Backbone Motions Research Articles

Pushing the Backbone in Protein-Protein Docking

Conformational changes of proteins that occur upon binding typically confound computational docking algorithms. In this study, we test computational methods to capture protein backbone conformational change related to binding. To address how well existing algorithms can sample bound-like backbones, wequery seven techniques including Monte Carlo-based sampling, molecular dynamics, and normal mode analysis. All methods tested rarely sample near-bound states from the unbound conformation. Nevertheless, the direction of the predicted motions overlapwith the actual conformational change. We next forced the backbone from the unbound toward the bound conformation to create a family of dockingenergy landscapes. Seventy percent of docking targets succeed when the unbound backbones is pushed to within 0.6Å of the bound. Current methods can capture an average of 22% of unbound-bound transitions through conformer selection methods and another 57% through induced-fit methodologies, delineating a stubborn gap (21%) in backbone motion not covered by any current approach.

Numerical study of propulsion performance in swimming fish using boundary element method

In this paper, hydrodynamic simulation of fish-like swimming for two types of aquatic animals including tuna fish and giant danio is presented. We employ an unsteady three-dimensional inviscid boundary element method including time stepping algorithm to capture the wake sheet and flow features around swimming fish in a straight course. At each time step, an unsteady Bernoulli equation was used to find the pressure distribution and thrust generated by the animal. To couple fluid solver with kinematic equations of flexible body, undulating motions of backbone were defined using a prescribed continuous function. Although the flexible motion mechanism controls the fish swimming but no structural model has been considered for the body, there is no fluid–solid interaction. To validate the model, we compare our results with the numerical work of Zhu et al. [1] and experimental results of Barrett et al. [2] and show that this methodology could be fast and reliable approach for the prediction of flexible propulsors.

Binding Mode and Induced Fit Predictions for Prospective Computational Drug Design.

Computer-aided drug design plays an important role in medicinal chemistry to obtain insights into molecular mechanisms and to prioritize design strategies. Although significant improvement has been made in structure based design, it still remains a key challenge to accurately model and predict induced fit mechanisms. Most of the current available techniques either do not provide sufficient protein conformational sampling or are too computationally demanding to fit an industrial setting. The current study presents a systematic and exhaustive investigation of predicting binding modes for a range of systems using PELE (Protein Energy Landscape Exploration), an efficient and fast protein-ligand sampling algorithm. The systems analyzed (cytochrome P, kinase, protease, and nuclear hormone receptor) exhibit different complexities of ligand induced fit mechanisms and protein dynamics. The results are compared with results from classical molecular dynamics simulations and (induced fit) docking. This study shows that ligand induced side chain rearrangements and smaller to medium backbone movements are captured well in PELE. Large secondary structure rearrangements, however, remain challenging for all employed techniques. Relevant binding modes (ligand heavy atom RMSD < 1.0 Å) can be obtained by the PELE method within a few hours of simulation, positioning PELE as a tool applicable for rapid drug design cycles.

Dynamics of Polydots: Soft Luminescent Polymeric Nanoparticles

The conformation and dynamics of luminescent polymers collapsed into nanoparticles or polydots were studied using fully atomistic molecular dynamics (MD) simulations, providing a first insight into their internal dynamics. Controlling the conformation and dynamics of confined polymers is essential for realization of the full potential of polydots in nanomedicine and biotechnology. Specifically, the shape and internal dynamics of polydots that consist of highly rigid dialkyl p-phenylene ethynylene (PPE) are probed as a function of temperature. At room temperature, the polydots are spherical without any correlations between the aromatic rings on the PPE backbone. With increasing temperature, they expand and become slightly aspherical; however, the polymers remain confined. The coherent dynamic structure factor reveals that the internal motion of the polymer backbone is arrested, and the side chains dominate the internal dynamics of the polydots. These new soft nanoparticles retain their overall shape and dy...

Calculated pKa Variations Expose Dynamic Allosteric Communication Networks.

