Probing the energy landscape of protein folding/unfolding transition states

Previous molecular dynamics (MD) simulations of the thermal denaturation of chymotrypsin inhibitor 2 (CI2) have provided atomic-resolution models of the transition state ensemble that is well supported by experimental studies. Here, we use simulations to further investigate the energy landscape around the transition state region. Nine structures within approximately 35 ps and 3 A C(alpha) RMSD of the transition state ensemble identified in a previous 498 K thermal denaturation simulation were quenched under the quasi-native conditions of 335 K and neutral pH. All of the structures underwent hydrophobically driven collapse in response to the drop in temperature. Structures less denatured than the transition state became structurally more native-like, while structures that were more denatured than the transition state tended to show additional loss of native structure. The structures in the immediate region of the transition state fluctuated between becoming more and less native-like. All of the starting structures had the same native-like topology and were quite similar (within 3.5 A C(alpha) RMSD). That the structures all shared native-like topology, yet diverged into either more or less native-like structures depending on which side of the transition state they occupied on the unfolding trajectory, indicates that topology alone does not dictate protein folding. Instead, our results suggest that a detailed interplay of packing interactions and interactions with water determine whether a partially denatured protein will become more native-like under refolding conditions.     

Reconstruction of the src-SH3 protein domain transition state ensemble using multiscale molecular dynamics simulations

We use an integrated computational approach to reconstruct accurately the transition state ensemble (TSE) for folding of the src-SH3 protein domain. We first identify putative TSE conformations from free energy surfaces generated by importance sampling molecular dynamics for a fully atomic, solvated model of the src-SH3 protein domain. These putative TSE conformations are then subjected to a folding analysis using a coarse-grained representation of the protein and rapid discrete molecular dynamics simulations. Those conformations that fold to the native conformation with a probability (P(fold)) of approximately 0.5, constitute the true transition state. Approximately 20% of the putative TSE structures were found to have a P(fold) near 0.5, indicating that, although correct TSE conformations are populated at the free energy barrier, there is a critical need to refine this ensemble. Our simulations indicate that the true TSE conformations are compact, with a well-defined central beta sheet, in good agreement with previous experimental and theoretical studies. A structured central beta sheet was found to be present in a number of pre-TSE conformations, however, indicating that this element, although required in the transition state, does not define it uniquely. An additional tight cluster of contacts between highly conserved residues belonging to the diverging turn and second beta-sheet of the protein emerged as being critical elements of the folding nucleus. A number of commonly used order parameters to identify the transition state for folding were investigated, with the number of native Cbeta contacts displaying the most satisfactory correlation with P(fold) values.     

Effect of glycerol–water binary mixtures on the structure and dynamics of protein solutions

We have performed 20?ns of fully atomistic molecular dynamics simulations of Hen Egg-White Lysozyme in 0, 10, 20, 30, and 100% by weight of glycerol in water to better understand the microscopic physics behind the bioprotection offered by glycerol to naturally occuring biological systems. The solvent exposure of protein surface residues changes when glycerol is introduced. The dynamic behavior of the protein, as quantified by the incoherent intermediate scattering function, shows a nonmonotonic dependence on glycerol content. The fluctuations of the protein residues with respect to each other were found to be similar in all water-containing solvents, but different from the pure glycerol case. The increase in the number of protein–glycerol hydrogen bonds in glycerol–water binary mixtures explains the slowing down of protein dynamics as the glycerol content increases. We also explored the dynamic behavior of the hydration layer. We show that the short length scale dynamics of this layer are insensitive to glycerol concentration. However, the long length scale behavior shows a significant dependence on glycerol content. We also provide insights into the behavior of bound and mobile water molecules.     

Scrutiny of chain-length and N-terminal effects in α-helix folding: a molecular dynamics study on polyalanine peptides

Protein folding remains an unsolved problem as main-chain, side-chain, and solvent interactions remain entangled and have been hard to resolve. Polyalanines are promising models to analyze protein folding initiation and propagation structurally as well as energetically. In the present work, the effect of chain-length and N-terminal residue stereochemistry in polyalanine peptides are investigated for their role in the nucleation of α-helical conformation. The end-protected polyalanine peptides, tetra-alanine, Ac-^LAla₄-NHMe (Ia) and Ac-^DAla-^LAla₃-NHMe (Ib), hexa-alanine, Ac-^LAla₆-NHMe (IIa) and Ac-^DAla-^LAla₅-NHMe (IIb), and octa-alanine, Ac-^LAla₈-NHMe (IIIa) and Ac-^DAla-^LAla₇-NHMe (IIIb), are assessed as chain-length and stereochemical-structure perturbed models. The appreciable variations in the sampling of α-helical conformation, including a sampling of α-helix folds, due to the cooperative effect of chain-length and N-terminal residue stereochemistry have been noted. The electrostatics of α-helical conformation rather than the conformational entropy of the main-chain appear to be decisive in the initiation of α-helix folding. The results of the present work will enhance our understanding on the nucleation of α-helical conformation in short peptides and aid in the design of novel peptides with α-helical structure that can modulate disease-related protein–protein interactions.     

