An extended dynamical hydration shell around proteins

The focus in protein folding has been very much on the protein backbone and sidechains. However, hydration waters make comparable contributions to the structure and energy of proteins. The coupling between fast hydration dynamics and protein dynamics is considered to play an important role in protein folding. Fundamental questions of protein hydration include, how far out into the solvent does the influence of the biomolecule reach, how is the water affected, and how are the properties of the hydration water influenced by the separation between protein molecules in solution? We show here that Terahertz spectroscopy directly probes such solvation dynamics around proteins, and determines the width of the dynamical hydration layer. We also investigate the dependence of solvation dynamics on protein concentration. We observe an unexpected nonmonotonic trend in the measured terahertz absorbance of the five helix bundle protein λ_6–85* as a function of the protein: water molar ratio. The trend can be explained by overlapping solvation layers around the proteins. Molecular dynamics simulations indicate water dynamics in the solvation layer around one protein to be distinct from bulk water out to ≈10 Å. At higher protein concentrations such that solvation layers overlap, the calculated absorption spectrum varies nonmonotonically, qualitatively consistent with the experimental observations. The experimental data suggest an influence on the correlated water network motion beyond 20 Å, greater than the pure structural correlation length usually observed.     

Microdynamics in stationary complex networks

Many complex systems, including networks, are not static but can display strong fluctuations at various time scales. Characterizing the dynamics in complex networks is thus of the utmost importance in the understanding of these networks and of the dynamical processes taking place on them. In this article, we study the example of the US airport network in the time period 1990–2000. We show that even if the statistical distributions of most indicators are stationary, an intense activity takes place at the local (“microscopic”) level, with many disappearing/appearing connections (links) between airports. We find that connections have a very broad distribution of lifetimes, and we introduce a set of metrics to characterize the links'' dynamics. We observe in particular that the links that disappear have essentially the same properties as the ones that appear, and that links that connect airports with very different traffic are very volatile. Motivated by this empirical study, we propose a model of dynamical networks, inspired from previous studies on firm growth, which reproduces most of the empirical observations both for the stationary statistical distributions and for the dynamical properties.     

Protein conformational dynamics in the mechanism of HIV-1 protease catalysis

We have used chemical protein synthesis and advanced physical methods to probe dynamics-function correlations for the HIV-1 protease, an enzyme that has received considerable attention as a target for the treatment of AIDS. Chemical synthesis was used to prepare a series of unique analogues of the HIV-1 protease in which the flexibility of the "flap" structures (residues 37-61 in each monomer of the homodimeric protein molecule) was systematically varied. These analogue enzymes were further studied by X-ray crystallography, NMR relaxation, and pulse-EPR methods, in conjunction with molecular dynamics simulations. We show that conformational isomerization in the flaps is correlated with structural reorganization of residues in the active site, and that it is preorganization of the active site that is a rate-limiting factor in catalysis.     

Dissection of complex protein dynamics in human thioredoxin

We report our direct study of complex protein dynamics in human thioredoxin by dissecting into elementary processes and determining their relevant time scales. By combining site-directed mutagenesis with femtosecond spectroscopy, we have distinguished four partly time-overlapped dynamical processes at the active site of thioredoxin. Using intrinsic tryptophan as a molecular probe and from mutation studies, we ascertained the negligible contribution to solvation by protein sidechains and observed that the hydration dynamics at the active site occur in 0.47-0.67 and 10.8-13.2 ps. With reduced and oxidized states, we determined the electron-transfer quenching dynamics between excited tryptophan and a nearby disulfide bond in 10-17.5 ps for three mutants. A robust dynamical process in 95-114 ps, present in both redox states and all mutants regardless of neighboring charged, polar, and hydrophobic residues around the probe, is attributed to the charge transfer reaction with its adjacent peptide bond. Site-directed mutations also revealed the electronic quenching dynamics by an aspartate residue at a hydrogen bond distance in 275-615 ps. The local rotational dynamics determined by the measurement of anisotropy changes with time unraveled a relatively rigid local configuration but implies that the protein fluctuates on the time scale of longer than nanoseconds. These results elucidate the temporal evolution of hydrating water motions, electron-transfer reactions, and local protein fluctuations at the active site, and show continuously synergistic dynamics of biological function over wide time scales.     

