Conformational transitions in eosinophil cationic protein: a molecular dynamics study in aqueous environment

Extensive molecular dynamics simulations have been performed on eosinophil cationic protein (ECP). The two structures found in the crystallographic dimer (ECPA and ECPB) have been independently simulated. A small difference in the pattern of the sidechain hydrogen bonds in the starting structure has resulted in interesting differences in the conformations accessed during the simulations. In one simulation (ECPB), a stable equilibrium conformation was obtained and in the other (ECPA), conformational transitions at the level of sidechain interactions were observed. The conformational transitions exhibit the involvement of the solvent (water) molecules with a pore-like construct in the equilibrium conformation and an opening for a large number of water molecules during the transition phase. The details of these transitions are examined in terms of intra-protein hydrogen bonds, protein-water networks and the residence times of water molecules on the polar atoms of the protein. These properties show some significant differences in the region between the N-terminal helix and the loop before the C-terminal strand as a function of different conformations accessed during the simulations. However, the stable hydrogen bonds, the protein-water networks, and the hydration patterns in most part of the protein including the active site are very much similar in both the simulations, indicating the fact that these are intrinsic properties of proteins.     

Energy landscape of a native protein: Jumping-among-minima model

We have investigated energy landscape of human lysozyme in its native state by using principal component analysis and a model, jumping-among-minima (JAM) model. These analyses are applied to 1 nsec molecular dynamics trajectory of the protein in water. An assumption embodied in the JAM model allows us to divide protein motions into intra-substate and inter-substate motions. By examining intra-substate motions, it is shown that energy surfaces of individual conformational substates are nearly harmonic and mutually similar. As a result of principal component analysis and JAM model analysis, protein motions are shown to consist of three types of collective modes, multiply hierarchical modes, singly hierarchical modes, and harmonic modes. Multiply hierarchical modes, the number of which accounts only for 0.5% of all modes, dominate contributions to total mean-square atomic fluctuation. Inter-substate motions are observed only in a small-dimensional subspace spanned by the axes of multiplyhierarchical and singly hierarchical modes. Inter-substate motions have two notable time components: faster component seen within 200 psec and slower component. The former involves transitions among the conformational substates of the low-level hierarchy, whereas the latter involves transitions of the higher level substates observed along the first four multiply hierarchical modes. We also discuss dependence of the subspace, which contains conformational substates, on time duration of simulation. Proteins 33:496–517, 1998. © 1998 Wiley-Liss, Inc.     

New biochemical insights into the dynamics of water molecules at the GMP or IMP binding site of human GMPR enzyme: A molecular dynamics study

Human GMP reductase (hGMPR) enzyme is involved in a cellular metabolic pathway, converting GMP into IMP, and also it is an important target for anti-leukemic agents. Present computational investigations explain dynamical behavior of water molecules during the conformational transition process from GMP to IMP using molecular dynamics simulations. Residues at substrate-binding site of cancerous protein (PDB Id. 2C6Q) are mostly more dynamic in nature than the normal protein (PDB Id. 2BLE). Nineteen conserved water molecules are identified at the GMP/IMP binding site and are classified as (i) conserved stable dynamic and (ii) infrequent dynamic. Water molecules W11, W14, and W16 are classified as conserved stable dynamic due to their immobile character, whereas remaining water molecules (W1, W2, W3, W4, W5, W7, W8, W9, W10, W12, W13, W15, W17, W18, and W19) are infrequent with dynamic nature. Entrance or displacement of these infrequent water molecules at GMP/IMP sites may occur due to forward and backward movement of reference residues involving ligands. Four water molecules of hGMPR-I and nine water molecules of hGMPR-II are observed in repetitive transitions from GMP to IMP pathway, which indicates discrimination between two isoforms of hGMPRs. Water molecules in cancerous protein are more dynamic and unstable compared to normal protein. These water molecules execute rare dynamical events at GMP binding site and could assist in detailed understanding of conformational transitions that influence the hGMPR's biological functionality. The present study should be of interest to the experimental community engaged in leukemia research and drug discovery for CML cancer.     

Protein-water displacement distributions

The statistical properties of fast protein-water motions are analyzed by dynamic neutron scattering experiments. Using isotopic exchange, one probes either protein or water hydrogen displacements. A moment analysis of the scattering function in the time domain yields model-independent information such as time-resolved mean square displacements and the Gauss-deviation. From the moments, one can reconstruct the displacement distribution. Hydration water displays two dynamical components, related to librational motions and anomalous diffusion along the protein surface. Rotational transitions of side chains, in particular of methyl groups, persist in the dehydrated and in the solvent-vitrified protein structure. The interaction with water induces further continuous protein motions on a small scale. Water acts as a plasticizer of displacements, which couple to functional processes such as open-closed transitions and ligand exchange.     