Dynamic Role of the G Protein in Stabilizing the Active State of the Adenosine A2A Receptor

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Controlling the regiospecificity and coupling of cytochrome P450cam: T185F mutant increases coupling and abolishes 3-hydroxynorcamphor product.

Cytochrome P450cam (P450CIA1) catalyzes the hydroxylation of camphor and several substrate analogues such as norcamphor and 1-methyl-norcamphor. Hydroxylation was found experimentally at the 3, 5, and 6 positions of norcamphor, but only at the 5 and 6 positions of 1-methyl-norcamphor. In the catalytic cycle, the hydroxylation of substrate is coupled to the consumption of NADH. For camphor, the degree of coupling is 100%, but for both norcamphor and 1-methyl-norcamphor, the efficiency is dramatically lowered to 12% and 50%, respectively. Based on an examination of the active site of P450cam, it appeared that mutating position 185 might dramatically alter the product specificity and coupling of hydroxylation of norcamphor by P450cam. Analysis of molecular dynamics trajectories of norcamphor bound to the T185F mutant of cytochrome P450cam predicted that hydroxylation at the 3 position should be abolished and that the coupling should be dramatically increased. This mutant was constructed and the product profile and coupling experimentally determined. The coupling was doubled, and hydroxylation at the 3 position was essentially abolished. Both of these results are in agreement with the prediction.     

真菌G蛋白信号调控蛋白的功能研究进展

G蛋白信号途径是真菌细胞信号转导网络的枢纽,在细胞的各种生物学调控过程中具有重要作用。G蛋白信号调控蛋白(Rgulators of G protein signaling,RGS)是一类重要的G蛋白信号调控因子,能通过促进G蛋白α亚基(Gα)偶联的GTP水解,使Gα和Gβγ亚基发生聚合,导致G蛋白失活,从而迅速关闭与G蛋白偶联的信号途径。自从第一个RGS蛋白在酿酒酵母中被鉴定以来,目前已经有30多个RGS蛋白在重要的模式真菌中被报道,包括构巢曲霉、绿僵菌、稻瘟病菌、玉米赤霉菌、轮枝镰孢菌、新型隐球菌和白色念珠菌等。RGS蛋白在真菌的营养菌丝生长、产孢、毒素和色素生产、致病性和有性生殖等过程中发挥着重要作用。本文对真菌中已报道RGS蛋白的功能进行了总结,对真菌RGS蛋白的结构特征和调控机制进行了评述。     

Electrostatic interaction-mediated conformational changes of adipocyte fatty acid binding protein probed by molecular dynamics simulation

Abstract

Adipocyte fatty acid binding protein (A-FABP) is a potential drug target for treatment of diabetes, obesity and atherosclerosis. Molecular dynamics (MD) simulations, principal component (PC) analysis and binding free energy calculations were combined to probe effect of electrostatic interactions of residues R78, R106 and R126 with inhibitors ZGB, ZGC and IBP on structural stability of three inhibitor/A-FABP complexes. The results indicate that mutation R126A produces significant influence on polar interactions of three inhibitors with A-FABP and these interactions are main force for driving the conformational change of A-FABP. Analyses on hydrogen bond interactions show that the decrease in hydrogen bonding interactions of residues R126 and Y128 with three inhibitors and the increase in that of K58 with inhibitors ZGC and IBP in the R126A mutated systems mostly regulate the conformational changes of A-FABP. This work shows that R126A can generate a significant perturbation on structural stability of A-FABP, which implies that R126 is of significance in inhibitor bindings. We expect that this study can provide a theoretical guidance for design of potent inhibitors targeting A-FABP.