Coupling of protein and hydration-water dynamics in biological membranes

The dynamical coupling between proteins and their hydration water is important for the understanding of macromolecular function in a cellular context. In the case of membrane proteins, the environment is heterogeneous, composed of lipids and hydration water, and the dynamical coupling might be more complex than in the case of the extensively studied soluble proteins. Here, we examine the dynamical coupling between a biological membrane, the purple membrane (PM), and its hydration water by a combination of elastic incoherent neutron scattering, specific deuteration, and molecular dynamics simulations. Examining completely deuterated PM, hydrated in H(2)O, allowed the direct experimental exploration of water dynamics. The study of natural abundance PM in D(2)O focused on membrane dynamics. The temperature-dependence of atomic mean-square displacements shows inflections at 120 K and 260 K for the membrane and at 200 K and 260 K for the hydration water. Because transition temperatures are different for PM and hydration water, we conclude that ps-ns hydration water dynamics are not directly coupled to membrane motions on the same time scale at temperatures <260 K. Molecular-dynamics simulations of hydrated PM in the temperature range from 100 to 296 K revealed an onset of hydration-water translational diffusion at approximately 200 K, but no transition in the PM at the same temperature. Our results suggest that, in contrast to soluble proteins, the dynamics of the membrane protein is not controlled by that of hydration water at temperatures <260 K. Lipid dynamics may have a stronger impact on membrane protein dynamics than hydration water.     

Functional conformational motions in the turnover cycle of cholesterol oxidase

Reexamining experimental data of single-molecule fluorescence correlation spectroscopy for cholesterol oxidase, we find that the existing Michaelis-Menten models with dynamical disorder cannot explain strong correlations between subsequent turnover cycles revealed in the diagonal feature in the joint statistical distribution of adjacent "on" times of this enzyme. We suggest that functional conformational motions representing ordered sequences of transitions between a set of conformational substates are involved, along with equilibrium conformational fluctuations in the turnover cycle of cholesterol oxidase. A two-channel model of single-enzyme dynamics, including a slow functional conformational motion in one of the channels, is proposed that allows us to reproduce such strong correlations.     

Tunable nonequilibrium dynamics of field quenches in spin ice

We present nonequilibrium physics in spin ice as a unique setting that combines kinematic constraints, emergent topological defects, and magnetic long-range Coulomb interactions. In spin ice, magnetic frustration leads to highly degenerate yet locally constrained ground states. Together, they form a highly unusual magnetic state—a “Coulomb phase”—whose excitations are point-like defects—magnetic monopoles—in the absence of which effectively no dynamics is possible. Hence, when they are sparse at low temperature, dynamics becomes very sluggish. When quenching the system from a monopole-rich to a monopole-poor state, a wealth of dynamical phenomena occur, the exposition of which is the subject of this article. Most notably, we find reaction diffusion behavior, slow dynamics owing to kinematic constraints, as well as a regime corresponding to the deposition of interacting dimers on a honeycomb lattice. We also identify potential avenues for detecting the magnetic monopoles in a regime of slow-moving monopoles. The interest in this model system is further enhanced by its large degree of tunability and the ease of probing it in experiment: With varying magnetic fields at different temperatures, geometric properties—including even the effective dimensionality of the system—can be varied. By monitoring magnetization, spin correlations or zero-field NMR, the dynamical properties of the system can be extracted in considerable detail. This establishes spin ice as a laboratory of choice for the study of tunable, slow dynamics.The nature and origin of unusual—in particular, slow—dynamics in disorder-free systems (1) are among the most fascinating aspects of disciplines as diverse as the physics of structural glasses and polymers (2), chemical reactions, and biological matter (3). Kinetically constrained models, following the original idea by Fredrickson and Andersen (4), represent one paradigm in which unusual dynamics is generated by short-distance ingredients alone without disorder (5). Another is provided by reaction-diffusion systems, in which spatial and temporal fluctuations feed off each other to provide a wide variety of dynamical phenomena (6) especially due to the slow decay of long wavelength fluctuations.Spin ice systems (7) allow combining both aspects, thanks to the nature of their emergent topological excitations, which take the form of magnetic monopoles (8, 9) with long-range Coulomb interactions. The ground-state correlations in these localized spin systems lead to kinematic constraints in the reaction-diffusion behavior of these mobile excitations (10, 11, 12).Understanding the dynamics of spin ice systems, and in particular proposing new ways to probe their out-of-equilibrium properties, is of direct experimental relevance. For instance, modeling the emergent excitations near equilibrium (13, 14) allowed gaining insight on the observed spin freezing at low temperatures (15, 16) and explaining in part the time scales measured in zero-field NMR (17). Despite the fast-paced progress, several open questions remain unanswered, in particular concerning the behavior of spin ice materials far from equilibrium, as evidenced for instance by recent low-temperature magnetic relaxation experiments (18–20).In this article, we study the strongly out-of-equilibrium behavior following a quench from a monopole-rich to a monopole-poor regime by means of varying an applied magnetic field. We uncover a wide range of dynamical regimes and we provide a theoretical understanding of their origin. We show that the initial evolution maps onto a deposition of dimers on a honeycomb lattice, in the presence of long-range Coulomb interactions between the “vacancies” (i.e., the uncovered sites). The long-time behavior can instead be understood as a dynamical arrest owing to the appearance of field-induced energy barriers to monopole motion. This regime can be seen as similar to conventional spin ice, but with a monopole hopping time exponentially sensitive to temperature: We have a Coulomb liquid in which time can pass arbitrarily slowly.We discuss how to probe these phenomena in experiment, showing how by monitoring magnetization, spin correlations, or zero-field NMR the dynamical properties of the system can be extracted in detail.Overall, this richness and versatility establish spin ice as a laboratory of choice for the study of slow dynamics arising from an interplay of frustration (local constraints), topological defects (monopoles), and magnetic long-range Coulomb interactions.     

