Side-chain Motions Research Articles

Vibrational Spectroscopy and 2D Correlation Analysis as a Probe of Structure in Ionomer Membrane Materials

Infrared spectroscopy is being applied to gain new insights into structural properties of fuel cell membrane materials and to aid in determinations of membrane water content. In the area of structure elucidation, two-dimensional (2D) correlation methods are being adapted in the analysis of spectral data sets collected during water uptake into fluorinated ionomer materials. Synchronous 2D correlation plots assist in the resolution of spectral regions complicated by overlapping bands and indicate distinctions in transformations centered within hydrophilic relative to more hydrophobic ionomer domains. Asynchronous 2D correlation plots reveal relationships in the dynamics of side chain and backbone motions that accompany changes in membrane hydration state.

Coupling between side chain interactions and binding pocket flexibility in HLA-B*44:02 molecules investigated by molecular dynamics simulations

MHC class I molecules present antigenic peptides to cytotoxic T-cells at the cell surface. Peptide loading of class I molecules in the endoplasmatic reticulum can involve interaction with the tapasin chaperone protein. The human class I allotype HLA-B*44:02 with an Asp at position 116 at the floor of the F pocket (which binds the peptide C-terminal residues) depends on tapasin for efficient peptide loading. However, HLA-B*44:05 (identical to B*44:02 except for tyrosine 116) can efficiently load peptides in the absence of tapasin. Both allotypes adopt very similar structures in the presence of the same peptide. Molecular dynamics simulations indicate a significantly higher conformational flexibility of the F pocket in the absence of a peptide for B*44:02 compared to B*44:05. Free energy simulations to open the F pocket indicate a molecular side chain switch mechanism that underlies the global opening motion. This side chain switch involves the rearrangement of salt bridges and hydrogen bonding of the basic arginine 97 with three acidic aspartate residues 114, 116 and 156 near the F pocket. A replica exchange simulation to specifically accelerate side chain motions demonstrates that the same side chain rearrangements induce global opening motions of the F pocket. In case of B*44:05 the free energy barrier for F pocket opening was significantly higher compared to B*44:02 and no associated side chain rearrangement was observed. Such coupling of local side chain rearrangements with global conformational changes might be the basis for allosteric changes in other class I allotypes as well as for allosteric changes in other proteins.

Measuring Entropy in Molecular Recognition by Proteins.

Molecular recognition by proteins is fundamental to the molecular basis of biology. Dissection of the thermodynamic landscape governing protein-ligand interactions has proven difficult because determination of various entropic contributions is quite challenging. Nuclear magnetic resonance relaxation measurements, theory, and simulations suggest that conformational entropy can be accessed through a dynamical proxy. Here, we review the relationship between measures of fast side-chain motion and the underlying conformational entropy. The dynamical proxy reveals that the contribution of conformational entropy can range from highly favorable to highly unfavorable and demonstrates the potential of this key thermodynamic variable to modulate protein-ligand interactions. The dynamical so-called entropy meter also refines the role of solvent entropy and directly determines the loss in rotational-translational entropy that occurs upon formation of high-affinity complexes. The ability to quantify the roles of entropy through an entropy meter based on measurable dynamical properties promises to highlight its role in protein function.

Contribution of weak S-H · · · O hydrogen bonds to the side chain motions in D,L-homocysteine on cooling.

Sulfhydryl groups play an important role in the formation of native structures of proteins and their biological functions. In the present work, we report for the first time the crystal structure of D,L-homocysteine and the results of a detailed study of the dynamics of its sulfhydryl group on cooling by precise single-crystal X-ray diffraction combined with polarized Raman spectroscopy of oriented single crystals. Although the crystal structures of both D,L-cysteine and D,L-homocysteine are layered, hydrogen bonds formed by -SH groups differ. In contrast with the crystal structure of D,L-cysteine with weak S-H · · · S hydrogen bonds between layers, D,L-homocysteine resembles the structures of amino acids with hydrophobic aliphatic side chains with no hydrogen bonds between the layers. The side chain of D,L-homocysteine forms a three-centered S-H · · · O hydrogen bond with carboxylate groups of two neighboring zwitterions. On cooling down, despite the shortening of the two S · · · O distances in the bifurcated S-H · · · O hydrogen bond, the wavenumber of the stretching vibrations of -SH groups increases. The same effect was also observed previously for other sulfhydryl containing amino acids, L-cysteine, and N-acetyl-L-cysteine on increasing pressure and is related to the strengthening of a three-centered bifurcated S-H · · · O hydrogen bond.

