites. Superior normal tissue sparing was observed in RSS5.0 for optic nerves and optic chiasm in brain and HN cases. RSS5.0 demonstrated improved dose sparing for cord and esophagus in lung cases, as well as penile bulb in prostate cases. The mean body dose was comparable for both techniques. For the WBHA cases, the target homogeneity was significantly degraded in RSS2.5 without distinct dose sparing for hippocampus, compared to HT1.0. Conclusions: Compared to the fixed jaw HT delivery, RSS combined with a larger jaw width provides faster treatment delivery and improved cranial-caudal target dose conformity. The target coverage achieved by RSS with a large jaw width is comparable to the fixed jaw HT delivery for common cancer sites, but may deteriorate for cases where complex geometry is present in the middle part of the target.« less

48 CFR 752.7027 - Personnel.

Code of Federal Regulations, 2010 CFR

2010-10-01

.... For use in all USAID services contracts involving performance overseas. Note that paragraphs (f) and... notification from the Contracting Officer of Cooperating Country clearance of any employee sent outside the... employees and dependents. See the clause of this contract entitled Physical Fitness. (c) Conformity to laws...

How maltose influences structural changes to bind to maltose-binding protein: results from umbrella sampling simulation.

PubMed

Mascarenhas, Nahren Manuel; Kästner, Johannes

2013-02-01

A well-studied periplasmic-binding protein involved in the abstraction of maltose is maltose-binding protein (MBP), which undergoes a ligand-induced conformational transition from an open (ligand-free) to a closed (ligand-bound) state. Umbrella sampling simulations have been us to estimate the free energy of binding of maltose to MBP and to trace the potential of mean force of the unbinding event using the center-of-mass distance between the protein and ligand as the reaction coordinate. The free energy thus obtained compares nicely with the experimentally measured value justifying our theoretical basis. Measurement of the domain angle (N-terminal-domain - hinge - C-terminal-domain) along the unbinding pathway established the existence of three different states. Starting from a closed state, the protein shifts to an open conformation during the initial unbinding event of the ligand then resides in a semi-open conformation and later resides predominantly in an open-state. These transitions along the ligand unbinding pathway have been captured in greater depth using principal component analysis. It is proposed that in mixed-model, both conformational selection and an induced-fit mechanism combine to the ligand recognition process in MBP. Copyright © 2012 Wiley Periodicals, Inc.

Constraints on parity violating conformal field theories in d = 3

NASA Astrophysics Data System (ADS)

Chowdhury, Subham Dutta; David, Justin R.; Prakash, Shiroman

2017-11-01

We derive constraints on three-point functions involving the stress tensor, T, and a conserved U(1) current, j, in 2+1 dimensional conformal field theories that violate parity, using conformal collider bounds introduced by Hofman and Maldacena. Conformal invariance allows parity-odd tensor-structures for the 〈 T T T〉 and 〈 jjT〉 correlation functions which are unique to three space-time dimensions. Let the parameters which determine the 〈 T T T〉 correlation function be t 4 and α T , where α T is the parity-violating contribution. Similarly let the parameters which determine 〈 jjT〉 correlation function be a 2, and α J , where α J is the parity-violating contribution. We show that the parameters ( t 4, α T ) and (a2, α J ) are bounded to lie inside a disc at the origin of the t 4 - α T plane and the a 2 - α J plane respectively. We then show that large N Chern-Simons theories coupled to a fundamental fermion/boson lie on the circle which bounds these discs. The `t Hooft coupling determines the location of these theories on the boundary circles.

Crystal structure of the complete integrin αVβ3 ectodomain plus an α/β transmembrane fragment

PubMed Central

Xiong, Jian-Ping; Mahalingham, Bhuvaneshwari; Alonso, Jose Luis; Borrelli, Laura Ann; Rui, Xianliang; Anand, Saurabh; Hyman, Bradley T.; Rysiok, Thomas; Müller-Pompalla, Dirk; Goodman, Simon L.

2009-01-01

We determined the crystal structure of 1TM-αVβ3, which represents the complete unconstrained ectodomain plus short C-terminal transmembrane stretches of the αV and β3 subunits. 1TM-αVβ3 is more compact and less active in solution when compared with ΔTM-αVβ3, which lacks the short C-terminal stretches. The structure reveals a bent conformation and defines the α–β interface between IE2 (EGF-like 2) and the thigh domains. Modifying this interface by site-directed mutagenesis leads to robust integrin activation. Fluorescent lifetime imaging microscopy of inactive full-length αVβ3 on live cells yields a donor–membrane acceptor distance, which is consistent with the bent conformation and does not change in the activated integrin. These data are the first direct demonstration of conformational coupling of the integrin leg and head domains, identify the IE2–thigh interface as a critical steric barrier in integrin activation, and suggest that inside-out activation in intact cells may involve conformational changes other than the postulated switch to a genu-linear state. PMID:19704023

Infrared Spectroscopy of Mobility-Selected H+-Gly-Pro-Gly-Gly (GPGG)

NASA Astrophysics Data System (ADS)

Masson, Antoine; Kamrath, Michael Z.; Perez, Marta A. S.; Glover, Matthew S.; Rothlisberger, U.; Clemmer, David E.; Rizzo, Thomas R.