Allosteric regulation of protein function, the process by which binding of an effector molecule provokes a functional response from a distal site, is critical for metabolic pathways. Yet, the way the allosteric signal is communicated remains elusive, especially in dynamic, entropically driven regulation mechanisms for which no major conformational changes are observed. To identify these dynamic allosteric communication networks, we have developed an approach that monitors the pKa variations of ionizable residues over the course of molecular dynamics simulations performed in the presence and absence of an allosteric regulator. As the pKa of ionizable residues depends on their environment, it represents a simple metric to monitor changes in several complex factors induced by binding an allosteric effector. These factors include Coulombic interactions, hydrogen bonding, and solvation, as well as backbone motions and side chain fluctuations. The predictions that can be made with this method concerning the roles of ionizable residues for allosteric communication can then be easily tested experimentally by changing the working pH of the protein or performing single point mutations. To demonstrate the method's validity, we have applied this approach to the subtle dynamic regulation mechanism observed for Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first enzyme of aromatic biosynthesis. We were able to identify key communication pathways linking the allosteric binding site to the active site of the enzyme and to validate these findings experimentally by reestablishing the catalytic activity of allosterically inhibited enzyme via modulation of the working pH, without compromising the binding affinity of the allosteric regulator.

Allostery through Protein Motion at Different Length and Time Scales

The conformational dynamics of proteins are often critical for mediating their functions. They are particularly important for ubiquitin, which must adapt its structure to interact with a plethora of cellular interaction partners. Through an in-depth relaxation dispersion (RD) analysis, we determined how backbone and side chain motions mutually affect one another through a process we term “population shuffling”. Intriguingly, these motions form an allosteric network that operates using concerted motion throughout the protein. This network is a key part of enabling multispecificity and suggests that ubiquitin regulation may be more complex than previously thought. In addition to concerted motions, simulations and crystal structures have suggested that population shuffling also operates between distant parts of the backbone, and may serve as another mechanism behind allosteric communication. In an effort to further elucidate the details of these motions, we have collected a large dataset of time-dependent Nuclear Overhauser Effect (NOE) measurements on both the backbone and side chains. Through explicit modeling of the effect that fast and slow motions have on the NOE, we gain even more insight into energy landscape of ubiquitin. By computationally combining residual dipolar coupling (RDC), RD, and NOE data our structural ensembles incorporate protein dynamics from timescales covering nine orders of magnitude.

Binding of the Single-Stranded DNA Binding Protein (gp32) of T4 Bacteriophage Induces Position-Specific Local Conformational Changes in DNA Lattices that can be Monitored by Fluorescent Probes

The single-stranded (ss) DNA binding protein (gp32) of the bacteriophage T4 DNA replication system plays a major role in integrating the functions of the sub-assemblies of the replication complex by interacting with different replication proteins. Substitution of fluorescent base analogues at defined positions in nucleic acid constructs can be used to study local conformational changes of the nucleic acid bases at specific sites, and Cy3/Cy5 dye-pairs inserted into the sugar-phosphate backbones can track the backbone motions of the DNA lattice during gp32 binding. By monitoring the fluorescence, circular dichroism, absorbance and FRET signals of these base and backbone probes, equilibrium and dynamic measurements can provide information about the molecular details of replication mechanisms. In the present study we use 6-methyl isoxanthopterin (6-MI), a fluorescent base analogue of guanine, to monitor local conformational changes of DNA lattices on gp32 binding. We find that aromatic amino acid residues of gp32 stacks with the bases of ssDNA lattices, depending on the specific positions of the bases within the binding domain of the gp32 molecule, and that Molecular Dynamics simulations are consistent with the experimental results. These results may shed light on how the hydrophobic pockets found in many protein-nucleic acid interaction complexes may stabilize the intercalation or stacking of aromatic amino acid residues with nucleic acid bases. Using internally labeled cyanine dyes, we have also investigated the double-stranded DNA melting properties of gp32 protein at a replication fork junction, and our results suggest that gp32 can enhance breathing at a fork junction if the ssDNA arm at the fork is of sufficient length to permit formation of cooperatively-bound gp32 clusters at the junction.

Differential backbone dynamics of companion helices in the extended helical coiled-coil domain of a bacterial chemoreceptor.

