Conformational Dynamics of apo‐GlnBP Revealed by Experimental and Computational Analysis

The glutamine binding protein (GlnBP) binds l ‐glutamine and cooperates with its cognate transporters during glutamine uptake. Crystal structure analysis has revealed an open and a closed conformation for apo‐ and holo‐GlnBP, respectively. However, the detailed conformational dynamics have remained unclear. Herein, we combined NMR spectroscopy, MD simulations, and single‐molecule FRET techniques to decipher the conformational dynamics of apo‐GlnBP. The NMR residual dipolar couplings of apo‐GlnBP were in good agreement with a MD‐derived structure ensemble consisting of four metastable states. The open and closed conformations are the two major states. This four‐state model was further validated by smFRET experiments and suggests the conformational selection mechanism in ligand recognition of GlnBP.     

抗脂解活性的人参十四肽的结构预测

本文对抗脂解活性的人参多肽的结构进行了分析. 在一级结构的基础上预测了其二级结构含有较多的α螺旋, 后接一段无规卷曲. 应用分子力学方法, 结合晶体数据库信息, 计算了人参中抗脂解十四肽分子的相对构家能量和原子坐标, 模拟了构象能量优化过程, 得到了可能的最低能量构象. 并利用计算机分子图形技术建立了相应的人参抗脂解十四肽的三维分子模型. 模型的空间结构表明构象分析结果与二级预测的结论很好地相符。     

Structure change of β-hairpin induced by turn optimization: an enhanced sampling molecular dynamics simulation study

Our previous study showed that for the tested polypeptides which have similar β-hairpin structures but different sequences, their folding free energy pathways are dominantly determined by the turn conformational propensity. In this study, we study how the turn conformational propensity affects the structure of hairpins. The folding of two mutants of GB1p peptide (GB1m2 and GB1m3), which have the optimized turn sequence ((6)DDATK(11)T?→?(6)NPATG(11)K) with native structures unsolved, were simulated using integrated tempering sampling molecular dynamics simulations and the predicted stable structures were compared to wild-type GB1p. It was observed that the turn optimization of GB1p generates a more favored 5-residue type I(') turn in addition to the 6-residue type I turn in wild-type GB1p. As a result two distinctly different hairpin structures are formed corresponding to the "misfolded" (M) and the "folded" (F) states. M state is a one-residue-shifted asymmetric β-hairpin structure whereas F state has the similar symmetric hairpin structure as wild-type GB1p. The formation of the favored type I(') turn has a small free energy barrier and leads to the shifted β-hairpin structure, following the modified "zipping" model. The presence of disfavored type I turn structure makes the folding of a β-hairpin consistent with the "hydrophobic-core-centric" model. On the other hand, the folding simulations on other two GB1p mutants (GB1r1 and GBr2), which have the position of the hydrophobic core cluster further away from the turn compared to wild-type GB1p, showed that moving the hydrophobic core cluster away from the turn region destabilizes but does not change the hairpin structure. Therefore, the present study showed that the turn conformational propensity is a key factor in affecting not only the folding pathways but also the stable structure of β-hairpins, and the turn conformational change induced by the turn optimization leads to significant changes of β-hairpin structure.     

Conformational analysis of dicarbonyl(methylcyclopentadienyl)(2-(diethylamino)-2-oxoethyl)iron(II): a spectroscopic and molecular mechanics study

A combination of NMR and IR spectroscopic techniques was used to examine the conformational preferences of the previously unreported oxaallyliron compound dicarbonyl(methylcyclopentadienyl)(2-(diethylamino)-2-oxoethyl)iron(II) (1). IR studies revealed that 1 existed in n-pentane solutions as an equilibrium between two or more exchanging conformers through a rotation about the Fe---C bond. An additional contribution to the conformational dynamics of 1 was identified due to the resonance component of the amide group. The resonance contribution manifested in the observed restricted rotation about the C---N amide bond. Molecular mechanics calculations were used to model the conformational processes. The calculations predicted that the resonance stabilized conformation was the energetically preferred structure of 1. This agrees with the experimental evidence that identified the influence of resonance on the conformation of 1.     