Hidden regularity and universal classification of fast side chain motions in proteins.

Proteins display characteristic dynamical signatures that appear to be universal across all proteins regardless of topology and size. Here, we systematically characterize the universal features of fast side chain motions in proteins by examining the conformational energy surfaces of individual residues obtained using enhanced sampling molecular dynamics simulation (618 free energy surfaces obtained from 0.94 μs MD simulation). The side chain conformational free energy surfaces obtained using the adaptive biasing force (ABF) method for a set of eight proteins with different molecular weights and secondary structures are used to determine the methyl axial NMR order parameters (O(axis)(2)), populations of side chain rotamer states (ρ), conformational entropies (S(conf)), probability fluxes, and activation energies for side chain inter-rotameric transitions. The free energy barriers separating side chain rotamer states range from 0.3 to 12 kcal/mol in all proteins and follow a trimodal distribution with an intense peak at ~5 kcal/mol and two shoulders at ~3 and ~7.5 kcal/mol, indicating that some barriers are more favored than others by proteins to maintain a balance between their conformational stability and flexibility. The origin and the influences of the trimodal barrier distribution on the distribution of O(axis)(2) and the side chain conformational entropy are discussed. A hierarchical grading of rotamer states based on the conformational free energy barriers, entropy, and probability flux reveals three distinct classes of side chains in proteins. A unique nonlinear correlation is established between O(axis)(2) and the side chain rotamer populations (ρ). The apparent universality in O(axis)(2) versus ρ correlation, trimodal barrier distribution, and distinct characteristics of three classes of side chains observed among all proteins indicates a hidden regularity (or commonality) in the dynamical heterogeneity of fast side chain motions in proteins.

Banding 2of NMR-derived methyl order parameters: Implications for protein dynamics

Our understanding of protein folding, stability, and function has begun to more explicitly incorporate dynamical aspects. Nuclear magnetic resonance has emerged as a powerful experimental method for obtaining comprehensive site-resolved insight into protein motion. It has been observed that methyl-group motion tends to cluster into three "classes" when expressed in terms of the popular Lipari-Szabo model-free squared generalized order parameter. Here the origins of the three classes or bands in the distribution of order parameters are examined. As a first step, a Bayesian based approach, which makes no a priori assumption about the existence or number of bands, is developed to detect the banding of Oaxis2 values derived either from NMR experiments or molecular dynamics simulations. The analysis is applied to seven proteins with extensive molecular dynamics simulations of these proteins in explicit water to examine the relationship between O2 and fine details of the motion of methyl bearing side chains. All of the proteins studied display banding, with some subtle differences. We propose a very simple yet plausible physical mechanism for banding. Finally, our Bayesian method is used to analyze the measured distributions of methyl group motions in the catabolite activating protein and several of its mutants in various liganded states and discuss the functional implications of the observed banding to protein dynamics and function.

Structural Determinants of Water Permeation through the Sodium-Galactose Transporter vSGLT

Sodium-glucose transporters (SGLTs) facilitate the movement of water across the cell membrane, playing a central role in cellular homeostasis. Here, we present a detailed analysis of the mechanism of water permeation through the inward-facing state of vSGLT based on nearly 10 μs of molecular dynamics simulations. These simulations reveal the transient formation of a continuous water channel through the transporter that permits water to permeate the protein. Trajectories in which spontaneous release of galactose is observed, as well as those in which galactose remains in the binding site, show that the permeation rate, although modulated by substrate occupancy, is not tightly coupled to substrate release. Using a, to our knowledge, novel channel-detection algorithm, we identify the key residues that control water flow through the transporter and show that solvent gating is regulated by side-chain motions in a small number of residues on the extracellular face. A sequence alignment reveals the presence of two insertion sites in mammalian SGLTs that flank these outer-gate residues. We hypothesize that the absence of these sites in vSGLT may account for the high water permeability values for vSGLT determined via simulation compared to the lower experimental estimates for mammalian SGLT1.