From the Cover: Quasiresonant amplification of planetary waves and recent Northern Hemisphere weather extremes

In recent years, the Northern Hemisphere has suffered several devastating regional summer weather extremes, such as the European heat wave in 2003, the Russian heat wave and the Indus river flood in Pakistan in 2010, and the heat wave in the United States in 2011. Here, we propose a common mechanism for the generation of persistent longitudinal planetary-scale high-amplitude patterns of the atmospheric circulation in the Northern Hemisphere midlatitudes. Those patterns—with zonal wave numbers m = 6, 7, or 8—are characteristic of the above extremes. We show that these patterns might result from trapping within midlatitude waveguides of free synoptic waves with zonal wave numbers k ≈ m. Usually, the quasistationary dynamical response with the above wave numbers m to climatological mean thermal and orographic forcing is weak. Such midlatitude waveguides, however, may favor a strong magnification of that response through quasiresonance.     

Active site dynamics of ribonuclease.

The stochastic boundary molecular dynamics method is used to study the structure, dynamics, and energetics of the solvated active site of bovine pancreatic ribonuclease A. Simulations of the native enzyme and of the enzyme complexed with the dinucleotide substrate CpA and the transition-state analog uridine vanadate are compared. Structural features and dynamical couplings for ribonuclease residues found in the simulation are consistent with experimental data. Water molecules, most of which are not observed in crystallographic studies, are shown to play an important role in the active site. Hydrogen bonding of residues with water molecules in the free enzyme is found to mimic the substrate-enzyme interactions of residues involved in binding. Networks of water stabilize the cluster of positively charged active site residues. Correlated fluctuations between the uridine vanadate complex and the distant lysine residues are mediated through water and may indicate a possible role for these residues in stabilizing the transition state.     