Belizeanic acid: a potent protein phosphatase 1 inhibitor belonging to the okadaic acid class, with an unusual skeleton

Belizeanic acid (BA), a novel metabolite belonging to the okadaic acid class of protein phosphatase inhibitors, was isolated from artificial cultures of the dinoflagellate Prorocentrum belizeanum. The structure and conformational behaviour of BA was elucidated by a combination of NMR spectroscopy and conformational analysis. The isolation of this metabolite, which possesses a simplified version of the okadaic acid skeleton, supports the biogenetic pathway previously reported for this class of compounds. BA showed potent inhibitory activity against protein phosphatase 1 (PP1) within the nM range. A plausible model for the interaction of BA with the PP1 binding pocket was derived from computational docking studies.     

pH-induced reversible conformational and morphological regulation of polyleucine grafted polyallylamine assembly in solution

One of the essential parts in the molecular mechanism of biological properties is the structural changes of proteins induced by stimuli. An amphiphilic copolymer, poly(L-leucine) grafted polyallylamine as a simple model of proteins, has been prepared by NCA polymerization with free amino groups of polyallylamine as an initiator. Here, we report the pH-induced reversible conformational and morphological regulation of the amphiphilic copolymer, whose hydrophobic peptide graft chains have no pH-sensitive groups, in an aqueous solution containing 50 vol % trifluoroethanol. The conformation of the poly(L-leucine) graft chain was found to be strongly pH dependent. Under acidic conditions, where electrostatic repulsion existed between the neighboring protonated amine moieties of the polyallylamine main chain, the rapid aggregation of the poly(l-leucine) graft chains was disturbed, and the peptide graft chains formed a beta-sheet structure owing to the intramolecular hydrogen bonding among the graft chains. Under this condition, the amphiphilic polymer formed amyloid-like fibrils, and then the fibrils grew into a planer plate composed of staked beta-sheets. On the other hand, under basic conditions, the poly(L-leucine) graft chains showed conformational transitions from a beta-sheet structure to an alpha-helical conformation owing to a distortion of the regular arrangement of the peptide graft chains by the conformational change of the polyallylamine main chain, whose amino groups were deprotonated. The conformational transition resulted in a disturbance of the regular sheet assembly of the amphiphilic copolymer and induced morphological changes to the amorphous globular aggregates. The pH-induced conformational and morphological changes of the poly(L-leucine) graft polyallylamine were reversible and synchronized with the protonation of the polyallylamine main chain.     

Characterization of hemoglobin immobilized on gamma-zirconium phosphate

The fact that different γ-zirconium phosphate (γ-ZrP) preintercalation method induced varied degree and type of conformational change of the adsorption protein was confirmed by characterization techniques including circular dichroism (CD), fourier transform infrared spectroscopy (FTIR) and X-ray powder diffraction (XRD) analysis. The results indicated that the association of hemoglobin with γ-ZrP preintercalated using butylamine was correlated with conformational change in the secondary structure of the protein. γ-ZrP which was preintercalated with tetra (n-butylammonium) hydroxide caused the conformational change of Hemoglobin in both the secondary structure and the tertiary structure. X-ray powder diffraction analysis was used to analyze the crystalline structure of the nanocomposites prepared by relamination. The adsorption isotherms of Hemoglobin on different matrices were set up and fitted with Langmuir and Freundlich equations.     

Prediction of the crystal structure of a cyclic tetrapeptide

Prediction of the known crystal structure of cyclic-L-Ser(O-t-Bu)-β-Ala-Gly-L-β-Asp(OMe) has been attempted by establishing the low-energy conformations of the isolated molecule by conformational analysis, and then regarding each of these as a rigid molecule, by establishing the favorable crystal packing arrangements by molecular packing analysis. The theoretical model closest to the observed structure was one of the lowest-energy solutions and was recognized as essentially correct by reference to the x-ray data. The limitations of the model follow from the fact that the side chain conformations are somewhat affected by packing interactions.     