Side Chain Conformational Averaging in Human Dihydrofolate Reductase

The three-dimensional structures of the dihydrofolate reductase enzymes from Escherichia coli (ecDHFR or ecE) and Homo sapiens (hDHFR or hE) are very similar, despite a rather low level of sequence identity. Whereas the active site loops of ecDHFR undergo major conformational rearrangements during progression through the reaction cycle, hDHFR remains fixed in a closed loop conformation in all of its catalytic intermediates. To elucidate the structural and dynamic differences between the human and E. coli enzymes, we conducted a comprehensive analysis of side chain flexibility and dynamics in complexes of hDHFR that represent intermediates in the major catalytic cycle. Nuclear magnetic resonance relaxation dispersion experiments show that, in marked contrast to the functionally important motions that feature prominently in the catalytic intermediates of ecDHFR, millisecond time scale fluctuations cannot be detected for hDHFR side chains. Ligand flux in hDHFR is thought to be mediated by conformational changes between a hinge-open state when the substrate/product-binding pocket is vacant and a hinge-closed state when this pocket is occupied. Comparison of X-ray structures of hinge-open and hinge-closed states shows that helix αF changes position by sliding between the two states. Analysis of χ1 rotamer populations derived from measurements of 3JCγCO and 3JCγN couplings indicates that many of the side chains that contact helix αF exhibit rotamer averaging that may facilitate the conformational change. The χ1 rotamer adopted by the Phe31 side chain depends upon whether the active site contains the substrate or product. In the holoenzyme (the binary complex of hDHFR with reduced nicotinamide adenine dinucleotide phosphate), a combination of hinge opening and a change in the Phe31 χ1 rotamer opens the active site to facilitate entry of the substrate. Overall, the data suggest that, unlike ecDHFR, hDHFR requires minimal backbone conformational rearrangement as it proceeds through its enzymatic cycle, but that ligand flux is brokered by more subtle conformational changes that depend on the side chain motions of critical residues.

Measuring Order in Regioregular Poly(3-hexylthiophene) with Solid-State 13C CPMAS NMR.

We report on measurements of order in semicrystalline, high molar mass poly(3-hexylthiophene) (P3HT) by solid-state 13C cross-polarization magic angle spinning (CPMAS) nuclear magnetic resonance (NMR) measurements. The relative degree of crystallinity was estimated for two films with different drying conditions via X-ray diffraction (XRD) and differential scanning calorimetry (DSC). Order determined by 13C NMR does not necessarily correlate with crystallinity, indicating that local order can occur in noncrystalline regions. Slow main chain dynamics influence the 13C NMR peak widths at lower temperatures (<0 °C), with side chain motions influencing the main chain motions. At higher temperatures (>0 °C), where narrower thiophene resonances are observed, these main chain conformation rearrangements occur on fast time scales (≪3 ms). This room-temperature dynamic disorder suggests that P3HT may be classified as a conformationally disordered (CONDIS) crystal.

Conformational Flexibility and Structure in High-Pressure Excited States of Apomyoglobin Revealed by SDSL-EPR