2015-09-01

We report the first results from a new instrument capable of acquiring infrared spectra of mobility-selected ions. This demonstration involves using ion mobility to first separate the protonated peptide Gly-Pro-Gly-Gly (GPGG) into two conformational families with collisional cross-sections of 93.8 and 96.8 Å2. After separation, each family is independently analyzed by acquiring the infrared predissociation spectrum of the H2-tagged molecules. The ion mobility and spectroscopic data combined with density functional theory (DFT) based molecular dynamics simulations confirm the presence of one major conformer per family, which arises from cis/ trans isomerization about the proline residue. We induce isomerization between the two conformers by using collisional activation in the drift tube and monitor the evolution of the ion distribution with ion mobility and infrared spectroscopy. While the cis-proline species is the preferred gas-phase structure, its relative population is smaller than that of the trans-proline species in the initial ion mobility drift distribution. This suggests that a portion of the trans-proline ion population is kinetically trapped as a higher energy conformer and may retain structural elements from solution.

Developing hybrid approaches to predict pKa values of ionizable groups

PubMed Central

Witham, Shawn; Talley, Kemper; Wang, Lin; Zhang, Zhe; Sarkar, Subhra; Gao, Daquan; Yang, Wei

2011-01-01

Accurate predictions of pKa values of titratable groups require taking into account all relevant processes associated with the ionization/deionization. Frequently, however, the ionization does not involve significant structural changes and the dominating effects are purely electrostatic in origin allowing accurate predictions to be made based on the electrostatic energy difference between ionized and neutral forms alone using a static structure. On another hand, if the change of the charge state is accompanied by a structural reorganization of the target protein, then the relevant conformational changes have to be taken into account in the pKa calculations. Here we report a hybrid approach that first predicts the titratable groups, which ionization is expected to cause conformational changes, termed “problematic” residues, then applies a special protocol on them, while the rest of the pKa’s are predicted with rigid backbone approach as implemented in multi-conformation continuum electrostatics (MCCE) method. The backbone representative conformations for “problematic” groups are generated with either molecular dynamics simulations with charged and uncharged amino acid or with ab-initio local segment modeling. The corresponding ensembles are then used to calculate the pKa of the “problematic” residues and then the results are averaged. PMID:21744395

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.

Fluorine Substitution in Neurotransmitters: Microwave Spectroscopy and Modelling of the Conformational Space and Non Bonding Interactions

NASA Astrophysics Data System (ADS)

Melandri, S.; Maris, A.; Merloni, A.

2011-06-01

Fluorine substitution in molecules is a common practice in bio-organic chemistry in order to modulate physicochemical properties and biological activity of molecules and an increasing number of drugs on the market contain fluorine, the presence of which is often of major importance to modify pharmacokinetics properties and molecular activity. The rationale for such a strategy is that fluorine is generally a stronger electron acceptor than the other halogen atoms and its size is intermediate between that of hydrogen and oxygen. We have studied two fluorinated analogs of 2-phenylethylamine (PEA), the prototype molecule for adrenergic neurotransmitters, namely: 4-Fluoro (4FPEA) and 2-Fluoro-2-phenylethylamine (2FPEA) by Molecular Beam Fourier Transform Microwave Spectroscopy in the frequency range 6-18 GHz and ab initio calculations at the MP2/6311++G** level. The aim is to obtain information on the spatial arrangement of the ethylamine side chain and the effects of fluorination on the energy landscape. The conformational space is dominated by low energy gauche conformations stabilized by weak interactions between the aminic hydrogens and the electron cloud of the benzene ring and anti conformations higher in energy. In 2FPEA the presence of the fluorine atom almost duplicate the number of possible conformation with respect to 4FPEA. We observed two conformers of 4FPEA and five conformers of 2FPEA which have been classified with the guide provided by accurate ab initio calculations. The identification of the conformational species was helped by the analysis of the quadrupole hyperfine pattern which is greatly influenced by the orientation of the amino group and acts as a fingerprint for each conformation. The orientation of the dipole moment within the principal axis frame and the order of stability of the different conformations are other independent pieces of evidence for the unambiguous assignment and identification of the conformers. The order of stability was found to be altered in both molecules with respect to the prototype PEA molecule, especially in the case of 2FPEA where we observe a stabilization of some of the anti forms and great destabilization of some of the gauche forms. These observations are in agreement with the results of the theoretical calculation and can be rationalized in terms of the effect of the fluorine atom on the electron density of the molecule and in particular on the electron cloud on the benzene ring.

Modelling Carpool and Transit Park-and-Ride Lots

DOT National Transportation Integrated Search

1997-01-01

Park-and-Ride (PnR) lots are an increasingly common element of many areas plans for air quality conformity. However, few, if any, travel models estimate the impacts of carpool PnR lots, and it is not at all clear that they always improve air quality....