Probing the conformational features of a phage display polypeptide sequence directed against single-walled carbon nanohorn surfaces

Single-walled carbon nanohorns (SWNHs) are interesting carbon nanostructures that have applications to science and technology. Using M13 phage display technology, polypeptides directed again SWNHs surfaces have been created for a number of nanotechnology and pharmaceutical purposes, yet the molecular mechanism of polypeptide sequence interaction and binding to SWNHs surfaces is not known. Recently, we identified a linear 12-AA M13 phage pIII sequence, NH-12-5-2 (DYFSSPYYEQLF), that binds with high affinity to SWNHs surfaces. To probe the structure of this pIII tail polypeptide further, we investigated the conformation of a model peptide representing the 12 AA NH-12-5-2 sequence. At neutral pH, the NH-12-5-2 model polypeptide is conformationally labile and exhibits two-state conformational exchange involving the D1-S5 N-terminal segment. Simultaneous with this conformational exchange process is the observation that the P6 residue exhibits imido ring conformational variation. In the presence of the structure-stabilizing solvent, TFE, or at pH 2.5, both the exchange process and Pro ring motion phenomena disappear, indicating that the structure of this peptide sequence can be stabilized by extrinsic factors. Interestingly, we observe NMR parameters (ROEs, (3)J coupling constants) for NH-12-5-2 in 90% v/v TFE that are consistent with the presence of a partial helical structure, similar to what was observed at low pH in our earlier CD experiments. We conclude that the NH-12-5-2 model polypeptide sequence possesses an inherent conformational instability that involves the D1-S5 sequence segment and the P6 residue but that this instability can be offset by extrinsic factors (e.g., charge neutralization, imido ring interconversion, and hydrophobic-hydrophobic interactions). These nonbonding interactions may play a role in the recognition and binding of this phage sequence region to SWNHs surfaces.     

Dependence of the conformation of nascent poly(oxymethylene) on the conditions of cationic polymerization of trioxane in solution

The conformational structure of nascent poly(oxymethylene) (POM) obtained by cationic polymerization of trioxane in nitrobenzene was investigated by i.r. spectroscopy. It was found that the conformational order of this POM depends considerably on the conditions of preparation. Under conditions of simultaneous polymerization and crystallization, “A” polymer, with long sequences of monomer units in regular G conformation, is obtained. Under conditions of successive polymerization and crystallization, the formation of conformational defects in the helical POM chains is favoured. Then, depending on the supersaturation, we obtained either (a) POM of “B” form with short sequences of monomer units in G conformation, or (b) POM of a mixed type, the i.r. spectrum being describable as a superposition of “A” and “B” spectra. The results indicate that, at comparatively high catalyst concentration, the thermodynamical approach for regulation of supermolecular structure of polymers can be applied successfully for regulation of the conformational order of nascent POM also. At lower concentration of active centres in the polymerizing system, kinetic factors affect considerably the conformational structure of nascent POM.     

Single molecule conformational dynamics of adenylate kinase: energy landscape, structural correlations, and transition state ensembles

We developed a coarse grained two-well model to study the single molecule protein conformational dynamics in microscopic detail at the residue level, overcoming the often encountered computational bottleneck. In particular, we explored the underlying conformational energy landscape of adenylate kinase, a crucial protein for signal transduction in the cell, and identified two major kinetic pathways for the conformational switch between open and closed states through either the intermediate state or the transient state. Based on the parameters fitted to the room-temperature experimental data, we predicted open and closed kinetic rates at the whole temperature ranges from 10 to 50 degrees C, which agree well with the experimental turnover numbers. After uncovering the underlying mechanism for conformational dynamics and exploring the structural correlations, we found the crucial dynamical interplay between the nucleoside monophosphate binding domain (NMP) and the ATP-binding domain (LID) in controlling the conformational switch. The key residues and contacts responsible for the conformational transitions are identified by following the time evolution of the two-dimensional spatial contact maps and characterizing the transition state as well as intermediate structure ensembles through phi value analysis. Our model provides a general framework to study the conformational dynamics of biomolecules and can be applied to many other systems.     