Proteins exist in an ensemble of conformations at equilibrium, and while higher energy (“excited”) states may play important functional roles, under normal conditions they are too sparsely populated to detect. The application of high hydrostatic pressure may be used to populate excited states, but characterization of the high-pressure conformational ensemble is complicated by the presence of multiple conformations exchanging on the μs-ms time scale. The method of site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) is ideally suited to explore such excited states. The intrinsic timescale of EPR (ns) is fast compared to that of conformational exchange, so the EPR spectrum captures a snapshot of conformational equilibrium frozen in time and can reveal the existence of multiple conformational substates. For each resolved substate, the spectral lineshape encodes information on the local backbone dynamics and tertiary fold. In the present work, a set of spin-labeled mutants of both holo- and apomyoglobin were studied in the pressure range 0 - 2 kbar. Many EPR spectra in the well-ordered holomyoglobin native state are essentially pressure independent, demonstrating that the internal motion of the spin label side chain is pressure insensitive. Thus, pressure-dependent spectral changes observed in more flexible systems can be directly interpreted in terms of protein compressibility rather than side chain effects. Pressurization of apomyoglobin up to 2 kbar populates a low-lying excited state that has been designated a “high-pressure molten globule,” although the work presented here shows it to be distinct from the conventional low pH molten globule that is an intermediate along the folding pathway. Interestingly, the EPR spectra of apomyoglobin at 2 kbar suggest the existence of a conformation with native-like dynamics within the ensemble of conformations that comprise the high-pressure molten globule.

Uncovering the Links Between Conformational Flexibility and Function for a Bacterial Voltage-Gated Sodium Channel

Voltage-gated sodium channels play essential roles in electrical signaling in the nervous system and are key pharmacological targets for a range of disorders. We have carried out fully-atomistic simulations on the multi-microsecond timescale to investigate sodium channel function. These simulations have produced complete unbiased free energy maps that reveal complex multi-ion conduction mechanisms for sodium (as well as potassium and calcium ions). They have also uncovered a surprising level of conformational flexibility of the channel, including concerted movements of the selectivity filter's EEEE ring sequence side chains, and significant changes in pore domain structure as a function of glutamate protonation states. We have observed asymmetrical rearrangements of the activation gate, resembling previously proposed inactivated structures, as well as helix bending involving residues critical for slow inactivation. We report how these structural changes regulate access to lipid-facing fenestrations and the binding of the local anesthetic and anticonvulsant drugs phenytoin and benzocaine, providing new insight into the molecular mechanisms of sodium channel inhibition.

Molecular Dynamics Studies of Ion Conduction in a Prokaryotic Channel

Recently, the crystal structures of putative bacterial, voltage-gated sodium channels have been published, giving an atomic scale view of the NaChBac family of voltage-gated sodium channels. The selectivity filters of the open pore structure of NavMs (McCusker et al (2012) Nature Comm. 3, 1102) and the closed pore structure of NavAb (Payandeh et al (2011) Nature 475, 353) differ only on the order of 1-2 A in diameter. Previously published studies aimed at predicting the mechanism of ion selectivity through free energy surfaces have not provided a consistent free energy surface. Electrophysiology studies suggest that sodium selectivity in the NaChBac family is slight when compared to the potassium selectivity in potassium-selective ion channels (Shaya et al (2011) PNAS 108, 12313). Multi-microsecond equilibrium simulations of NavAb by Chakrabarti et al. suggest that conformational movements of the glutamate side chains are correlated to sodium movement through the selectivity filter (PNAS (2013) 110, 11331).We present microsecond timescale molecular dynamics simulations of ion conduction through a truncated model of the NavAb pore as a function of applied voltage, in solutions containing sodium, potassium, and both, and as a function of concentration. The model NavAb is constructed from the NavAb pore in which the S5 and S6 helices are truncated, creating an open pore, and embedded in a neon support. The S5 and S6 helices and the support are restrained, but the pore helices and selectivity filter are unrestrained. We show that conformational distribution of the glutamate side chains is voltage dependent. The dependence of selectivity on voltage, concentration, and cations present is demonstrated. Additionally, it is apparent that the rate of and predominant mechanism of conduction is not only dependent upon the voltage and cation but also upon the direction of current.

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

Ligand binding at the extracellular domain of pentameric ligand-gated ion channels initiates a relay of conformational changes that culminates at the gate within the transmembrane domain. The interface between the two domains is a key structural entity that governs gating. Molecular events in signal transduction at the interface are poorly defined because of its intrinsically dynamic nature combined with functional modulation by membrane lipid and water vestibules. Here we used electron paramagnetic resonance spectroscopy to delineate protein motions underlying Gloeobacter violaceus ligand-gated ion channel gating in a membrane environment and report the interface conformation in the closed and the desensitized states. Extensive intrasubunit interactions were observed in the closed state that are weakened upon desensitization and replaced by newer intersubunit contacts. Gating involves major rearrangements of the interfacial loops, accompanied by reorganization of the protein-lipid-water interface. These structural changes may serve as targets for modulation of gating by lipids, alcohols, and amphipathic drug molecules.