Dynamical magnetostructural properties of Anabaena ferredoxin

A mixed quantum/classical investigation of the dynamical magnetostructural properties, that is, "magnetodynamics," of oxidized Anabaena PCC7119 ferredoxin is carried out at room temperature in two distinct conformational states. This protein hosts a [2Fe-2S] cluster in which two iron centers are antiferromagnetically coupled to an overall low-spin electronic ground state that has a genuine multireference character. To study the magnetodynamics of this prosthetic group, an approximate spin projection method is formulated in the framework of density functional theory that allows for multideterminant ab initio molecular dynamics simulations to be carried out efficiently. By using this scheme, the influence of both thermal fluctuations and conformational motion on the structure of the [2Fe-2S] cluster and on the dynamics of the antiferromagnetic coupling constant, J(t), has been investigated. In addition to demonstrating how sensitively the shape of the [2Fe-2S] core itself is affected by hydrogen bonding, the analyses reveal a complex dynamical coupling of J to both local vibrations and large-amplitude motion. It is shown that this interplay can be understood in terms of specific vibrational modes and distinct hydrogen-bonding patterns between the iron-sulfur cluster and the protein backbone, respectively. This implies going beyond the Goodenough-Kanamori rules for angular magnetostructural correlations of oxidized iron-sulfur prosthetic groups.     

Surface residues dynamically organize water bridges to enhance electron transfer between proteins

Cellular energy production depends on electron transfer (ET) between proteins. In this theoretical study, we investigate the impact of structural and conformational variations on the electronic coupling between the redox proteins methylamine dehydrogenase and amicyanin from Paracoccus denitrificans. We used molecular dynamics simulations to generate configurations over a duration of 40 ns (sampled at 100-fs intervals) in conjunction with an ET pathway analysis to estimate the ET coupling strength of each configuration. In the wild-type complex, we find that the most frequently occurring molecular configurations afford superior electronic coupling due to the consistent presence of a water molecule hydrogen-bonded between the donor and acceptor sites. We attribute the persistence of this water bridge to a “molecular breakwater” composed of several hydrophobic residues surrounding the acceptor site. The breakwater supports the function of nearby solvent-organizing residues by limiting the exchange of water molecules between the sterically constrained ET region and the more turbulent surrounding bulk. When the breakwater is affected by a mutation, bulk solvent molecules disrupt the water bridge, resulting in reduced electronic coupling that is consistent with recent experimental findings. Our analysis suggests that, in addition to enabling the association and docking of the proteins, surface residues stabilize and control interprotein solvent dynamics in a concerted way.     

A complexity measure for selective matching of signals by the brain.

We have previously derived a theoretical measure of neural complexity (CN) in an attempt to characterize functional connectivity in the brain. CN measures the amount and heterogeneity of statistical correlations within a neural system in terms of the mutual information between subsets of its units. CN was initially used to characterize the functional connectivity of a neural system isolated from the environment. In the present paper, we introduce a related statistical measure, matching complexity (CM), which reflects the change in CN that occurs after a neural system receives signals from the environment. CM measures how well the ensemble of intrinsic correlations within a neural system fits the statistical structure of the sensory input. We show that CM is low when the intrinsic connectivity of a simulated cortical area is randomly organized. Conversely, CM is high when the intrinsic connectivity is modified so as to differentially amplify those intrinsic correlations that happen to be enhanced by sensory input. When the input is represented by an individual stimulus, a positive value of CM indicates that the limited mutual information between sensory sheets sampling the stimulus and the rest of the brain triggers a large increase in the mutual information between many functionally specialized subsets within the brain. In this way, a complex brain can deal with context and go "beyond the information given."     

Reassessing a sparse energetic network within a single protein domain

Understanding the molecular principles that govern allosteric communication is an important goal in protein science. One way allostery could be transmitted is via sparse energetic networks of residues, and one such evolutionary conserved network was identified in the PDZ domain family of proteins by multiple sequence alignment [Lockless SW, Ranganathan R (1999) Science 286:295-299]. We have reassessed the energetic coupling of these residues by double mutant cycles together with ligand binding and stability experiments and found that coupling is not a special property of the coevolved network of residues in PDZ domains. The observed coupling for ligand binding is better explained by a distance relationship, where residues close in space are more likely to couple than distal residues. Our study demonstrates that statistical coupling from sequence analysis is not necessarily a reporter of energetic coupling and allostery.     

