Structure and energy landscape of a photoswitchable peptide: a replica exchange molecular dynamics study

A replica exchange molecular dynamics (REMD) simulation of a bicyclic azobenzene peptide in explicit dimethyl sulfoxide solution is presented in order to characterize the conformational structures and energy landscape of a photoswitchable peptide. It is shown that an enhanced-sampling technique such as the REMD method is essential to obtain a converged conformational sampling of the peptide at room temperature. This is because conventional MD simulations of less than approximately 100-ns length are either trapped in local minima (at 295 K) or-if run at high temperature-do not resemble the room-temperature REMD results. Calculating various nuclear Overhauser effects (NOEs) and (3)J-couplings, a good overall agreement between the REMD simulations and the NMR experiments of Renner et al. (Biopolymers 2000;54:501-514) is found. In particular, the REMD study confirms the general picture drawn by Renner et al. that the trans-isomer of the azobenzene peptide exhibits a well-defined structure, while the cis-isomer is a conformational heterogeneous system; that is, the trans-isomer occurs in 2 well-defined conformers, while the cis-isomer represents an energetically frustrated system that leads to an ensemble of conformational structures. Employing a principal component analysis of the REMD data, the free energy landscape of the systems is studied at various temperatures. The implications for the folding and unfolding pathways of the system are discussed.     

Application of biasing‐potential replica‐exchange simulations for loop modeling and refinement of proteins in explicit solvent

Comparative or homology modeling of a target protein based on sequence similarity to a protein with known structure is widely used to provide structural models of proteins. Depending on the target‐template similarity these model structures may contain regions of limited structural accuracy. In principle, molecular dynamics (MD) simulations can be used to refine protein model structures and also to model loop regions that connect structurally conserved regions but it is limited by the currently accessible simulation time scales. A recently developed biasing potential replica exchange (BP‐REMD) method was used to refine loops and complete decoy protein structures at atomic resolution including explicit solvent. In standard REMD simulations several replicas of a system are run in parallel at different temperatures allowing exchanges at preset time intervals. In a BP‐REMD simulation replicas are controlled by various levels of a biasing potential to reduce the energy barriers associated with peptide backbone dihedral transitions. The method requires much fewer replicas for efficient sampling compared with T‐REMD. Application of the approach to several protein loops indicated improved conformational sampling of backbone dihedral angle of loop residues compared to conventional MD simulations. BP‐REMD refinement simulations on several test cases starting from decoy structures deviating significantly from the native structure resulted in final structures in much closer agreement with experiment compared to conventional MD simulations. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Conformational sampling by NMR solution structures calculated with the program DIANA evaluated by comparison with long-time molecular dynamics calculations in explicit water

The NMR solution structure of bovine pancreatic trypsin inhibitor (BPTI) obtained by distance geometry calculations with the program DIANA is compared with groups of conformers generated by molecular dynamics (MD) simulations in explicit water at ambient temperature and pressure. The MD simulations started from a single conformer and were free or restrained either by the experimental NOE distance restraints or by time-averaged restraints; the groups of conformers were collected either in 10 ps intervals during 200 ps periods of simulation, or in 50 ps intervals during a 1 ns period of simulation. Overall, these comparisons show that the standard protein structure determination protocol with the program DIANA provides a picture of the protein structure that is in agreement with MD simulations using “realistic” potential functions over a nanosecond timescale. For well-constrained molecular regions there is a trend in the free MD simulation of duration 1 ns that the sampling of the conformation space is slightly increased relative to the DIANA calculations. In contrast, for surface-exposed side-chains that are less extensively constrained by the NMR data, the DIANA conformers tend to sample larger regions of conformational space than conformers selected from any of the MD trajectories. Additional insights into the behavior of surface side-chains come from comparison of the MD runs of 200 ps or 1 ns duration. In this time range the sampling of conformation space by the protein surface depends strongly on the length of the simulation, which indicates that significant side-chain transitions occur on the nanosecond timescale and that much longer simulations will be needed to obtain statistically significant data on side-chain dynamics.     