Understanding Side-Chain Motions using Statistical Torsion Angle Potential

To understand protein functions, an accurate protein structure is required. Because protein side-chain affects protein-protein interaction and protein-molecule interaction in tertiary protein structure, side-chain mobility is particularly important for understanding protein function. In general, determination of side-chain population is experimentally difficult, so we used computational method: Statistical Torsion Angle Potential (STAP) with Φ-χ1, Ψ-χ1 and χ1-χ2 combinations and simulated annealing protocol are used to predict side-chain mobility. Order parameters (S2) of methyl carbon and their correlation coefficients are evaluated for accuracy check. The computational prediction of χ1, χ2 rotamer population is performed for 17 target proteins. Seven target proteins are used for training set to find optimal values of van der Waals and STAP force constant. Ten target proteins are used for test set. This study can be used to characterize the χ1, χ2 conformation of exposed residues and to understand side-chain motions.

Comparison of Side-Chain Motion of Calbindin D-9K in its Four Calcium Binding States by Molecular Dynamics Simulation

Calbindin D-9k, a small single domain protein found predominantly in tissues involved in the uptake and transport of calcium, consists of a single pair of a helix-loop-helix motif(called EF-hand)that binds calcium with the ligands provided by the loop residues and helical residues immediately adjacent to the loop. It exits in four calcium binding states: a doubly loaded state(a state with a calcium atom in each of its two binding sites), two singly loaded states (a state with calcium in its first binding site only and a state with calcium in its second binding site) and an apo-state (a state with no calcium atom). Experiments have shown that calcium binding occurs in a positive cooperative fashion. This fact is also supported by computational studies on dynamics of backbone of the protein. Studies of the methyl side chain dynamics of the doubly loaded state of the protein by molecular dynamics simulation further enhances the point. To further investigate by computation, the molecular dynamics simulation approach has been used to study the side chain dynamics of all four calcium binding states of the protein. In the study, the different kinds of force fields, especially the AMBER (a molecular dynamics simulation suit)force fields, and different kinds of water models are employed in the GPU environment.

Motions and mechanics: investigating conformational transitions in multi-domain proteins with coarse-grain simulations

Protein flexibility is an essential aspect of protein function, and proteic activity involves a wide range of structural changes, varying from small side-chain movements to large-scale domain motion. In order to understand how these large-scale rearrangements impact on proteins internal dynamics and mechanics, we carried out coarse-grain simulations on a set of proteins presenting conformational changes due to domain–domain motions, and investigated the resulting variations of their mechanical properties. These changes are highly heterogeneous along the protein sequence, and our results show that the residues undergoing important variations of their force constant occupy key positions for protein function, as they are mostly located in the ligand-binding site or on the domain–domain interface.

Water scaffolding in collagen: Implications on protein dynamics as revealed by solid‐state NMR

Solid-state NMR studies of collagen samples of various origins confirm that the amplitude of collagen backbone and sidechain motions increases significantly on increasing the water content. This conclusion is supported by the changes observed in three different NMR observables: (i) the linewidth dependence on the 1H decoupling frequency; (ii) 13C CSA changes for the peptide carbonyl groups, and (iii) dephasing rates of 1H-13C dipolar couplings. In particular, a nearly threefold increase in motional amplitudes of the backbone librations about C-Cα or N-Cα bonds was found on increasing the added water content up to 47 wt%D2 O. On the basis of the frequencies of NMR observables involved, the timescale of the protein motions dependent on the added water content is estimated to be of the order of microseconds. This estimate agrees with that from wideline T2(1)H NMR measurements. Also, our wideline 1H NMR measurements revealed that the timescale of the microsecond motions in proteins reduces significantly on increasing the added water content, i.e., an ∼15-fold increase in protein motional frequencies is observed on increasing the added water content to 45 wt% D2 O. The observed changes in collagen dynamics is attributed to the increase in water translational diffusion on increasing the amount of added water, which leads to more frequent "bound water/free water" exchange on the protein surface, accompanied by the breakage and formation of new hydrogen bonds with polar functionalities of protein.