Cytoplasmic domains of transmembrane bacterial chemoreceptors are largely extended four-helix coiled coils. Previous observations suggested the domain was structurally dynamic. We probed directly backbone dynamics of this domain of the transmembrane chemoreceptor Tar from Escherichia coli using site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. Spin labels were positioned on solvent-exposed helical faces because EPR spectra for such positions reflect primarily polypeptide backbone movements. We acquired spectra for spin-labeled, intact receptor homodimers solubilized in detergent or inserted into native E. coli lipid bilayers in Nanodiscs, characterizing 16 positions distributed throughout the cytoplasmic domain and on both helices of its helical hairpins, one amino terminal to the membrane-distal tight turn (N-helix), and the other carboxyl terminal (C-helix). Detergent solubilization increased backbone dynamics for much of the domain, suggesting that loss of receptor activities upon solubilization reflects wide-spread destabilization. For receptors in either condition, we observed an unanticipated difference between the N- and C-helices. For bilayer-inserted receptors, EPR spectra from sites in the membrane-distal protein-interaction region and throughout the C-helix were typical of well-structured helices. In contrast, for approximately two-thirds of the N-helix, from its origin as the AS-2 helix of the membrane-proximal HAMP domain to the beginning of the membrane-distal protein-interaction region, spectra had a significantly mobile component, estimated by spectral deconvolution to average approximately 15%. Differential helical dynamics suggests a four-helix bundle organization with a pair of core scaffold helices and two more dynamic partner helices. This newly observed feature of chemoreceptor structure could be involved in receptor function.

Dynamics of proton exchange in a model phosphonic acid-functionalized polymer

The dynamics and mechanism of proton exchange in phosphonic acid-functionalized polymers were studied using poly(vinyl-phosphonic acid) (PVPA) as a model system along with quantum chemical calculations and Born–Oppenheimer molecular dynamics (BOMD) simulations at the B3LYP/TZVP level as model calculations. This theoretical study began with searching for the smallest, most active polymer segments and their intermediate conformations which could be involved in the local proton-exchange process. The B3LYP/TZVP results confirmed that a low local dielectric environment and excess proton conditions are required to generate the intermediate conformations, and the shapes of the potential energy curves of the proton exchange between the two phosphonic acid functional groups are sensitive to the local conformational changes. In contrast, a high local dielectric environment increases the energy barriers, thereby preventing the proton from returning to the original functional group. Based on the static results, a mechanism for the proton exchange between the two functional groups involving fluctuations in the local dielectric environment and a local conformational change was proposed. The BOMD results confirmed the proposed mechanism by showing that the activation energies for the proton exchange in the hydrogen bond between two immobilized phosphonic acid moieties, obtained from the exponential relaxation behaviors of the envelopes of the velocity autocorrelation functions and the 1H Nuclear Magnetic Resonance (NMR) line-shape analyses, are too low to be the rate-determining process. Instead, coupled librational motion in the backbone which leads to the interconversion between the two intermediate conformations possesses higher activation energy, and therefore represents one of the most important rate-determining processes. These findings suggested that the rate of the proton exchange in the model phosphonic acid-functionalized polymer is determined by the polymer mobility which, in this case, is the large-amplitude librational motion of the vinyl backbone. © 2015 Wiley Periodicals, Inc.

Rotational Dynamics of Spin-Labeled Polyacid Chain Segments in Polyelectrolyte Complexes Studied by CW EPR Spectroscopy

A nitroxide spin label has been covalently linked to the weak polyacid poly(ethylene-alt-maleic acid) (P(E-alt-MA)) to study the rotational mobility of the polyacid backbone in polyelectrolyte complexes (PEC) formed with the oppositely charged strong polycation poly(diallyldimethylammonium chloride) (PDADMAC) in dependence on the pH of the dispersion and the temperature. The rotational mobility of the polyacid chain segments has been determined by simulation of the line shape of the continuous wave (CW) electron paramagnetic resonance (EPR) spectra using the microscopic order/macroscopic disorder (MOMD) model of restricted rotational diffusion. The study has shown that the diffusion coefficient characterizing the rotational motions of the polyacid backbone is significantly smaller at low degree of dissociation at pH 4 than at high degree of dissociation at pH 7 and pH 10.