Conformational transitions in protein-protein association: binding of fasciculin-2 to acetylcholinesterase

The neurotoxin fasciculin-2 (FAS2) is a picomolar inhibitor of synaptic acetylcholinesterase (AChE). The dynamics of binding between FAS2 and AChE is influenced by conformational fluctuations both before and after protein encounter. Submicrosecond molecular dynamics trajectories of apo forms of fasciculin, corresponding to different conformational substates, are reported here with reference to the conformational changes of loop I of this three-fingered toxin. This highly flexible loop exhibits an ensemble of conformations within each substate corresponding to its functions. The high energy barrier found between the two major substates leads to transitions that are slow on the timescale of the diffusional encounter of noninteracting FAS2 and AChE. The more stable of the two apo substates may not be the one observed in the complex with AChE. It seems likely that the more stable apo form binds rapidly to AChE and conformational readjustments then occur in the resulting encounter complex.     

A Physical Picture of Protein Dynamics and Conformational Changes

A physical model is reviewed which explains different aspects of protein dynamics consistently. At low temperatures, the molecules are frozen in conformational substates. Their average energy is 3/2RT. Solid-state vibrations occur on a time scale of femtoseconds to nanoseconds. Above a characteristic temperature, often called the dynamical transition temperature, slow modes of motions can be observed occurring on a time scale between about 140 and 1 ns. These motions are overdamped, quasidiffusive, and involve collective motions of segments of the size of an α-helix. Molecules performing these types of motion are in the “flexible state”. This state is reached by thermal activation. It is shown that these motions are essential for conformational relaxation. Based on this picture, a new approach is proposed to understand conformational changes. It connects structural fluctuations and conformational transitions.     

Theoretical probes of conformational fluctuations in S-peptide and RNase A/3′–UMP enzyme product complex

The dynamic properties of the RNase A/3′–UMP enzyme/product complex and the S-peptide of RNase A have been investigated by molecular dynamics simulations using suitable generalization of ideas introduced to probe the energy landscape in structural glasses. We introduce two measures, namely, the kinetic energy fluctuation metric and the force metric, both of which are used to calculate the time needed for sampling the conformation space of the molecules. The calculation of the fluctuation metric requires a single trajectory whereas the force metric is computed using two independent trajectories. The vacuum MD simulations show that for both systems the time required for kinetic energy equipartitioning is surprisingly long even at high temperatures. We show that the force metric is a powerful means of probing the nature and relative importance of conformational substates which determine the dynamics at low temperatures. In particular the time dependence of the non-bonded force metric is used to demonstrate that at low temperatures the system is predominantly localized hi a single cluster of conformational substates. The force metric is used to show that relaxation of long range (in sequence space) interactions must be mediated by a sequence of local dihedral angle transitions. We also argue that the time needed for compact structure formation is intimately related to the time needed for the relaxation of the dihedral angle degrees of freedom. The tame for non-bonded interactions, which drive protein molecules to fold under appropriate conditions, to relax becomes extremely long as the temperature is lowered suggesting that the formation of maximally compact structure hi proteins must be a very slow process. © 1993 Wiley-Liss, Inc.     

Hierarchical Conformational Analysis of Native Lysozyme Based on Sub-Millisecond Molecular Dynamics Simulations

Hierarchical organization of free energy landscape (FEL) for native globular proteins has been widely accepted by the biophysics community. However, FEL of native proteins is usually projected onto one or a few dimensions. Here we generated collectively 0.2 milli-second molecular dynamics simulation trajectories in explicit solvent for hen egg white lysozyme (HEWL), and carried out detailed conformational analysis based on backbone torsional degrees of freedom (DOF). Our results demonstrated that at micro-second and coarser temporal resolutions, FEL of HEWL exhibits hub-like topology with crystal structures occupying the dominant structural ensemble that serves as the hub of conformational transitions. However, at 100ns and finer temporal resolutions, conformational substates of HEWL exhibit network-like topology, crystal structures are associated with kinetic traps that are important but not dominant ensembles. Backbone torsional state transitions on time scales ranging from nanoseconds to beyond microseconds were found to be associated with various types of molecular interactions. Even at nanoseconds temporal resolution, the number of conformational substates that are of statistical significance is quite limited. These observations suggest that detailed analysis of conformational substates at multiple temporal resolutions is both important and feasible. Transition state ensembles among various conformational substates at microsecond temporal resolution were observed to be considerably disordered. Life times of these transition state ensembles are found to be nearly independent of the time scales of the participating torsional DOFs.     