Use of essential and molecular dynamics to study gammaB-crystallin unfolding after non-enzymic post-translational modifications

Essential and Molecular Dynamics (ED/MD) have been used to model the conformational changes of a protein implicated in a conformational disease--cataract, the largest cause of blindness in the world-after non-enzymic post-translational modification. Cyanate modification did not significantly alter flexibility, while the Schiff's base adduct produced a more flexible N-terminal domain, and intra-secondary structure regions, than either the cyanate adduct or the native structure. Glycation also increased linker flexibility and disrupted the charge network. A number of post-translational adducts showed structural disruption around Cys15 and increased linker flexibility; this may be important in subsequent protein aggregation. Our modelling results are in accord with experimental evidence, and show that ED/MD is a useful tool in modelling conformational changes in proteins implicated in disease processes.     

Specific and nonspecific effects of glycosylation

Glycosylation regulates vital cellular processes and dramatically influences protein folding and stability. In particular, experiments have demonstrated that asparagine (N)-linked disaccharides drive a "conformational switch" in a model peptide. The present work investigates this conformational switch via extensive atomically detailed replica exchange molecular dynamics simulations in explicit solvent. To distinguish the effects of specific and nonspecific interactions upon the peptide conformational ensemble, these simulations considered model peptides that were N-linked to a disaccharide and to a steric crowder of the same shape. The simulations are remarkably consistent with experiment and provide detailed insight into the peptide structure ensemble. They suggest that steric crowding by N-linked disaccharides excludes extended conformations, but does not significantly impact the tetrahedral structure of the surrounding solvent or otherwise alter the peptide free energy surface. However, the combination of steric crowding with specific hydrogen bonds and hydrophobic stacking interactions more dramatically impacts the peptide ensemble and stabilizes new structures.     

Switching in molecular transport junctions: polarization response

We discuss several proposed explanations for the switching and negative differential resistance (NDR) behavior seen in some molecular junctions. Several theoretical models are discussed, and we present results of electronic structure calculations on a series of substituted oligo(phenylene ethynylene) molecules. It is shown that a previously proposed polaron model is successful in predicting NDR behavior, and the model is elaborated with image charge effects and parameters from electronic structure calculations. This model now incorporates substituent effects and includes the effects of conformational change, charging, and image charge stabilization.     

Atomistic Brownian dynamics simulation of peptide phosphorylation

We report the implementation of an all-atom Brownian dynamics simulation model of peptides using the constraint algorithm LINCS. The algorithm has been added as a part of UHBD. It uses adaptive time steps to achieve a balance between computational speed and stability. The algorithm was applied to study the effect of phosphorylation on the conformational preference of the peptide Gly-Ser-Ser-Ser. We find that the middle serine residue experiences considerable conformational change from the C(7eq) to the alpha(R) structure upon phosphorylation. NMR (3)J coupling constants were also computed from the Brownian trajectories using the Karplus equation. The calculated (3)J results agree reasonably well with experimental data for phosphorylated peptide but less so for doubly charged phosphorylated one.     

Hidden protein folding pathways in free-energy landscapes uncovered by network analysis

A network analysis is used to uncover hidden folding pathways in free-energy landscapes usually defined in terms of such arbitrary order parameters as root-mean-square deviation from the native structure, radius of gyration, etc. The analysis has been applied to molecular dynamics (MD) trajectories of the B-domain of staphylococcal protein A, generated with the coarse-grained united-residue (UNRES) force field in a broad range of temperatures (270K ≤ T ≤ 325K). Thousands of folding pathways have been identified at each temperature. Out of these many folding pathways, several most probable ones were selected for investigation of the conformational transitions during protein folding. Unlike other conformational space network (CSN) methods, a node in the CSN variant implemented in this work is defined according to the nativelikeness class of the structure, which defines the similarity of segments of the compared structures in terms of secondary-structure, contact-pattern, and local geometry, as well as the overall geometric similarity of the conformation under consideration to that of the reference (experimental) structure. Our previous findings, regarding the folding model and conformations found at the folding-transition temperature for protein A (Maisuradze et al., J. Am. Chem. Soc. 132, 9444, 2010), were confirmed by the conformational space network analysis. In the methodology and in the analysis of the results, the shortest path identified by using the shortest-path algorithm corresponds to the most probable folding pathway in the conformational space network.     