Protein dynamics. Direct observation of hierarchical protein dynamics.

One of the fundamental challenges of physical biology is to understand the relationship between protein dynamics and function. At physiological temperatures, functional motions arise from the complex interplay of thermal motions of proteins and their environments. Here, we determine the hierarchy in the protein conformational energy landscape that underlies these motions, based on a series of temperature-dependent magic-angle spinning multinuclear nuclear-magnetic-resonance relaxation measurements in a hydrated nanocrystalline protein. The results support strong coupling between protein and solvent dynamics above 160 kelvin, with fast solvent motions, slow protein side-chain motions, and fast protein backbone motions being activated consecutively. Low activation energy, small-amplitude local motions dominate at low temperatures, with larger-amplitude, anisotropic, and functionally relevant motions involving entire peptide units becoming dominant at temperatures above 220 kelvin.

A hierarchy of protein motions

Protein Dynamics Functioning proteins are not static but explore complex conformational energy landscapes. Lewandowski et al. used multinuclear solid-state nuclear magnetic resonance experiments to measure protein motion over a broad range of temperatures and time scales. Above 160 K there was a strong coupling between solvent and protein motion. The hierarchy of motions as the temperature increased revealed the dynamic modes that relate solvent, sidechain, and backbone motion. Science , this issue p. [578][1] [1]: /lookup/volpage/348/578?iss=6234

A wrench in the works of human acetylcholinesterase: soman induced conformational changes revealed by molecular dynamics simulations.

Irreversible inactivation of human acetylcholinesterase (hAChE) by organophosphorous pesticides (OPs) and chemical weapon agents (CWA) has severe morbidity and mortality consequences. We present data from quantum mechanics/molecular mechanics (QM/MM) and 80 classical molecular dynamics (MD) simulations of the apo and soman-adducted forms of hAChE to investigate the effects on the dynamics and protein structure when the catalytic Serine 203 is phosphonylated. We find that the soman phosphonylation of the active site Ser203 follows a water assisted addition-elimination mechanism with the elimination of the fluoride ion being the highest energy barrier at 6.5 kcal/mole. We observe soman-dependent changes in backbone and sidechain motions compared to the apo form of the protein. These alterations restrict the soman-adducted hAChE to a structural state that is primed for the soman adduct to be cleaved and removed from the active site. The altered motions and resulting structures provide alternative pathways into and out of the hAChE active site. In the soman-adducted protein both side and back door pathways are viable for soman adduct access. Correlation analysis of the apo and soman adducted MD trajectories shows that the correlation of gorge entrance and back door motion is disrupted when hAChE is adducted. This supports the hypothesis that substrate and product can use two different pathways as entry and exit sites in the apo form of the protein. These alternative pathways have important implications for the rational design of medical countermeasures.

On the relationship between NMR-derived amide order parameters and protein backbone entropy changes.

Molecular dynamics simulations are used to analyze the relationship between NMR-derived squared generalized order parameters of amide NH groups and backbone entropy. Amide order parameters (O(2) NH ) are largely determined by the secondary structure and average values appear unrelated to the overall flexibility of the protein. However, analysis of the more flexible subset (O(2) NH < 0.8) shows that these report both on the local flexibility of the protein and on a different component of the conformational entropy than that reported by the side chain methyl axis order parameters, O(2) axis . A calibration curve for backbone entropy vs. O(2) NH is developed, which accounts for both correlations between amide group motions of different residues, and correlations between backbone and side chain motions. This calibration curve can be used with experimental values of O(2) NH changes obtained by NMR relaxation measurements to extract backbone entropy changes, for example, upon ligand binding. In conjunction with our previous calibration for side chain entropy derived from measured O(2) axis values this provides a prescription for determination of the total protein conformational entropy changes from NMR relaxation measurements.

Molecular dynamics explorations of active site structure in designed and evolved enzymes.

