Functional Dynamics of Hexameric Helicase Probed by Hydrogen Exchange and Simulation

The biological function of large macromolecular assemblies depends on their structure and their dynamics over a broad range of timescales; for this reason, it is a significant challenge to investigate these assemblies using conventional experimental techniques. One of the most promising experimental techniques is hydrogen-deuterium exchange detected by mass spectrometry. Here, we describe to our knowledge a new computational method for quantitative interpretation of deuterium exchange kinetics and apply it to a hexameric viral helicase P4 that unwinds and translocates RNA into a virus capsid at the expense of ATP hydrolysis. Room-temperature dynamics probed by a hundred nanoseconds of all-atom molecular dynamics simulations is sufficient to predict the exchange kinetics of most sequence fragments and provide a residue-level interpretation of the low-resolution experimental results. The strategy presented here is also a valuable tool to validate experimental data, e.g., assignments, and to probe mechanisms that cannot be observed by x-ray crystallography, or that occur over timescales longer than those that can be realistically simulated, such as the opening of the hexameric ring.     

Beta‐barrel models of soluble amyloid beta oligomers and annular protofibrils

Both soluble and membrane‐bound prefibrillar assemblies of Abeta (Aβ) peptides have been associated with Alzheimer's disease (AD). The size and nature of these assemblies vary greatly and are affected by many factors. Here, we present models of soluble hexameric assemblies of Aβ42 and suggest how they can lead to larger assemblies and eventually to fibrils. The common element in most of these assemblies is a six‐stranded β‐barrel formed by the last third of Aβ42, which is composed of hydrophobic residues and glycines. The hydrophobic core β‐barrels of the hexameric models are shielded from water by the N‐terminus and central segments. These more hydrophilic segments were modeled to have either predominantly β or predominantly α secondary structure. Molecular dynamics simulations were performed to analyze stabilities of the models. The hexameric models were used as starting points from which larger soluble assemblies of 12 and 36 subunits were modeled. These models were developed to be consistent with numerous experimental results. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Functional visualization of viral molecular motor by hydrogen-deuterium exchange reveals transient states

Molecular motors undergo cyclical conformational changes and convert chemical energy into mechanical work. The conformational dynamics of a viral packaging motor, the hexameric helicase P4 of dsRNA bacteriophage phi8, was visualized by hydrogen-deuterium exchange and high-resolution mass spectrometry. Concerted changes of exchange kinetics revealed a cooperative unit that dynamically links ATP-binding sites and the central RNA-binding channel. The cooperative unit is compatible with a structure-based model in which translocation is mediated by a swiveling helix. Deuterium labeling also revealed the transition state associated with RNA loading, which proceeds via opening of the hexameric ring. The loading mechanism is similar to that of other hexameric helicases. Hydrogen-deuterium exchange provides an important link between time-resolved spectroscopic observations and high-resolution structural snapshots of molecular machines.     

Ligand escape pathways and (un)binding free energy calculations for the hexameric insulin-phenol complex

Cooperative binding of phenolic species to insulin hexamers is known to stabilize pharmaceutical preparations of the hormone. Phenol dissociation is rapid on hexamer dissolution timescales, and phenol unbinding upon dilution is likely the first step in the conversion of (pharmaceutical) hexameric insulin to the active monomeric form upon injection. However, a clear understanding of the determinants of the rates of phenol unbinding remains obscure, chiefly because residues implicated in phenol exchange as determined by NMR are not all associated with likely unbinding routes suggested by the best-resolved hexamer structures. We apply random acceleration molecular dynamics simulation to identify potential escape routes of phenol from hydrophobic cavities in the hexameric insulin-phenol complex. We find three major pathways, which provide new insights into (un)binding mechanisms for phenol. We identify several residues directly participating in escape events that serve to resolve ambiguities from recent NMR experiments. Reaction coordinates for dissociation of phenol are developed based on these exit pathways. Potentials of mean force along the reaction coordinate for each pathway are resolved using multiple independent steered molecular dynamics simulations with second-order cumulant expansion of Jarzynski's equality. Our results for ΔF agree reasonably well within the range of known experimental and previous simulation magnitudes of this quantity. Based on structural analysis and energetic barriers for each pathway, we suggest a plausible preferred mechanism of phenolic exchange that differs from previous mechanisms. Several weakly-bound metastable states are also observed for the first time in the phenol dissociation reaction.     

How does it really move? Recent progress in the investigation of protein nanosecond dynamics by NMR and simulation

Nuclear magnetic resonance (NMR) spin relaxation experiments currently probe molecular motions on timescales from picoseconds to nanoseconds. The detailed interpretation of these motions in atomic detail benefits from complementarity with the results from molecular dynamics (MD) simulations. In this mini-review, we describe the recent developments in experimental techniques to study the backbone dynamics from ¹⁵N relaxation and side-chain dynamics from ¹³C relaxation, discuss the different analysis approaches from model-free to dynamics detectors, and highlight the many ways that NMR relaxation experiments and MD simulations can be used together to improve the interpretation and gain insights into protein dynamics.     