COMMON CONFORMATIONAL EFFECTS IN THE P53 PROTEIN OF VINYL CHLORIDE-INDUCED MUTATIONS. (R826685)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Alternating phenylene and furan/pyrrole/thiophene units-based oligomers: A computational study of the structures and optoelectronic properties

NASA Astrophysics Data System (ADS)

Sahu, Harikrishna; Shukla, Rishabh; Goswami, Juri; Gaur, Priyank; Panda, Aditya N.

2018-01-01

Structural and optoelectronic properties of phenylene-furan, phenylene-pyrrole and phenylene-thiophene oligomers are reported using density functional theory methods. Studies reveal that stabilities of conformers change with increasing chain length, and helical conformers are energetically feasible for large oligomers of the studied systems, due to stacking interactions between adjacent helical turns. Absorption spectra of helices are dominated by multiple number of electronic transitions other than the S0 →S1 , involving orbitals other than the HOMO/LUMO. All studied helices are optically active having similar pattern of negative and positive peaks in the CD spectra.

Structure of the Response Regulator PhoP from Mycobacterium tuberculosis Reveals a Dimer Through the Receiver Domain

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

S Menon; S Wang

The PhoP protein from Mycobacterium tuberculosis is a response regulator of the OmpR/PhoB subfamily, whose structure consists of an N-terminal receiver domain and a C-terminal DNA-binding domain. How the DNA-binding activities are regulated by phosphorylation of the receiver domain remains unclear due to a lack of structural information on the full-length proteins. Here we report the crystal structure of the full-length PhoP of M. tuberculosis. Unlike other known structures of full-length proteins of the same subfamily, PhoP forms a dimer through its receiver domain with the dimer interface involving {alpha}4-{beta}5-{alpha}5, a common interface for activated receiver domain dimers. However, themore » switch residues, Thr99 and Tyr118, are in a conformation resembling those of nonactivated receiver domains. The Tyr118 side chain is involved in the dimer interface interactions. The receiver domain is tethered to the DNA-binding domain through a flexible linker and does not impose structural constraints on the DNA-binding domain. This structure suggests that phosphorylation likely facilitates/stabilizes receiver domain dimerization, bringing the DNA-binding domains to close proximity, thereby increasing their binding affinity for direct repeat DNA sequences.« less

Discovery of a Kelch-like ECH-associated protein 1-inhibitory tetrapeptide and its structural characterization

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

Sogabe, Satoshi; Sakamoto, Kotaro; Kamada, Yusuke

Keap1 constitutively binds to the transcription factor Nrf2 to promote its degradation, resulting in negative modulation of genes involved in cellular protection against oxidative stress. Keap1 is increasingly recognized as an attractive target for treating diseases involving oxidative stress, including cancer, atherosclerosis, diabetes, arthritis, and neurodegeneration. We used phage-display peptide screening to identify a tetrapeptide showing moderate binding affinity, which inhibits the interaction between Nrf2 and Keap1. The tetrapeptide does not include an ETGE motif, which is a commonly found consensus sequence in known peptidic inhibitors. In addition to affinity parameters, IC{sub 50}, K{sub D}, and thermodynamic parameters, the crystalmore » structure of the complex was determined to elucidate the binding conformation. The binding interactions resemble those of known small-molecule inhibitors as opposed to those of substrates and peptidic inhibitors. Although the tetrapeptide's affinity is not very high, our results may help facilitate the designing of small-molecule inhibitors during lead generation in drug discovery. - Highlights: • Keap1 inhibitory tetrapeptide with moderate affinity was discovered. • Crystal structure of the complex showed the unique binding mode. • Structural information gives a valuable insight for design of therapeutic compounds.« less

pH-dependent structural change of the extracellular sensor domain of the DraK histidine kinase from Streptomyces coelicolor.

PubMed

Yeo, Kwon Joo; Kim, Eun Hye; Hwang, Eunha; Han, Young-Hyun; Eo, Yumi; Kim, Hyun Jung; Kwon, Ohsuk; Hong, Young-Soo; Cheong, Chaejoon; Cheong, Hae-Kap

2013-02-15

Recently, the DraR/DraK (Sco3063/Sco3062) two-component system (TCS) of Streptomycescoelicolor has been reported to be involved in the differential regulation of antibiotic biosynthesis. However, it has not been shown that under which conditions and how the DraR/DraK TCS is activated to initiate the signal transduction process. Therefore, to understand the sensing mechanism, structural study of the sensory domain of DraK is highly required. Here, we report the biochemical and biophysical properties of the extracellular sensory domain (ESD) of DraK. We observed a reversible pH-dependent conformational change of the ESD in a pH range of 2.5-10. Size-exclusion chromatography and AUC (analytical ultracentrifugation) data indicated that the ESD is predominantly monomeric in solution and exists in equilibrium between monomer and dimer states in acidic condition. Using NMR (nuclear magnetic resonance) and CD (circular dichroism) spectroscopy, our findings suggest that the structure of the ESD at low pH is more structured than that at high pH. In particular, the glutamate at position 83 is an important residue for the pH-dependent conformational change. These results suggest that this pH-dependent conformational change of ESD may be involved in signal transduction process of DraR/DraK TCS. Copyright © 2013 Elsevier Inc. All rights reserved.