This Account describes the use of molecular dynamics (MD) simulations to reveal how mutations alter the structure and organization of enzyme active sites. As proposed by Pauling about 70 years ago and elaborated by many others since then, biocatalysis is efficient when functional groups in the active site of an enzyme are in optimal positions for transition state stabilization. Changes in mechanism and covalent interactions are often critical parts of enzyme catalysis. We describe our explorations of the dynamical preorganization of active sites using MD, studying the fluctuations between active and inactive conformations normally concealed to static crystallography. MD shows how the various arrangements of active site residues influence the free energy of the transition state and relates the populations of the catalytic conformational ensemble to the enzyme activity. This Account is organized around three case studies from our laboratory. We first describe the importance of dynamics in evaluating a series of computationally designed and experimentally evolved enzymes for the Kemp elimination, a popular subject in the enzyme design field. We find that the dynamics of the active site is influenced not only by the original sequence design and subsequent mutations but also by the nature of the ligand present in the active site. In the second example, we show how microsecond MD has been used to uncover the role of remote mutations in the active site dynamics and catalysis of a transesterase, LovD. This enzyme was evolved by Tang at UCLA and Codexis, Inc., and is a useful commercial catalyst for the production of the drug simvastatin. X-ray analysis of inactive and active mutants did not reveal differences in the active sites, but relatively long time scale MD in solution showed that the active site of the wild-type enzyme preorganizes only upon binding of the acyl carrier protein (ACP) that delivers the natural acyl group to the active site. In the absence of bound ACP, a noncatalytic arrangement of the catalytic triad is dominant. Unnatural truncated substrates are inactive because of the lack of protein-protein interactions provided by the ACP. Directed evolution is able to gradually restore the catalytic organization of the active site by motion of the protein backbone that alters the active site geometry. In the third case, we demonstrate the key role of MD in combination with crystallography to identify the origins of substrate-dependent stereoselectivities in a number of Codexis-engineered ketoreductases, one of which is used commercially for the production of the antibiotic sulopenem. Here, mutations alter the shape of the active site as well as the accessibility of water to different regions of it. Each of these examples reveals something different about how mutations can influence enzyme activity and shows that directed evolution, like natural evolution, can increase catalytic activity in a variety of remarkable and often subtle ways.

Proline puckering parameters for collagen structure simulations

Collagen is made of triple helices rich in proline residues, and hence is influenced by the conformational motions of prolines. Because the backbone motions of prolines are restricted by the helical structures, the only side chain motion—proline puckering—becomes an influential factor that may affect the stability of collagen structures. In molecular simulations, a proper proline puckering population is desired so to yield valid results of the collagen properties. Here we design the proline puckering parameters in order to yield suitable proline puckering populations as demonstrated in the experimental results. We test these parameters in collagen and the proline dipeptide simulations. Compared with the results of the PDB and the quantum calculations, we propose the proline puckering parameters for the selected collagen model simulations.

Molecular dynamics of spider dragline silk fiber investigated by 2H MAS NMR.

The molecular dynamics of the proteins that comprise spider dragline silk were investigated with solid-state (2)H magic angle spinning (MAS) NMR line shape and spin-lattice relaxation time (T1) analysis. The experiments were performed on (2)H/(13)C/(15)N-enriched N. clavipes dragline silk fibers. The silk protein side-chain and backbone dynamics were probed for Ala-rich regions (β-sheet and 31-helical domains) in both native (dry) and supercontracted (wet) spider silk. In native (dry) silk fibers, the side chains in all Ala containing regions undergo similar fast methyl rotations (>10(9) s(-1)), while the backbone remains essentially static (<10(2) s(-1)). When the silk is wet and supercontracted, the presence of water initiates fast side-chain and backbone motions for a fraction of the β-sheet region and 31-helicies. β-Sheet subregion 1 ascribed to the poly(Ala) core exhibits slower dynamics, while β-sheet subregion 2 present in the interfacial, primarily poly(Gly-Ala) region that links the β-sheets to disordered 31-helical motifs, exhibits faster motions when the silk is supercontracted. Particularly notable is the observation of microsecond backbone motions for β-sheet subregion 2 and 31-helicies. It is proposed that these microsecond backbone motions lead to hydrogen-bond disruption in β-sheet subregion 2 and helps to explain the decrease in silk stiffness when the silk is wet and supercontracted. In addition, water mobilizes and softens 31-helical motifs, contributing to the increased extensibility observed when the silk is in a supercontracted state. The present study provides critical insight into the supercontraction mechanism and corresponding changes in mechanical properties observed for spider dragline silks.