Exploring the energy landscape of protein folding using replica-exchange and conventional molecular dynamics simulations

Two independent replica-exchange molecular dynamics (REMD) simulations with an explicit water model were performed of the Trp-cage mini-protein. In the first REMD simulation, the replicas started from the native conformation, while in the second they started from a nonnative conformation. Initially, the first simulation yielded results qualitatively similar to those of two previously published REMD simulations: the protein appeared to be over-stabilized, with the predicted melting temperature 50-150K higher than the experimental value of 315K. However, as the first REMD simulation progressed, the protein unfolded at all temperatures. In our second REMD simulation, which starts from a nonnative conformation, there was no evidence of significant folding. Transitions from the unfolded to the folded state did not occur on the timescale of these simulations, despite the expected improvement in sampling of REMD over conventional molecular dynamics (MD) simulations. The combined 1.42 micros of simulation time was insufficient for REMD simulations with different starting structures to converge. Conventional MD simulations at a range of temperatures were also performed. In contrast to REMD, the conventional MD simulations provide an estimate of Tm in good agreement with experiment. Furthermore, the conventional MD is a fraction of the cost of REMD and continuous, realistic pathways of the unfolding process at atomic resolution are obtained.     

The investigation of the secondary structure propensities and free-energy landscapes of peptide ligands by replica exchange molecular dynamics simulations

The conformational states of two peptide sequences that bind to staphylococcal enterotoxin B are sampled by replica exchange molecular dynamic (REMD) simulations in explicit water. REMD simulations were treated with 52 replicas in the range of 280–501 K for both peptides. The conformational ensembles of both peptides are dominated by random coil, bend and turn structures with a small amount of helical structures for each temperature. In addition, while an insignificant presence of β-bridge structures were observed for both peptides, the β-sheet structure was observed only for peptide 3. The results obtained from simulations at 300 K are consistent with the experimental results obtained from circular dichroism spectroscopy. From the analysis of REMD results, we also calculated hydrophobic and hydrophilic solvent accessible surface areas for both peptides, and it was observed that the hydrophobic segments of the peptides tend to form bend or turn structures. Moreover, the free-energy landscapes of both peptides were obtained by principal component analysis to understand how the secondary structural properties change according to their complex space. From the free-energy analysis, we have found several minima for both peptides at decreased temperature. For these obvious minima of both peptides, it was observed that the random coil, bend and turn structures are still dominant and the helix, β-bridge or β-sheet structures can appear or disappear with respect to minima. On the other hand, when we compare the results of REMD with conventional MD simulations for these peptides, the configurations of peptide 3 might be trapped in energy minima during the conventional MD simulations. Hence, it can be said that the REMD simulations have provided a sufficiently high sampling efficiency.     

A dynamic model of long-range conformational adaptations triggered by nucleotide binding in GroEL-GroES

The molecular chaperone, GroEL, essential for correct protein folding in E. coli, is composed of 14 identical subunits organized in two interacting rings, each providing a folding chamber for non‐native substrate proteins. The oligomeric assembly shows positive cooperativity within each ring and negative cooperativity between the rings. Although it is well known that ATP and long‐range allosteric interactions drive the functional cycle of GroEL, an atomic resolution view of how ligand binding modulates conformational adaptations over long distances remains a major challenge. Moreover, little is known on the relation between equilibrium dynamics at physiological temperatures and the allosteric transitions in GroEL. Here we present multiple all‐atom molecular dynamics simulations of the GroEL‐GroES assemblies at different stages of the functional cycle. Combined with an extensive analysis of the complete set of experimentally available structures, principal component analysis and conformer plots, we provide an explicit evaluation of the accessible conformational space of unliganded GroEL. Our results suggest the presence of pre‐existing conformers at the equatorial domain level, and a shift of the conformational ensemble upon ATP‐binding. At the inter‐ring interface the simulations capture a remarkable offset motion of helix D triggered by ATP‐binding to the folding active ring. The reorientation of helix D, previously only observed upon GroES association, correlates with a change of the internal dynamics in the opposite ring. This work contributes to the understanding of the molecular mechanisms in GroEL and highlights the ability of all‐atom MD simulations to model long‐range structural changes and allosteric events in large systems. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Conformational dynamics of α-1 acid glycoprotein (AGP) in cancer: A comparative study of glycosylated and unglycosylated AGP