Large-scale networks of hydration water molecules around proteins investigated by cryogenic X-ray crystallography.

The structures at protein-water interface, i.e. the hydration structure of proteins, have been investigated by cryogenic X-ray crystal structure analyses. Hydration structures appeared far clearer at cryogenic temperature than at ambient temperature, presumably because the motions of hydration water molecules were quenched by cooling. Based on the structural models obtained, the hydration structures were systematically analyzed with respect to the amount of water molecules, the interaction modes between water molecules and proteins, the local and the global distribution of them on the surface of proteins. The standard tetrahedral interaction geometry of water in bulk retained at the interface and enabled the three-dimensional chain connection of hydrogen bonds between hydration water molecules and polar protein atoms. Large-scale networks of hydrogen bonds covering the entire surface of proteins were quite flexible to accommodate to the large-scale conformational changes of proteins and seemed to have great influences on the dynamics and function of proteins. The present observation may provide a new concept for discussing the dynamics of proteins in aqueous solution.     

Interplay between local protein interactions and water bridging of a proton antenna carboxylate cluster

Proteins that bind protons at cell membrane interfaces often expose to the bulk clusters of carboxylate and histidine sidechains that capture protons transiently and, in proton transporters, deliver protons to an internal site. The protonation-coupled dynamics of bulk-exposed carboxylate clusters, also known as proton antennas, is poorly described. An essential open question is how water-mediated bridges between sidechains of the cluster respond to protonation change and facilitate transient proton storage. To address this question, here I studied the protonation-coupled dynamics at the proton-binding antenna of PsbO, a small extrinsinc subunit of the photosystem II complex, with atomistic molecular dynamics simulations and systematic graph-based analyses of dynamic protein and protein-water hydrogen-bond networks. The protonation of specific carboxylate groups is found to impact the dynamics of their local protein-water hydrogen-bond clusters. Regardless of the protonation state considered for PsbO, carboxylate pairs that can sample direct hydrogen bonding, or bridge via short hydrogen-bonded water chains, anchor to nearby basic or polar protein sidechains. As a result, carboxylic sidechains of the hypothesized antenna cluster are part of dynamic hydrogen bond networks that may rearrange rapidly when the protonation changes.     

Starting‐structure dependence of nanosecond timescale intersubstate transitions and reproducibility of MD‐derived order parameters

Factors affecting the accuracy of molecular dynamics (MD) simulations are investigated by comparing generalized order parameters for backbone NH vectors of the B3 immunoglobulin‐binding domain of streptococcal protein G (GB3) derived from simulations with values obtained from NMR spin relaxation (Yao L, Grishaev A, Cornilescu G, Bax A, J Am Chem Soc 2010;132:4295‐4309.). Choices for many parameters of the simulations, such as buffer volume, water model, or salt concentration, have only minor influences on the resulting order parameters. In contrast, seemingly minor conformational differences in starting structures, such as orientations of sidechain hydroxyl groups, resulting from applying different protonation algorithms to the same structure, have major effects on backbone dynamics. Some, but not all, of these effects are mitigated by increased sampling in simulations. Most discrepancies between simulated and experimental results occur for residues located at the ends of secondary structures and involve large amplitude nanosecond timescale transitions between distinct conformational substates. These transitions result in autocorrelation functions for bond vector reorientation that do not converge when calculated over individual simulation blocks, typically of length similar to the overall rotational diffusion time. A test for convergence before averaging the order parameters from different blocks results in better agreement between order parameters calculated from different sets of simulations and with NMR‐derived order parameters. Thus, MD‐derived order parameters are more strongly affected by transitions between conformational substates than by fluctuations within individual substates themselves, while conformational differences in the starting structures affect the frequency and scale of such transitions. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Water mobility, denaturation and the glass transition in proteins

A quantitative mechanism is presented that links protein denaturation and the protein-water glass transition through an energy criterion for the onset of mobility of strong protein-water bonds. Differences in the zero point vibrational energy in the ordered and disordered bonded states allow direct prediction of the two transition temperatures. While the onset of water mobility induces the same change in heat capacity for both transitions, the order-disorder transition of denaturation also predicts the observed excess enthalpy gain. The kinetics of the water and protein components through the glass transition are predicted and compared with dielectric spectroscopy observations. The energetic approach provides a consistent mechanism for processes such as refolding and aggregation of proteins involved in protein maintenance and adaptability, as the conformational constraints of strong water-amide bonds are lost with increased molecular mobility. Moreover, we suggest that the ordered state of peptide-water bonds is induced at the point of protein synthesis and could play a key role in the function of proteins through the enhancement of electronic activity by ferroelectric domains in the protein hydration shell, which is lost upon denaturation.     