Communicated by Ramaswamy H. Sarma     

Estimation of pH effect on the structure and stability of kinase domain of human integrin-linked kinase

Integrin-linked kinase (ILK) is an evolutionarily conserved Ser/Thr protein kinase, involved in many physiological functions such as signal transduction, actin rearrangement, cell proliferation, migration, polarisation, angiogenesis and apoptosis. An increased expression of ILK is associated with different cancers and thus considered as an attractive target for cancer therapy. We have successfully cloned, expressed and purified the kinase domain (193–446 residues) of ILK. To see the effect of pH on the structure and conformation, we performed circular diachroism, fluorescence and absorbance measurements in a wide range of pH conditions. We observed that within the range of pH 7.5–11.0, ILK_193–446 maintains its both secondary and tertiary structures. While visible aggregates were observed under the acidic pH 2.0–5.5 conditions, in order to complement these observations, we have performed molecular dynamics simulations of this kinase domain by mimicking diverse pH conditions which enabled us to see conformational preferences of the protein under such conditions. A significant correlation between the spectroscopic and molecular dynamics simulation was observed. These findings are useful to understand the conformation of ILK protein under certain pH condition which may be further implicated in the drug design and discovery.     

Smoluchowski dynamics of the vnd/NK‐2 homeodomain from Drosophila melanogaster: First‐order mode‐coupling approximation

This work is the first in a series devoted to applying mode coupling diffusion theory to the derivation of local dynamics properties of proteins in solution. The first‐order mode‐coupling approximation, or optimized Rouse–Zimm local dynamics (ORZLD), is applied here to derive the rotational dynamics of the bonds and compare the calculated with the experimental nmr ¹⁵N spin–lattice relaxation time behavior of the vnd/NK‐2 homeodomain from Drosophila melanogaster. The starting point for the calculations is the experimental three‐dimensional structure of the homeodomain determined by multidimensional nmr spectroscopy. The results of the computations are compared with experimentally measured ¹⁵N spin–lattice relaxation times T₁, at 34.5 and 60.8 MHz, to check the first‐order approximation. To estimate the relative importance of internal and overall rotation, both rigid and fluctuating dynamic models are examined, with fluctuations evaluated using molecular dynamics (MD) simulations. The correlation times for the fundamental bond vector time correlation function and for the second‐order bond orientational TCF are obtained as a function of the residue number for vnd/NK‐2. The stability of the corresponding local dynamics pattern for the fluctuating structure as a function of the length of the MD trajectory is presented. Diffusive dynamics, which is essentially free of model parameters even at first order in the mode‐coupling diffusion approach, confirm that local dynamics of proteins can be described in terms of rotational diffusion of a fluctuating quasi‐rigid structure. The comparison with the nmr data shows that the first‐order mode coupling diffusion approximation accounts for the correct order of magnitude of the results and of important qualitative aspects of the data sensitive to conformational changes. Indications are obtained from this study to efficiently extend the theory to higher order in the mode‐coupling expansion. These results demonstrate the promise of the mode‐coupling approach, where the local dynamics of proteins is described in terms of rotational diffusion of a fluctuating quasi‐rigid structure, to analyze nmr spin–lattice relaxation behavior. © 1999 John Wiley & Sons, Inc. Biopoly 49: 235–254, 1999     

DSP1, a Drosophila HMG protein, is involved in spatiotemporal expression of the homoeotic gene Sex combs reduced

BACKGROUND INFORMATION: The Pc-G (Polycomb group) and trx-G (trithorax group) genes play a key role in the regulation of the homoeotic genes. The homoeotic gene Scr (Sex combs reduced) contained in the Antennapedia complex specifies segmental identity of the labial and prothoracic segments in Drosophila. Regulation of Scr requires the action of different enhancer elements spread over several kilobases. We previously identified an HMGB (high mobility group)-like protein DSP1 (dorsal switch protein 1), which works like a trx-G protein for the normal Scr expression. RESULTS: In the present study, we attempted to characterize the regulatory sequences involved in the maintenance of the Scr activation by DSP1. We report here, using a transgenic line for the Scr10.0XbaI-regulatory element, that lack of DSP1 affects the function of a reporter gene in legs' imaginal discs but not in embryos. We show by immunolocalization that DSP1 is recruited on polytene chromosomes to the insertion site of the transgene. Moreover, using chromatin immunoprecipitation experiments, we identify two regions of 1 kb in Scr10.0XbaI as the main DSP1 targets. CONCLUSION: These results provide strong evidence that the Scr gene expression is influenced by direct interaction between DSP1 and two Scr regulation elements. In addition, our results show that this interaction undergoes dynamic changes during development.