α-1 acid glycoprotein (AGP) is one of the most abundant plasma proteins. It fulfills two important functions: immunomodulation, and binding to various drugs and receptors. These different functions are closely associated and modulated via changes in glycosylation and cancer missense mutations. From a structural point of view, glycans alter the local biophysical properties of the protein leading to a diverse ligand-binding spectrum. However, glycans can typically not be observed in the resolved X-ray crystallography structure of AGP due to their high flexibility and microheterogeneity, so limiting our understanding of AGP's conformational dynamics 70 years after its discovery. We here investigate how mutations and glycosylation interfere with AGP's conformational dynamics changing its biophysical behavior, by using molecular dynamics (MD) simulations and sequence-based dynamics predictions. The MD trajectories show that glycosylation decreases the local backbone flexibility of AGP and increases the flexibility of distant regions through allosteric effects. We observe that mutations near the glycosylation site affect glycan's conformational preferences. Thus, we conclude that mutations control glycan dynamics which modulates the protein's backbone flexibility directly affecting its accessibility. These findings may assist in the drug design targeting AGP's glycosylation and mutations in cancer.     

Ligand Docking to Intermediate and Close-To-Bound Conformers Generated by an Elastic Network Model Based Algorithm for Highly Flexible Proteins

Incorporating receptor flexibility in small ligand-protein docking still poses a challenge for proteins undergoing large conformational changes. In the absence of bound structures, sampling conformers that are accessible by apo state may facilitate docking and drug design studies. For this aim, we developed an unbiased conformational search algorithm, by integrating global modes from elastic network model, clustering and energy minimization with implicit solvation. Our dataset consists of five diverse proteins with apo to complex RMSDs 4.7–15 Å. Applying this iterative algorithm on apo structures, conformers close to the bound-state (RMSD 1.4–3.8 Å), as well as the intermediate states were generated. Dockings to a sequence of conformers consisting of a closed structure and its “parents” up to the apo were performed to compare binding poses on different states of the receptor. For two periplasmic binding proteins and biotin carboxylase that exhibit hinge-type closure of two dynamics domains, the best pose was obtained for the conformer closest to the bound structure (ligand RMSDs 1.5–2 Å). In contrast, the best pose for adenylate kinase corresponded to an intermediate state with partially closed LID domain and open NMP domain, in line with recent studies (ligand RMSD 2.9 Å). The docking of a helical peptide to calmodulin was the most challenging case due to the complexity of its 15 Å transition, for which a two-stage procedure was necessary. The technique was first applied on the extended calmodulin to generate intermediate conformers; then peptide docking and a second generation stage on the complex were performed, which in turn yielded a final peptide RMSD of 2.9 Å. Our algorithm is effective in producing conformational states based on the apo state. This study underlines the importance of such intermediate states for ligand docking to proteins undergoing large transitions.     

Structural diversity and changes in conformational equilibria of biantennary complex-type N-glycans in water revealed by replica-exchange molecular dynamics simulation

Structural diversity of N-glycans is essential for specific binding to their receptor proteins. To gain insights into structural and dynamic aspects in atomic detail not normally accessible by experiment, we here perform extensive molecular-dynamics simulations of N-glycans in solution using the replica-exchange method. The simulations show that five distinct conformers exist in solution for the N-glycans with and without bisecting GlcNAc. Importantly, the population sizes of three of the conformers are drastically reduced upon the introduction of bisecting GlcNAc. This is caused by a local hydrogen-bond rearrangement proximal to the bisecting GlcNAc. These simulations show that an N-glycan modification like the bisecting GlcNAc selects a certain “key” (or group of “keys”) within the framework of the “bunch of keys” mechanism. Hence, the range of specific glycan-protein interactions and affinity changes need to be understood in terms of the structural diversity of glycans and the alteration of conformational equilibria by core modification.     