Transition networks for modeling the kinetics of conformational change in macromolecules

The kinetics and thermodynamics of complex transitions in biomolecules can be modeled in terms of a network of transitions between the relevant conformational substates. Such a transition network, which overcomes the fundamental limitations of reaction-coordinate-based methods, can be constructed either based on the features of the energy landscape, or from molecular dynamics simulations. Energy-landscape-based networks are generated with the aid of automated path-optimization methods, and, using graph-theoretical adaptive methods, can now be constructed for large molecules such as proteins. Dynamics-based networks, also called Markov State Models, can be interpreted and adaptively improved using statistical concepts, such as the mean first passage time, reactive flux and sampling error analysis. This makes transition networks powerful tools for understanding large-scale conformational changes.     

Computer simulations of cyclic enkephalin analogues

Molecular dynamics simulations and energy minimization studies of cyclic enkephalin analogues incorporating retro-inverso modifications have been carried out. The dynamic trajectories are analyzed in terms of the relative mobility of the 14-membered rings, conformational transitions among equilibrium states, and hydrogen-bonding patterns. The cyclization of the molecules reduces the motion of the ring structures substantially. Time-correlated conformational transitions resulting in the reorientation of peptide units are observed. Hydrogen bonds form principally C7 structures. Because of the incorporation of retro-inverso residues, C6 and C8 structures are also formed. Starting conformations for energy minimizations were obtained from the molecular dynamics simulations and from a systematic search of the conformational space available to the molecules. Several minimum energy backbone and side-chain conformations were found for each analogue. The effect of retro-inverso residues on hydrogen-bonding patterns and backbone conformations is discussed.     

Ionic channels with conformational substates.

Recent studies of protein dynamics suggest that ionic channels can assume many conformational substates. Long-lived substates have been directly observed in single-channel current records. In many cases, however, the lifetimes of conformational states will be far below the theoretical limit of time resolution of single-channel experiments. The existence of such hidden substates may strongly influence the observable (time-averaged) properties of a channel, such as the concentration dependence of conductance. A channel exhibiting fast, voltage-dependent transitions between different conductance states may behave as an intrinsic rectifier. In the presence of more than one permeable ion species, coupling between ionic fluxes may occur, even when the channel has only a single ion-binding site. In special situations the rate of ion translocation becomes limited by the rate of conformational transitions, meaning that the channel approaches the kinetic behavior of a carrier. As a result of the strong coulombic interaction between an ion in a binding site and polar groups of the protein, rate constants of conformational transitions may depend on the occupancy of the binding site. Under this condition a nonequilibrium distribution of conformational states is created when ions are driven through the channel by an external force. This may lead to an apparent violation of microscopic reversibility, i.e., to a situation in which the frequency of transitions from state A to state B is no longer equal to the transition frequency from state B to state A.     

Molecular dynamics study of water penetration in staphylococcal nuclease

The ionization properties of Lys and Glu residues buried in the hydrophobic core of staphylococcal nuclease (SN) suggest that the interior of this protein behaves as a highly polarizable medium with an apparent dielectric constant near 10. This has been rationalized previously in terms of localized conformational relaxation concomitant with the ionization of the internal residue, and with contributions by internal water molecules. Paradoxically, the crystal structure of the SN V66E variant shows internal water molecules and the structure of the V66K variant does not. To assess the structural and dynamical character of interior water molecules in SN, a series of 10-ns-long molecular dynamics (MD) simulations was performed with wild-type SN, and with the V66E and V66K variants with Glu66 and Lys66 in the neutral form. Internal water molecules were identified based on their coordination state and characterized in terms of their residence times, average location, dipole moment fluctuations, hydrogen bonding interactions, and interaction energies. The locations of the water molecules that have residence times of several nanoseconds and display small mean-square displacements agree well with the locations of crystallographically observed water molecules. Additional, relatively disordered water molecules that are not observed crystallographically were found in internal hydrophobic locations. All of the interior water molecules that were analyzed in detail displayed a distribution of interaction energies with higher mean value and narrower width than a bulk water molecule. This underscores the importance of protein dynamics for hydration of the protein interior. Further analysis of the MD trajectories revealed that the fluctuations in the protein structure (especially the loop elements) can strongly influence protein hydration by changing the patterns or strengths of hydrogen bonding interactions between water molecules and the protein. To investigate the dynamical response of the protein to burial of charged groups in the protein interior, MD simulations were performed with Glu66 and Lys66 in the charged state. Overall, the MD simulations suggest that a conformational change rather than internal water molecules is the dominant determinant of the high apparent polarizability of the protein interior.     