Assessing the acid-base and conformational properties of histidine residues in human prion protein (125-228) by means of pK(a) calculations and molecular dynamics simulations.

PubMed

Langella, Emma; Improta, Roberto; Crescenzi, Orlando; Barone, Vincenzo

2006-07-01

A thorough study of the acid-base behavior of the four histidines and the other titratable residues of the structured domain of human prion protein (125-228) is presented. By using multi-tautomer electrostatic calculations, average titration curves have been built for all titratable residues, using the whole bundles of NMR structures determined at pH 4.5 and 7.0. According to our results, (1) only histidine residues are likely to be involved in the first steps of the pH-driven conformational transition of prion protein; (2) the pK(a)'s of His140 and His177 are approximately 7.0, whereas those of His155 and His187 are < 5.5. 10-ns long molecular dynamics simulations have been performed on five different models, corresponding to the most significant combinations of histidine protonation states. A critical comparison between the available NMR structures and our computational results (1) confirms that His155 and His187 are the residues whose protonation is involved in the conformational rearrangement of huPrP in mildly acidic condition, and (2) shows how their protonation leads to the destructuration of the C-terminal part of HB and to the loss of the last turn of HA that represent the crucial microscopic steps of the rearrangement. (c) 2006 Wiley-Liss, Inc.

Structure and photochemistry of a saccharyl thiotetrazole.

PubMed

Ismael, A; Borba, A; Henriques, M S C; Paixão, J A; Fausto, R; Cristiano, M L S

2015-01-02

The molecular structure and photochemistry of 5-thiosaccharyl-1-methyltetrazole (TSMT) were studied by means of matrix-isolation FTIR spectroscopy, X-ray crystallography, and theoretical calculations. The calculations predicted two conformers of TSMT that differ in energy by more than 15 kJ mol(-1). The infrared spectrum of TSMT isolated in solid argon was fully assigned on the basis of the spectrum calculated (O3LYP/6-311++G(3df,3pd)) for the most stable conformer. In the crystal, TSMT molecules were found to assume the same conformation as for the isolated molecule, with each molecule forming four hydrogen bonds with three neighboring molecules, leading to a network of TSMT oligomers. Upon UV (λ = 265 nm) irradiation of the matrix-isolated TSMT, two photodegradation pathways were observed, both arising from cleavage of the tetrazolyl ring. Pathway a involves cleavage of the N1-N2 and N3-N4 bonds with extrusion of N2, leading to photostable diazirine and thiocarbodiimide derivatives. The photostability of the photoproduced diazirine under the conditions used precluded its rearrangement to the nitrile imine, as reported for 5-phenyltetrazole by Bégué et al. ( J. Am. Chem. Soc. 2012 , 134 , 5339 ). Pathway b involves cleavage of the C5-N1 and N4-N3 bonds, leading to a thiocyanate and methyl azide, the latter undergoing subsequent fragmentation to give CNH.

The concept and evolution of involved site radiation therapy for lymphoma.

PubMed

Specht, Lena; Yahalom, Joachim

2015-10-01

We describe the development of radiation therapy for lymphoma from extended field radiotherapy of the past to modern conformal treatment with involved site radiation therapy based on advanced imaging, three-dimensional treatment planning and advanced treatment delivery techniques. Today, radiation therapy is part of the multimodality treatment of lymphoma, and the irradiated tissue volume is much smaller than before, leading to highly significant reductions in the risks of long-term complications.

Three-dimensional conformal radiation for esophageal squamous cell carcinoma with involved-field irradiation may deliver considerable doses of incidental nodal irradiation.

PubMed

Ji, Kai; Zhao, Lujun; Yang, Chengwen; Meng, Maobin; Wang, Ping

2012-11-27

To quantify the incidental irradiation dose to esophageal lymph node stations when irradiating T1-4N0M0 thoracic esophageal squamous cell carcinoma (ESCC) patients with a dose of 60 Gy/30f. Thirty-nine patients with medically inoperable T1-4N0M0 thoracic ESCC were treated with three-dimensional conformal radiation (3DCRT) with involved-field radiation (IFI). The conformal clinical target volume (CTV) was re-created using a 3-cm margin in the proximal and distal direction beyond the barium esophagogram, endoscopic examination and CT scan defined the gross tumor volume (GTV) and a 0.5-cm margin in the lateral and anteroposterior directions of the CT scan-defined GTV. The PTV encompassed 1-cm proximal and distal margins and 0.5-cm radial margin based on the CTV. Nodal regions were delineated using the Japanese Society for Esophageal Diseases (JSED) guidelines and an EORTC-ROG expert opinion. The equivalent uniform dose (EUD) and other dosimetric parameters were calculated for each nodal station. Nodal regions with a metastasis rate greater than 5% were considered a high-risk lymph node subgroup. Under a 60 Gy dosage, the median D mean and EUD was greater than 40 Gy in most high-risk nodal regions except for regions of 104, 106tb-R in upper-thoracic ESCC and 101, 104-R, 105, 106rec-L, 2, 3&7 in middle-thoracic ESCC and 107, 3&7 in lower-thoracic ESCC. In the regions with an EUD less than 40 Gy, most incidental irradiation doses were significantly associated with esophageal tumor length and location. Lymph node stations near ESCC receive considerable incidental irradiation doses with involved-field irradiation that may contribute to the elimination of subclinical lesions.