Searching the conformational complexity and binding properties of HDAC6 through docking and molecular dynamic simulations

Histone deacetylases (HDACs) are a family of proteins involved in the deacetylation of histones and other non-histones substrates. HDAC6 belongs to class II and shares similar biological functions with others of its class. Nevertheless, its three-dimensional structure that involves the catalytic site remains unknown for exploring the ligand recognition properties. Therefore, in this contribution, homology modeling, 100-ns-long Molecular Dynamics (MD) simulation and docking calculations were combined to explore the conformational complexity and binding properties of the catalytic domain 2 from HDAC6 (DD2-HDAC6), for which activity and affinity toward five different ligands have been reported. Clustering analysis allowed identifying the most populated conformers present during the MD simulation, which were used as starting models to perform docking calculations with five DD2-HDAC6 inhibitors: Cay10603 (CAY), Rocilinostat (RCT), Tubastatin A (TBA), Tubacin (TBC), and Nexturastat (NXT), and then were also submitted to 100-ns-long MD simulations. Docking calculations revealed that the five inhibitors bind at the DD2-HDAC6 binding site with the lowest binding free energy, the same binding mode is maintained along the 100-ns-long MD simulations. Overall, our results provide structural information about the molecular flexibility of apo and holo DD2-HDAC6 states as well as insight of the map of interactions between DD2-HDAC6 and five well-known DD2-HDAC6 inhibitors allowing structural details to guide the drug design. Finally, we highlight the importance of combining different theoretical approaches to provide suitable structural models for structure-based drug design.     

Advanced replica-exchange sampling to study the flexibility and plasticity of peptides and proteins

Molecular dynamics (MD) simulations are ideally suited to investigate protein and peptide plasticity and flexibility simultaneously at high spatial (atomic) and high time resolution. However, the applicability is still limited by the force field accuracy and by the maximum simulation time that can be routinely achieved in current MD simulations. In order to improve the sampling the replica-exchange (REMD) methodology has become popular and is now the most widely applied advanced sampling approach. Many variants of the REMD method have been designed to reduce the computational demand or to enhance sampling along specific sets of conformational variables. An overview on recent methodological advances and discussion of specific aims and advantages of the approaches will be given. Applications in the area of free energy simulations and advanced sampling of intrinsically disordered peptides and proteins will also be discussed. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.     

Multi‐scale characterization of the energy landscape of proteins with application to the C3D/Efb‐C complex

We present a novel multi‐level methodology to explore and characterize the low energy landscape and the thermodynamics of proteins. Traditional conformational search methods typically explore only a small portion of the conformational space of proteins and are hard to apply to large proteins due to the large amount of calculations required. In our multi‐scale approach, we first provide an initial characterization of the equilibrium state ensemble of a protein using an efficient computational conformational sampling method. We then enrich the obtained ensemble by performing short Molecular Dynamics (MD) simulations on selected conformations from the ensembles as starting points. To facilitate the analysis of the results, we project the resulting conformations on a low‐dimensional landscape to efficiently focus on important interactions and examine low energy regions. This methodology provides a more extensive sampling of the low energy landscape than an MD simulation starting from a single crystal structure as it explores multiple trajectories of the protein. This enables us to obtain a broader view of the dynamics of proteins and it can help in understanding complex binding, improving docking results and more. In this work, we apply the methodology to provide an extensive characterization of the bound complexes of the C3d fragment of human Complement component C3 and one of its powerful bacterial inhibitors, the inhibitory domain of Staphylococcus aureus extra‐cellular fibrinogen‐binding domain (Efb‐C) and two of its mutants. We characterize several important interactions along the binding interface and define low free energy regions in the three complexes. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Protein flexibility: multiple molecular dynamics simulations of insulin chain B