Influence of diamine structure on the low temperature dielectric relaxation of some poly(ether imide)s

ABSTRACTBroadband dielectric relaxation spectra are reported on a range of poly(ether imide) polymers in which the chemical structure of the diamine used to create the polymer is systematically varied with the anhydride structure based on 2,2‐bis‐[4‐(3′,4′‐dicarboxyphenoxy)phenyl]hexafluoroisopropylidine dianhydride. In all the polymers examined, a dipole relaxation was observed below room temperature. The magnitude and activation energy associated with the relaxation process varied with the chemical structure reflecting the effects of steric hindrance on the conformational change associated with the NC and COC linkages. Values of the activation energies varied between 29 and 34 kJ/mol−1, and are consistent with the observed relaxation being associated with constrained local oscillatory motions of small elements of the polymer backbone. The glass transition temperatures of these polymers are in the range 195–243°C and are associated with the large scale motion of the polymer backbone. Changes in the backbone structure influence the extent of inter chain–chain interaction and are reflected in the amplitude of the relaxation process and the high frequency limiting dielectric permittivity ε∞ values which are important when these polymers are used in thin film electronic applications. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 41684.

General order parameter based correlation analysis of protein backbone motions between experimental NMR relaxation measurements and molecular dynamics simulations

Internal backbone dynamic motions are essential for different protein functions and occur on a wide range of time scales, from femtoseconds to seconds. Molecular dynamic (MD) simulations and nuclear magnetic resonance (NMR) spin relaxation measurements are valuable tools to gain access to fast (nanosecond) internal motions. However, there exist few reports on correlation analysis between MD and NMR relaxation data. Here, backbone relaxation measurements of 15N-labeled SH3 (Src homology 3) domain proteins in aqueous buffer were used to generate general order parameters (S2) using a model-free approach. Simultaneously, 80 ns MD simulations of SH3 domain proteins in a defined hydrated box at neutral pH were conducted and the general order parameters (S2) were derived from the MD trajectory. Correlation analysis using the Gromos force field indicated that S2 values from NMR relaxation measurements and MD simulations were significantly different. MD simulations were performed on models with different charge states for three histidine residues, and with different water models, which were SPC (simple point charge) water model and SPC/E (extended simple point charge) water model. S2 parameters from MD simulations with charges for all three histidines and with the SPC/E water model correlated well with S2 calculated from the experimental NMR relaxation measurements, in a site-specific manner.

Caution is required in interpretation of mutations in the voltage sensing domain of voltage gated channels as evidence for gating mechanisms.

The gating mechanism of voltage sensitive ion channels is generally considered to be the motion of the S4 transmembrane segment of the voltage sensing domains (VSD). The primary supporting evidence came from R→C mutations on the S4 transmembrane segment of the VSD, followed by reaction with a methanethiosulfonate (MTS) reagent. The cys side chain is –SH (reactive form –S−); the arginine side chain is much larger, leaving space big enough to accommodate the MTS sulfonate head group. The cavity created by the mutation has space for up to seven more water molecules than were present in wild type, which could be displaced irreversibly by the MTS reagent. Our quantum calculations show there is major reorientation of three aromatic residues that face into the cavity in response to proton displacement within the VSD. Two phenylalanines reorient sufficiently to shield/unshield the cysteine from the intracellular and extracellular ends, depending on the proton positions, and a tyrosine forms a hydrogen bond to the cysteine sulfur with its side chain –OH. These could produce the results of the experiments that have been interpreted as evidence for physical motion of the S4 segment, without physical motion of the S4 backbone. The computations strongly suggest that the interpretation of cysteine substitution reaction experiments be re-examined in the light of these considerations.