Modeling of polyvinylpyrrolidone and polyvinylimidazole in aqueous solution

We present the results of a molecular modeling investigation of iso-, syndio-, and atactic polyvinylpyrrolidone (PVP) and polyvinylimidazole (PVI) 20-mers in aqueous and ionic solutions at ambient conditions using the AMBER force filed in conjunction with the SPC/E water model. The PVP-water and PVI-water interactions were parameterized using solution properties of monomer-analogous molecules. Simulation of isolated oligomers in aqueous solution provided the structural (pair correlation functions, coordination numbers) and dynamic (residence times) characteristics of the polymer-water ‘interface’. Analysis of the oligomer conformational statistics permitted the construction of polymer conformations in an aqueous environment and the computation of characteristic ratios and structure factors. Finally, the oligomer conformational behavior was studied in the presence of sodium benzenesulfonate using the particle mesh Ewald technique.     

固有无序超嗜热菌FlgM蛋白在两种不同温度下的构象变化特征

基于分子动力学模拟方法比较了超嗜热菌FlgM 蛋白在常温(293 K)和生理温度(358 K)下的结构特征.基于GROMACS软件包, 采用OPLS-AA分子力场和TIP3P水模型, 对超嗜热菌FlgM 蛋白在293 和358 K进行了2组独立的长时间分子动力学模拟, 每组体系模拟时间为1500 ns. 主要分析了两种温度下超嗜热菌FlgM蛋白的二级结构特征、整体构象变化及半无序化区域和结构化区域的构象特征. 研究结果表明: 在常温下, N端具有一定程度的螺旋成分, 但在生理温度下, 超嗜热菌FlgM 蛋白的结构变得松散, 螺旋结构减少, 构象稳定性减弱, H1 螺旋散开, FlgM 蛋白构象灵活性增强, 不稳定程度增加. 这些不同温度的结构变化说明: 半无序化区域(N端)在非结合状态下有一定的螺旋结构, 但该段螺旋的稳定性随温度升高而降低. 超嗜热菌FlgM蛋白会通过增加结构的无序程度使结构更加灵活, 以适应高温, 从而使该类固有无序蛋白更好地行使其功能, 如提高同其他成分的结合速率等.     

Determination of conformational preferences of dipeptides using vibrational spectroscopy

The NMR coupling constants ((3)J(H(N), H(alpha))) of dipeptides indicate that the backbone conformational preferences vary strikingly among dipeptides. These preferences are similar to those of residues in small peptides, denatured proteins, and the coil regions of native proteins. Detailed characterization of the conformational preferences of dipeptides is therefore of fundamental importance for understanding protein structure and folding. Here, we studied the conformational preferences of 13 dipeptides using infrared and Raman spectroscopy. The main advantage of vibrational spectroscopy over NMR spectroscopy is in its much shorter time scale, which enables the determination of the conformational preferences of short-lived states. Accuracy of structure determination using vibrational spectroscopy depends critically on identification of the vibrational parameters that are sensitive to changes in conformation. We show that the frequencies of the amide I band and the A12 ratio of the amide I components of dipeptides correlate with the (3)J(H(N), H(alpha)). These two infrared vibrational parameters are thus analogous to (3)J(H(N), H(alpha)), indicators for the preference for the dihedral angle phi. We also show that the intensities of the components of the amide III bands in infrared spectra and the intensities of the skeletal vibrations in Raman spectra are indicators of populations of the P(II), beta, and alpha(R) conformations. The results show that alanine dipeptide adopts predominantly a PII conformation. The population of the beta conformation increases in valine dipeptides. The populations of the alpha(R) conformation are generally small. These data are in accord with the electrostatic screening model of conformational preferences.