Implementation of a standard format for GPS common view data

NASA Technical Reports Server (NTRS)

Weiss, Marc A.; Thomas, Claudine

1995-01-01

A new format for standardizing common view time transfer data, recommended by the Consultative Committee for the Definition of the Second, is being implemented in receivers commonly used for contributing data for the generation of International Atomic Time. We discuss three aspects of this new format that potentially improve GPS common-view time transfer: (1) the standard specifies the method for treating short term data, (2) it presents data in consistent formats including needed terms not previously available, and (3) the standard includes a header of parameters important for the GPS common-view process. In coordination with the release of firmware conforming to this new format the Bureau International des Poids et Mesures will release future international track schedules consistent with the new standard.

Conformational analysis of a quinolonic ribonucleoside with anti-HSV-1 activity

NASA Astrophysics Data System (ADS)

Yoneda, Julliane D.; Velloso, Marcia Helena R.; Leal, Kátia Z.; Azeredo, Rodrigo B. de V.; Sugiura, Makiko; Albuquerque, Magaly G.; Santos, Fernanda da C.; Souza, Maria Cecília B. V. de; Cunha, Anna Claudia; Seidl, Peter R.; Alencastro, Ricardo B. de; Ferreira, Vitor F.

2011-01-01

The infections caused by the Herpes Simplex Virus are one of the most common sources of diseases in adults and several natural nucleoside analogues are currently used in the treatment of these infections. In vitro tests of a series of quinolonic ribonucleosides derivatives synthesized by part of our group indicated that some of them have antiviral activity against HSV-1. The conformational analysis of bioactive compounds is extremely important in order to better understand their chemical structures and biological activity. In this work, we have carried out a nuclear relaxation NMR study of 6-Me ribonucleoside derivative in order to determine if the syn or anti conformation is preferential. The NMR analysis permits the determination of inter-atomic distances by using techniques which are based on nuclear relaxation and related phenomena. Those techniques are non-selective longitudinal or spin-lattice relaxation rates and NULL pulse sequence, which allow the determination of distances between pairs of hydrogen atoms. The results of NMR studies were compared with those obtained by molecular modeling.

Rapid vapor deposition of highly conformal silica nanolaminates.

PubMed

Hausmann, Dennis; Becker, Jill; Wang, Shenglong; Gordon, Roy G

2002-10-11

Highly uniform and conformal coatings can be made by the alternating exposures of a surface to vapors of two reactants, in a process commonly called atomic layer deposition (ALD). The application of ALD has, however, been limited because of slow deposition rates, with a theoretical maximum of one monolayer per cycle. We show that alternating exposure of a surface to vapors of trimethylaluminum and tris(tert-butoxy)silanol deposits highly conformal layers of amorphous silicon dioxide and aluminum oxide nanolaminates at rates of 12 nanometers (more than 32 monolayers) per cycle. This process allows for the uniform lining or filling of long, narrow holes. We propose that these ALD layers grow by a previously unknown catalytic mechanism that also operates during the rapid ALD of many other metal silicates. This process should allow improved production of many devices, such as trench insulation between transistors in microelectronics, planar waveguides, microelectromechanical structures, multilayer optical filters, and protective layers against diffusion, oxidation, or corrosion.

Large interdomain rearrangement triggered by suppression of micro- to millisecond dynamics in bacterial Enzyme I

NASA Astrophysics Data System (ADS)

Venditti, Vincenzo; Tugarinov, Vitali; Schwieters, Charles D.; Grishaev, Alexander; Clore, G. Marius

2015-01-01

Enzyme I (EI), the first component of the bacterial phosphotransfer signal transduction system, undergoes one of the largest substrate-induced interdomain rearrangements documented to date. Here we characterize the perturbations generated by two small molecules, the natural substrate phosphoenolpyruvate and the inhibitor α-ketoglutarate, on the structure and dynamics of EI using NMR, small-angle X-ray scattering and biochemical techniques. The results indicate unambiguously that the open-to-closed conformational switch of EI is triggered by complete suppression of micro- to millisecond dynamics within the C-terminal domain of EI. Indeed, we show that a ligand-induced transition from a dynamic to a more rigid conformational state of the C-terminal domain stabilizes the interface between the N- and C-terminal domains observed in the structure of the closed state, thereby promoting the resulting conformational switch and autophosphorylation of EI. The mechanisms described here may be common to several other multidomain proteins and allosteric systems.

Electron-Transfer Dynamics for a Donor-Bridge-Acceptor Complex in Ionic Liquids.