Thermal-activated protein mobility and its correlation with catalysis in thermophilic alcohol dehydrogenase

Temperature-dependent hydrogen-deuterium (H/D) exchange of the thermophilic alcohol dehydrogenase (htADH) has been studied by using liquid chromatography-coupled mass spectrometry. Analysis of the changes in H/D exchange patterns for the protein-derived peptides suggests that some regions of htADH are in a rigid conformational substate at reduced temperatures with limited cooperative protein motion. The enzyme undergoes two discrete transitions at approximately 30 and 45 degrees C to attain a more dynamic conformational substate. Four of the five peptides exhibiting the transition above 40 degrees C are in direct contact with the cofactor, and the NAD(+)-binding affinity is also altered in this temperature range, implicating a change in the mobility of the cofactor-binding domain >45 degrees C. By contrast, the five peptides exhibiting the transition at 30 degrees C reside in the substrate-binding domain. This transition coincides with a change in the activation energy of k(cat) for hydride transfer, leading to a linear correlation between k(cat) and the weighted average exchange rate constant k(HX(WA)) for the five peptides. These observations indicate a direct coupling between hydride transfer and protein mobility in htADH, and that an increased mobility is at least partially responsible for the reduced E(act) at high temperature. The data provide support for the hypothesis that protein dynamics play a key role in controlling hydrogen tunneling at enzyme active sites.     

Quaternary dynamics and plasticity underlie small heat shock protein chaperone function

Small Heat Shock Proteins (sHSPs) are a diverse family of molecular chaperones that prevent protein aggregation by binding clients destabilized during cellular stress. Here we probe the architecture and dynamics of complexes formed between an oligomeric sHSP and client by employing unique mass spectrometry strategies. We observe over 300 different stoichiometries of interaction, demonstrating that an ensemble of structures underlies the protection these chaperones confer to unfolding clients. This astonishing heterogeneity not only makes the system quite distinct in behavior to ATP-dependent chaperones, but also renders it intractable by conventional structural biology approaches. We find that thermally regulated quaternary dynamics of the sHSP establish and maintain the plasticity of the system. This extends the paradigm that intrinsic dynamics are crucial to protein function to include equilibrium fluctuations in quaternary structure, and suggests they are integral to the sHSPs’ role in the cellular protein homeostasis network.     

Impaired protein conformational landscapes as revealed in anomalous Arrhenius prefactors

A growing body of data supports a role for protein motion in enzyme catalysis. In particular, the ability of enzymes to sample catalytically relevant conformational substates has been invoked to model kinetic and spectroscopic data. However, direct experimental links between rapidly interconverting conformations and the chemical steps of catalysis remain rare. We report here on the kinetic analysis and characterization of the hydride transfer step catalyzed by a series of mutant thermophilic alcohol dehydrogenases (ht-ADH), presenting evidence for Arrhenius prefactor values that become enormously elevated above an expected value of approximately 10¹³ s^-1 when the enzyme operates below its optimal temperature range. Restoration of normal Arrhenius behavior in the ht-ADH reaction occurs at elevated temperatures. A simple model, in which reduced temperature alters the ability of the ht-ADH variants to sample the catalytically relevant region of conformational space, can reproduce the available data. These findings indicate an impaired landscape that has been generated by the combined condition of reduced temperature and mutation at a single, active-site hydrophobic side chain. The broader implication is that optimal enzyme function requires the maintenance of a relatively smooth landscape that minimizes low energy traps.     

Molecular Docking and Virtual Screening of an Influenza Virus Inhibitor That Disrupts Protein–Protein Interactions

Influenza is an acute respiratory infection caused by the influenza virus, but few drugs are available for its treatment. Consequently, researchers have been engaged in efforts to discover new antiviral mechanisms that can lay the foundation for novel anti-influenza drugs. The viral RNA-dependent RNA polymerase (RdRp) is an enzyme that plays an indispensable role in the viral infection process, which is directly linked to the survival of the virus. Methods of inhibiting PB1–PB2 (basic polymerase 1–basic polymerase 2) interactions, which are a key part of RdRp enzyme activity, are integral in the design of novel antiviral drugs, a specific PB1–PB2 interactions inhibitor has not been reported. We have screened Enamine’s database and conducted a parallel screening of multiple docking schemes, followed by simulations of molecular dynamics to determine the structure of a stable ligand—PB1 complex. We also calculated the free energy of binding between the screened compounds and PB1 protein. Ultimately, we screened and identified a potential PB1–PB2 inhibitor using the ADMET prediction model.