Specialized Dynamical Properties of Promiscuous Residues Revealed by Simulated Conformational Ensembles

The ability to interact with different partners is one of the most important features in proteins. Proteins that bind a large number of partners (hubs) have been often associated with intrinsic disorder. However, many examples exist of hubs with an ordered structure, and evidence of a general mechanism promoting promiscuity in ordered proteins is still elusive. An intriguing hypothesis is that promiscuous binding sites have specific dynamical properties, distinct from the rest of the interface and pre-existing in the protein isolated state. Here, we present the first comprehensive study of the intrinsic dynamics of promiscuous residues in a large protein data set. Different computational methods, from coarse-grained elastic models to geometry-based sampling methods and to full-atom Molecular Dynamics simulations, were used to generate conformational ensembles for the isolated proteins. The flexibility and dynamic correlations of interface residues with a different degree of binding promiscuity were calculated and compared considering side chain and backbone motions, the latter both on a local and on a global scale. The study revealed that (a) promiscuous residues tend to be more flexible than nonpromiscuous ones, (b) this additional flexibility has a higher degree of organization, and (c) evolutionary conservation and binding promiscuity have opposite effects on intrinsic dynamics. Findings on simulated ensembles were also validated on ensembles of experimental structures extracted from the Protein Data Bank (PDB). Additionally, the low occurrence of single nucleotide polymorphisms observed for promiscuous residues indicated a tendency to preserve binding diversity at these positions. A case study on two ubiquitin-like proteins exemplifies how binding promiscuity in evolutionary related proteins can be modulated by the fine-tuning of the interface dynamics. The interplay between promiscuity and flexibility highlighted here can inspire new directions in protein–protein interaction prediction and design methods.

Microscopic Insights into the NMR Relaxation-Based Protein Conformational Entropy Meter

Conformational entropy is a potentially important thermodynamic parameter contributing to protein function. Quantitative measures of conformational entropy are necessary for an understanding of its role but have been difficult to obtain. An empirical method that utilizes changes in conformational dynamics as a proxy for changes in conformational entropy has recently been introduced. Here we probe the microscopic origins of the link between conformational dynamics and conformational entropy using molecular dynamics simulations. Simulation of seven proteins gave an excellent correlation with measures of side-chain motion derived from NMR relaxation. The simulations show that the motion of methyl-bearing side chains are sufficiently coupled to that of other side chains to serve as excellent reporters of the overall side-chain conformational entropy. These results tend to validate the use of experimentally accessible measures of methyl motion--the NMR-derived generalized order parameters--as a proxy from which to derive changes in protein conformational entropy.

A Simple Method to Measure Protein Side-Chain Mobility Using NMR Chemical Shifts

Protein side-chain motions are involved in many important biological processes including enzymatic catalysis, allosteric regulation, and the mediation of protein-protein, protein-DNA, protein-RNA, and protein-cofactor interactions. NMR spectroscopy has long been used to provide insights into the motions of side-chain groups. Currently, the method of choice for studying side-chain dynamics by NMR is the measurement of methyl (2)H autorelaxation. Methyl (2)H autorelaxation exhibits simple relaxation mechanisms and can be straightforwardly converted to meaningful dynamic parameters. However, methyl groups can only be found in 6 of 19 side-chain bearing amino acids. Consequently, only a sparse assessment of protein side-chain dynamics is possible. Therefore, there is a significant interest in developing novel methods of studying side-chain motions that can be applied to all types of side-chains. Here, we show how side-chain chemical shifts can be used to determine the magnitude of fast side-chain motions in proteins. The chemical shift method is applicable to all side-chain bearing residues and does not require any additional measurements beyond standard NMR experiments for backbone and side-chain assignments.