PubMed

DeVine, Jessalyn A; Labib, Marena; Harries, Megan E; Rached, Rouba Abdel Malak; Issa, Joseph; Wishart, James F; Castner, Edward W

2015-08-27

Intramolecular photoinduced electron transfer from an N,N-dimethyl-p-phenylenediamine donor bridged by a diproline spacer to a coumarin 343 acceptor was studied using time-resolved fluorescence measurements in three ionic liquids and in acetonitrile. The three ionic liquids have the bis[(trifluoromethyl)sulfonyl]amide anion paired with the tributylmethylammonium, 1-butyl-1-methylpyrrolidinium, and 1-decyl-1-methylpyrrolidinium cations. The dynamics in the two-proline donor-bridge-acceptor complex are compared to those observed for the same donor and acceptor connected by a single proline bridge, studied previously by Lee et al. (J. Phys. Chem. C 2012, 116, 5197). The increased conformational freedom afforded by the second bridging proline resulted in multiple energetically accessible conformations. The multiple conformations have significant variations in donor-acceptor electronic coupling, leading to dynamics that include both adiabatic and nonadiabatic contributions. In common with the single-proline bridged complex, the intramolecular electron transfer in the two-proline system was found to be in the Marcus inverted regime.

2010 Award for Outstanding Doctoral Thesis Research in Biological Physics Talk: How the Genome Folds

NASA Astrophysics Data System (ADS)

Lieberman-Aiden, Erez

2011-03-01

I describe Hi-C, a novel technology for probing the three-dimensional architecture of whole genomes by coupling proximity-based ligation with massively parallel sequencing. Working with collaborators at the Broad Institute and UMass Medical School, we used Hi-C to construct spatial proximity maps of the human genome at a resolution of 1Mb. These maps confirm the presence of chromosome territories and the spatial proximity of small, gene-rich chromosomes. We identified an additional level of genome organization that is characterized by the spatial segregation of open and closed chromatin to form two genome-wide compartments. At the megabase scale, the chromatin conformation is consistent with a fractal globule, a knot-free conformation that enables maximally dense packing while preserving the ability to easily fold and unfold any genomic locus. The fractal globule is distinct from the more commonly used globular equilibrium model. Our results demonstrate the power of Hi-C to map the dynamic conformations of whole genomes.

Conformational Changes in the Hepatitis B Virus Core Protein Are Consistent with a Role for Allostery in Virus Assembly

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

Packianathan, Charles; Katen, Sarah P.; Dann, III, Charles E.

2010-01-12

In infected cells, virus components must be organized at the right place and time to ensure assembly of infectious virions. From a different perspective, assembly must be prevented until all components are available. Hypothetically, this can be achieved by allosterically controlling assembly. Consistent with this hypothesis, here we show that the structure of the hepatitis B virus (HBV) core protein dimer, which can spontaneously self-assemble, is incompatible with capsid assembly. Systematic differences between core protein dimer and capsid conformations demonstrate linkage between the intradimer interface and interdimer contact surface. These structures also provide explanations for the capsid-dimer selectivity of somemore » antibodies and the activities of assembly effectors. Solution studies suggest that the assembly-inactive state is more accurately an ensemble of conformations. Simulations show that allostery supports controlled assembly and results in capsids that are resistant to dissociation. We propose that allostery, as demonstrated in HBV, is common to most self-assembling viruses.« less

Calculation of Cyclodextrin Binding Affinities: Energy, Entropy, and Implications for Drug Design

PubMed Central

Chen, Wei; Chang, Chia-En; Gilson, Michael K.

2004-01-01

The second generation Mining Minima method yields binding affinities accurate to within 0.8 kcal/mol for the associations of α-, β-, and γ-cyclodextrin with benzene, resorcinol, flurbiprofen, naproxen, and nabumetone. These calculations require hours to a day on a commodity computer. The calculations also indicate that the changes in configurational entropy upon binding oppose association by as much as 24 kcal/mol and result primarily from a narrowing of energy wells in the bound versus the free state, rather than from a drop in the number of distinct low-energy conformations on binding. Also, the configurational entropy is found to vary substantially among the bound conformations of a given cyclodextrin-guest complex. This result suggests that the configurational entropy must be accounted for to reliably rank docked conformations in both host-guest and ligand-protein complexes. In close analogy with the common experimental observation of entropy-enthalpy compensation, the computed entropy changes show a near-linear relationship with the changes in mean potential plus solvation energy. PMID:15339804

The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility.

PubMed

Lamontanara, Allan Joaquim; Georgeon, Sandrine; Tria, Giancarlo; Svergun, Dmitri I; Hantschel, Oliver

2014-11-17

The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility

NASA Astrophysics Data System (ADS)

Lamontanara, Allan Joaquim; Georgeon, Sandrine; Tria, Giancarlo; Svergun, Dmitri I.; Hantschel, Oliver

2014-11-01

The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

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

PubMed

Frederick, Thomas E; Peng, Jeffrey W

2018-01-01

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

Geometric Patterns for Neighboring Bases Near the Stacked State in Nucleic Acid Strands.