Multiple molecular dynamics simulations totaling more than 100 ns were performed on chain B of insulin in explicit solvent at 300 K and 400 K. Despite some individual variations, a comparison of the protein dynamics of each simulation showed similar trends and most structures were consistent with NMR experimental values, even at the elevated temperature. The importance of packing interactions in determining the conformational transitions of the protein was observed, sometimes resulting in conformations induced by localized hydrophobic interactions. The high temperature simulation generated a more diverse range of structures with similar elements of secondary structure and populated conformations to the simulations at room temperature. A broad sampling of the conformational space of insulin chain B illustrated a wide range of conformational states with many transitions at room temperature in addition to the conformational states observed experimentally. The T-state conformation associated with insulin activity was consistently present and a possible mechanism of behavior was suggested.     

Reproducible polypeptide folding and structure prediction using molecular dynamics simulations

The folding of a polypeptide from an extended state to a well-defined conformation is studied using microsecond classical molecular dynamics (MD) simulations and replica exchange molecular dynamics (REMD) simulations in explicit solvent and in vacuo. It is shown that the solvated peptide folds many times in the REMD simulations but only a few times in the conventional simulations. From the folding events in the classical simulations we estimate an approximate folding time of 1-2 micros. The REMD simulations allow enough sampling to deduce a detailed Gibbs free energy landscape in three dimensions. The global minimum of the energy landscape corresponds to the native state of the peptide as determined previously by nuclear magnetic resonance (NMR) experiments. Starting from an extended state it takes about 50 ns before the native structure appears in the REMD simulations, about an order of magnitude faster than conventional MD. The calculated melting curve is in good qualitative agreement with experiment. In vacuo, the peptide collapses rapidly to a conformation that is substantially different from the native state in solvent.     

Interaction mechanism of insulin with ZnO nanoparticles by replica exchange molecular dynamics simulation

The interaction of ZnO nanoparticles with biological molecules such as proteins is one of the most important and challenging problems in molecular biology. Molecular dynamics (MD) simulations are useful technique for understating the mechanism of various interactions of proteins and nanoparticles. In the present work, the interaction mechanism of insulin with ZnO nanoparticles was studied. Simulation methods including MD and replica exchange molecular dynamics (REMD) and their conditions were surveyed. According to the results obtained by REMD simulation, it was found that insulin interacts with ZnO nanoparticle surface via its polar and charged amino acids. Unfolding insulin at ZnO nanoparticle surface, the terminal parts of its chains play the main role. Due to the linkage between chain of insulin and chain of disulfide bonds, opposite directional movements of N terminal part of chain A (toward nanoparticle surface) and N termini of chain B (toward solution) make insulin unfolding. In unfolding of insulin at this condition, its helix structures convert to random coils at terminal parts chains.     

Markov state modeling of membrane transport proteins

The flux of ions and molecules in and out of the cell is vital for maintaining the basis of various biological processes. The permeation of substrates across the cellular membrane is mediated through the function of specialized integral membrane proteins commonly known as membrane transporters. These proteins undergo a series of structural rearrangements that allow a primary substrate binding site to be accessed from either side of the membrane at a given time. Structural insights provided by experimentally resolved structures of membrane transporters have aided in the biophysical characterization of these important molecular drug targets. However, characterizing the transitions between conformational states remains challenging to achieve both experimentally and computationally. Though molecular dynamics simulations are a powerful approach to provide atomistic resolution of protein dynamics, a recurring challenge is its ability to efficiently obtain relevant timescales of large conformational transitions as exhibited in transporters. One approach to overcome this difficulty is to adaptively guide the simulation to favor exploration of the conformational landscape, otherwise known as adaptive sampling. Furthermore, such sampling is greatly benefited by the statistical analysis of Markov state models. Historically, the use of Markov state models has been effective in quantifying slow dynamics or long timescale behaviors such as protein folding. Here, we review recent implementations of adaptive sampling and Markov state models to not only address current limitations of molecular dynamics simulations, but to also highlight how Markov state modeling can be applied to investigate the structure–function mechanisms of large, complex membrane transporters.     