Conformational dynamics and thermodynamics of protein-ligand binding studied by NMR relaxation

Protein conformational dynamics can be critical for ligand binding in two ways that relate to kinetics and thermodynamics respectively. First, conformational transitions between different substates can control access to the binding site (kinetics). Secondly, differences between free and ligand-bound states in their conformational fluctuations contribute to the entropy of ligand binding (thermodynamics). In the present paper, I focus on the second topic, summarizing our recent results on the role of conformational entropy in ligand binding to Gal3C (the carbohydrate-recognition domain of galectin-3). NMR relaxation experiments provide a unique probe of conformational entropy by characterizing bond-vector fluctuations at atomic resolution. By monitoring differences between the free and ligand-bound states in their backbone and side chain order parameters, we have estimated the contributions from conformational entropy to the free energy of binding. Overall, the conformational entropy of Gal3C increases upon ligand binding, thereby contributing favourably to the binding affinity. Comparisons with the results from isothermal titration calorimetry indicate that the conformational entropy is comparable in magnitude to the enthalpy of binding. Furthermore, there are significant differences in the dynamic response to binding of different ligands, despite the fact that the protein structure is virtually identical in the different protein-ligand complexes. Thus both affinity and specificity of ligand binding to Gal3C appear to depend in part on subtle differences in the conformational fluctuations that reflect the complex interplay between structure, dynamics and ligand interactions.     

Temperature dependence of protein-hydration hydrodynamics by molecular dynamics simulations

The dynamics of water molecules near the protein surface are different from those of bulk water and influence the structure and dynamics of the protein itself. To elucidate the temperature dependence hydration dynamics of water molecules, we present results from the molecular dynamic simulation of the water molecules surrounding two proteins (Carboxypeptidase inhibitor and Ovomucoid) at seven different temperatures (T=273 to 303 K, in increments of 5 K). Translational diffusion coefficients of the surface water and bulk water molecules were estimated from 2 ns molecular dynamics simulation trajectories. Temperature dependence of the estimated bulk water diffusion closely reflects the experimental values, while hydration water diffusion is retarded significantly due to the protein. Protein surface induced scaling of translational dynamics of the hydration waters is uniform over the temperature range studied, suggesting the importance protein-water interactions.     

Instantaneous Normal Modes as an Unforced Reaction Coordinate for Protein Conformational Transitions

We present a novel sampling approach to explore large protein conformational transitions by determining unique substates from instantaneous normal modes calculated from an elastic network model, and applied to a progression of atomistic molecular dynamics snapshots. This unbiased sampling scheme allows us to direct the path sampling between the conformational end states over simulation timescales that are greatly reduced relative to the known experimental timescales. We use adenylate kinase as a test system to show that instantaneous normal modes can be used to identify substates that drive the structural fluctuations of adenylate kinase from its closed to open conformations, in which we observe 16 complete transitions in 4 μs of simulation time, reducing the timescale over conventional simulation timescales by two orders of magnitude. Analysis shows that the unbiased determination of substates is consistent with known pathways determined experimentally.     

PABMB Lecture. Protein dynamics, folding and misfolding: from basic physical chemistry to human conformational diseases.

Proteins exhibit a variety of motions ranging from amino acid side-chain rotations to the motions of large domains. Recognition of their conformational flexibility has led to the view that protein molecules undergo fast dynamic interconversion between different conformational substates. This proposal has received support from a wide variety of experimental techniques and from computer simulations of protein dynamics. More recently, studies of the subunit dissociation of oligomeric proteins induced by hydrostatic pressure have shown that the characteristic times for subunit exchange between oligomers and for interconversion between different conformations may be rather slow (hours or days). In such cases, proteins cannot be treated as an ensemble of rapidly interconverting conformational substates, but rather as a persistently heterogeneous population of different long-lived conformers. This is reminiscent of the deterministic behavior exhibited by macroscopic bodies, and may have important implications for our understanding of protein folding and biological functions. Here, we propose that the deterministic behavior of proteins may be closely related to the genesis of conformational diseases, a class of pathological conditions that includes transmissible spongiform encephalopathies, Alzheimer's disease and other amyloidosis.