PubMed

Sedova, Ada; Banavali, Nilesh K

2017-03-14

Structural variation in base stacking has been analyzed frequently in isolated double helical contexts for nucleic acids, but not as often in nonhelical geometries or in complex biomolecular environments. In this study, conformations of two neighboring bases near their stacked state in any environment are comprehensively characterized for single-strand dinucleotide (SSD) nucleic acid crystal structure conformations. An ensemble clustering method is used to identify a reduced set of representative stacking geometries based on pairwise distances between select atoms in consecutive bases, with multiple separable conformational clusters obtained for categories divided by nucleic acid type (DNA/RNA), SSD sequence, stacking face orientation, and the presence or absence of a protein environment. For both DNA and RNA, SSD conformations are observed that are either close to the A-form, or close to the B-form, or intermediate between the two forms, or further away from either form, illustrating the local structural heterogeneity near the stacked state. Among this large variety of distinct conformations, several common stacking patterns are observed between DNA and RNA, and between nucleic acids in isolation or in complex with proteins, suggesting that these might be stable stacking orientations. Noncanonical face/face orientations of the two bases are also observed for neighboring bases in the same strand, but their frequency is much lower, with multiple SSD sequences across categories showing no occurrences of such unusual stacked conformations. The resulting reduced set of stacking geometries is directly useful for stacking-energy comparisons between empirical force fields, prediction of plausible localized variations in single-strand structures near their canonical states, and identification of analogous stacking patterns in newly solved nucleic acid containing structures.

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

Optimal Mass Transport for Shape Matching and Comparison

PubMed Central

Su, Zhengyu; Wang, Yalin; Shi, Rui; Zeng, Wei; Sun, Jian; Luo, Feng; Gu, Xianfeng

2015-01-01

Surface based 3D shape analysis plays a fundamental role in computer vision and medical imaging. This work proposes to use optimal mass transport map for shape matching and comparison, focusing on two important applications including surface registration and shape space. The computation of the optimal mass transport map is based on Monge-Brenier theory, in comparison to the conventional method based on Monge-Kantorovich theory, this method significantly improves the efficiency by reducing computational complexity from O(n2) to O(n). For surface registration problem, one commonly used approach is to use conformal map to convert the shapes into some canonical space. Although conformal mappings have small angle distortions, they may introduce large area distortions which are likely to cause numerical instability thus resulting failures of shape analysis. This work proposes to compose the conformal map with the optimal mass transport map to get the unique area-preserving map, which is intrinsic to the Riemannian metric, unique, and diffeomorphic. For shape space study, this work introduces a novel Riemannian framework, Conformal Wasserstein Shape Space, by combing conformal geometry and optimal mass transport theory. In our work, all metric surfaces with the disk topology are mapped to the unit planar disk by a conformal mapping, which pushes the area element on the surface to a probability measure on the disk. The optimal mass transport provides a map from the shape space of all topological disks with metrics to the Wasserstein space of the disk and the pullback Wasserstein metric equips the shape space with a Riemannian metric. We validate our work by numerous experiments and comparisons with prior approaches and the experimental results demonstrate the efficiency and efficacy of our proposed approach. PMID:26440265

Comparison of the helical tomotherapy against the multileaf collimator-based intensity-modulated radiotherapy and 3D conformal radiation modalities in lung cancer radiotherapy

PubMed Central

Mavroidis, P; Shi, C; Plataniotis, G A; Delichas, M G; Costa Ferreira, B; Rodriguez, S; Lind, B K; Papanikolaou, N

2011-01-01

Objectives The aim of this study was to compare three-dimensional (3D) conformal radiotherapy and the two different forms of IMRT in lung cancer radiotherapy. Methods Cases of four lung cancer patients were investigated by developing a 3D conformal treatment plan, a linac MLC-based step-and-shoot IMRT plan and an HT plan for each case. With the use of the complication-free tumour control probability (P+) index and the uniform dose concept as the common prescription point of the plans, the different treatment plans were compared based on radiobiological measures. Results The applied plan evaluation method shows the MLC-based IMRT and the HT treatment plans are almost equivalent over the clinically useful dose prescription range; however, the 3D conformal plan inferior. At the optimal dose levels, the 3D conformal treatment plans give an average P+ of 48.1% for a effective uniform dose to the internal target volume (ITV) of 62.4 Gy, whereas the corresponding MLC-based IMRT treatment plans are more effective by an average ΔP+ of 27.0% for a Δ effective uniform dose of 16.3 Gy. Similarly, the HT treatment plans are more effective than the 3D-conformal plans by an average ΔP+ of 23.8% for a Δ effective uniform dose of 11.6 Gy. Conclusion A radiobiological treatment plan evaluation can provide a closer association of the delivered treatment with the clinical outcome by taking into account the dose–response relations of the irradiated tumours and normal tissues. The use of P – effective uniform dose diagrams can complement the traditional tools of evaluation to compare and effectively evaluate different treatment plans. PMID:20858664

Dual regulatory switch confers tighter control on HtrA2 proteolytic activity.