Amyloid beta-protein monomer folding: free-energy surfaces reveal alloform-specific differences

Alloform-specific differences in structural dynamics between amyloid β-protein (Aβ) 40 and Aβ42 appear to underlie the pathogenesis of Alzheimer's disease. To elucidate these differences, we performed microsecond timescale replica-exchange molecular dynamics simulations to sample the conformational space of the Aβ monomer and constructed its free-energy surface. We find that neither peptide monomer is unstructured, but rather that each may be described as a unique statistical coil in which five relatively independent folding units exist, comprising residues 1-5, 10-13, 17-22, 28-37, and 39-42, which are connected by four turn structures. The free-energy surfaces of both peptides are characterized by two large basins, comprising conformers with either substantial α-helix or β-sheet content. Conformational transitions within and between these basins are rapid. The two additional hydrophobic residues at the Aβ42 C-terminus, Ile41 and Ala42, significantly increase contacts within the C-terminus, and between the C-terminus and the central hydrophobic cluster (Leu17-Ala21). As a result, the β-structure of Aβ42 is more stable than that of Aβ40, and the conformational equilibrium in Aβ42 shifts towards β-structure. These results suggest that drugs stabilizing α-helical Aβ conformers (or destabilizing the β-sheet state) would block formation of neurotoxic oligomers. The atomic-resolution conformer structures determined in our simulations may serve as useful targets for this purpose. The conformers also provide starting points for simulations of Aβ oligomerization—a process postulated to be the key pathogenetic event in Alzheimer's disease.     

Mass-weighted molecular dynamics simulation and conformational analysis of polypeptide.

Atomic motions in protein molecules have been studied by molecular dynamics (MD) simulations; dynamics simulation methods have also been employed in conformational studies of polypeptide molecules. It was found that when atomic masses are weighted, the molecular dynamics method can significantly increase the sampling of dihedral conformation space in such studies, compared to a conventional MD simulation of the same total simulation time length. Herein the theoretical study of molecular conformation sampling by the molecular dynamics-based simulation method in which atomic masses are weighted is reported in detail; moreover, a numerical scheme for analyzing the extensive conformational sampling in the simulation of a tetrapeptide amide molecule is presented. From numerical analyses of the mass-weighted molecular dynamics trajectories of backbone dihedral angles, low-resolution structures covering the entire backbone dihedral conformation space of the molecule were determined, and the distribution of rotationally stable conformations in this space were analyzed quantitatively. The theoretical analyses based on the computer simulation and numerical analytical methods suggest that distinctive regimes in the conformational space of the peptide molecule can be identified.     

A "structural" water molecule in the family of fatty acid binding proteins

A single water molecule (w135), buried within the structure of rat intestinal fatty acid binding protein (I-FABP), is investigated by NMR, molecular dynamics simulations, and analysis of known crystal structures. An ordered water molecule was found in structurally analogous position in 24 crystal structures of nine different members of the family of fatty acid binding proteins. There is a remarkable conservation of the local structure near the w135 binding site among different proteins from this family. NMR cross-relaxation measurements imply that w135 is present in the I-FABP:ANS (1-sulfonato-8-(1')anilinonaphthalene) complex in solution with the residence time of >300 ps. Mean-square positional fluctuations of w135 oxygen observed in MD simulations (0.18 and 0.13 A2) are comparable in magnitude to fluctuations exhibited by the backbone atoms and result from highly constrained binding pocket as revealed by Voronoi volumes (averages of 27.0 +/- 1.8 A3 and 24.7 +/- 2.2 A3 for the two simulations). Escape of w135 from its binding pocket was observed only in one MD simulation. The escape process was initiated by interactions with external water molecules and was accompanied by large deformations in beta-strands D and E. Immediately before the release, w135 assumed three distinct states that differ in hydrogen bonding topology and persisted for about 15 ps each. Computer simulations suggest that escape of w135 from the I-FABP matrix is primarily determined by conformational fluctuations of the protein backbone and interactions with external water molecules.