PubMed

Singh, Nitu; D'Souza, Areetha; Cholleti, Anuradha; Sastry, G Madhavi; Bose, Kakoli

2014-05-01

High-temperature requirement protease A2 (HtrA2), a multitasking serine protease that is involved in critical biological functions and pathogenicity, such as apoptosis and cancer, is a potent therapeutic target. It is established that the C-terminal post-synaptic density protein, Drosophila disc large tumor suppressor, zonula occludens-1 protein (PDZ) domain of HtrA2 plays pivotal role in allosteric modulation, substrate binding and activation, as commonly reported in other members of this family. Interestingly, HtrA2 exhibits an additional level of functional modulation through its unique N-terminus, as is evident from 'inhibitor of apoptosis proteins' binding and cleavage. This phenomenon emphasizes multiple activation mechanisms, which so far remain elusive. Using conformational dynamics, binding kinetics and enzymology studies, we addressed this complex behavior with respect to defining its global mode of regulation and activity. Our findings distinctly demonstrate a novel N-terminal ligand-mediated triggering of an allosteric switch essential for transforming HtrA2 to a proteolytically competent state in a PDZ-independent yet synergistic activation process. Dynamic analyses suggested that it occurs through a series of coordinated structural reorganizations at distal regulatory loops (L3, LD, L1), leading to a population shift towards the relaxed conformer. This precise synergistic coordination among different domains might be physiologically relevant to enable tighter control upon HtrA2 activation for fostering its diverse cellular functions. Understanding this complex rheostatic dual switch mechanism offers an opportunity for targeting various disease conditions with tailored site-specific effector molecules. © 2014 FEBS.

Differential binding of prohibitin-2 to estrogen receptor α and to drug-resistant ERα mutants

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

Chigira, Takeru, E-mail: 8120661875@mail.ecc.u-tokyo.ac.jp; Nagatoishi, Satoru, E-mail: nagatoishi@bioeng.t.u-tokyo.ac.jp; Tsumoto, Kouhei, E-mail: tsumoto@bioeng.t.u-tokyo.ac.jp

2015-08-07

Endocrine resistance is one of the most challenging problems in estrogen receptor alpha (ERα)-positive breast cancer. The transcriptional activity of ERα is controlled by several coregulators, including prohibitin-2 (PHB2). Because of its ability to repress the transcriptional activity of activated ERα, PHB2 is a promising antiproliferative agent. In this study, were analyzed the interaction of PHB2 with ERα and three mutants (Y537S, D538G, and E380Q) that are frequently associated with a lack of sensitivity to hormonal treatments, to help advance novel drug discovery. PHB2 bound to ERα wild-type (WT), Y537S, and D538G, but did not bind to E380Q. The bindingmore » thermodynamics of Y537S and D538G to PHB2 were favorably altered entropically compared with those of WT to PHB2. Our results show that PHB2 binds to the ligand binding domain of ERα with a conformational change in the helix 12 of ERα. - Highlights: • Molten globule-likeness of an ERα repressor Prohibitin-2 (PHB2) is identified. • The thermodynamics is validated for the interaction between ERα and PHB2. • PHB2 binds to Y537S and D538G mutants of ERα commonly found in breast cancer. • ERα WT and mutants showed different thermodynamic parameters in the binding to PHB2. • ERα binds to PHB2 with conformational change involving packing of helix 12.« less

A 3D-structural model of unsulfated chondroitin from high-field NMR: 4-sulfation has little effect on backbone conformation

PubMed Central

Sattelle, Benedict M.; Shakeri, Javad; Roberts, Ian S.; Almond, Andrew

2010-01-01

The glycosaminoglycan chondroitin sulfate is essential in human health and disease but exactly how sulfation dictates its 3D-strucutre at the atomic level is unclear. To address this, we have purified homogenous oligosaccharides of unsulfated chondroitin (with and without 15N-enrichment) and analysed them by high-field NMR to make a comparison published chondroitin sulfate and hyaluronan 3D-structures. The result is the first full assignment of the tetrasaccharide and an experimental 3D-model of the hexasaccharide (PDB code 2KQO). In common with hyaluronan, we confirm that the amide proton is not involved in strong, persistent inter-residue hydrogen bonds. However, in contrast to hyaluronan, a hydrogen bond is not inferred between the hexosamine OH-4 and the glucuronic acid O5 atoms across the β(1→3) glycosidic linkage. The unsulfated chondroitin bond geometry differs slightly from hyaluronan by rotation about the β(1→3) ψ dihedral (as previously predicted by simulation), while the β(1→4) linkage is unaffected. Furthermore, comparison shows that this glycosidic linkage geometry is similar in chondroitin-4-sulfate. We therefore hypothesise that both hexosamine OH-4 and OH-6 atoms are solvent exposed in chondroitin, explaining why it is amenable to sulfation and hyaluronan is not, and also that 4-sulfation has little effect on backbone conformation. Our conclusions exemplify the value of the 3D-model presented here and progress our understanding of glycosaminoglycan molecular properties. PMID:20022001