Probing protein dynamics using temperature jump relaxation spectroscopy

There have been recent advances in initiating and perturbing chemical reactions on very fast timescales, as short as picoseconds, thus making it feasible to study a vast range of chemical kinetics problems that heretofore could not be studied. One such approach is the rapid heating of water solutions using laser excitation. Laser-induced temperature jump relaxation spectroscopy can be used to determine the dynamics of protein motion, an area largely unstudied for want of suitable experimental and theoretical probes, despite the obvious importance of dynamics to protein function. Coupled with suitable spectroscopic probes of structure, relaxation spectroscopy can follow the motion of protein atoms over an enormous time range, from picoseconds to minutes (or longer), and with substantial structural specificity.     

Effect of Membrane Thickness on Conformational Sampling of Phospholamban from Computer Simulations

The conformational sampling of monomeric, membrane-bound phospholamban is described from computer simulations. Phospholamban (PLB) plays a key role as a regulator of sarcoplasmic reticulum calcium ATPase. An implicit membrane model is used in conjunction with replica exchange molecular dynamics simulations to reach μs-ms timescales. The implicit membrane model was also used to study the effect of different membrane thicknesses by scaling the low-dielectric region. The conformational sampling with the membrane model mimicking dipalmitoylphosphatidylcholine bilayers is in good agreement overall with experimental measurements, but consists of a wide variety of different conformations including structures not described previously. The conformational ensemble shifts significantly in the presence of thinner or thicker membranes. This has implications for the structure and dynamics of PLB in physiological membranes and offers what we believe to be a new interpretation of previous experimental measurements of PLB in detergents and microsomal membrane.     

Millisecond Timescale Dynamics of Human Liver Fatty Acid Binding Protein: Testing of Its Relevance to the Ligand Entry Process

For over a decade, scientists have been attempting to know more about the conformational dynamics of fatty acid binding proteins (FABPs), to answer the puzzling question of how ligands could access the internalized binding site(s). Conformational exchange of FABPs on the microsecond to millisecond timescales has been found in many FABPs and offers an important hypothesis for the ligand entry mechanism. Despite the potential significance, the validity of this hypothesis has not been verified yet. In this study, the slow dynamics of human liver fatty acid binding protein (hLFABP) that was shown previously to be highly flexible on millisecond timescales was quantitatively characterized in detail. In addition, the interaction between hLFABP and 1,8-ANS was studied using NMR spectroscopy, and the kinetic rate of ANS association to hLFABP was measured. We believe the current result excludes the possibility that the intrinsic millisecond dynamics of hLFABP represents a critical conformational reorganization process required for ligand entry, but implies that it may represent the exchange between the apo-state and a state resembling the singly-bound conformation. Furthermore, we suggest these results show that the ligand-entry related functional dynamics could occur on the microsecond/submicrosecond timescales, highly encouraging future computational studies on this topic.     

αB-crystallin polydispersity is a consequence of unbiased quaternary dynamics

The inherent heterogeneity of many protein assemblies complicates characterization of their structure and dynamics, as most biophysical techniques require homogeneous preparations of isolated components. For this reason, quantitative studies of the molecular chaperone αB-crystallin, which populates a range of interconverting oligomeric states, have been difficult, and the physicochemical basis for its polydispersity has remained unknown. Here, we perform mass spectrometry experiments to study αB-crystallin and extract detailed information as to its oligomeric distribution and exchange of subunits under a range of conditions. This allows a determination of the thermodynamic and kinetic parameters that govern the polydisperse ensemble and enables the construction of a simple energy profile for oligomerization. We find that the quaternary structure and dynamics of the protein can be explained using a simple model with just two oligomer-independent interactions (i.e., interactions that are energetically identical in all oligomers from 10mers to 40mers) between constituent monomers. As such, the distribution of oligomers is governed purely by the dynamics of individual monomers. This provides a new means for understanding the polydispersity of αB-crystallin and a framework for interrogating other heterogeneous protein assemblies.     

Is there chaos in the brain? I. Concepts of nonlinear dynamics and methods of investigation

In the light of results obtained during the last two decades in a number of laboratories, it appears that some of the tools of nonlinear dynamics, first developed and improved for the physical sciences and engineering, are well-suited for studies of biological phenomena. In particular it has become clear that the different regimes of activities undergone by nerve cells, neural assemblies and behavioural patterns, the linkage between them, and their modifications over time, cannot be fully understood in the context of even integrative physiology, without using these new techniques. This report, which is the first of two related papers, is aimed at introducing the non expert to the fundamental aspects of nonlinear dynamics, the most spectacular aspect of which is chaos theory. After a general history and definition of chaos the principles of analysis of time series in phase space and the general properties of chaotic trajectories will be described as will be the classical measures which allow a process to be classified as chaotic in ideal systems and models. We will then proceed to show how these methods need to be adapted for handling experimental time series; the dangers and pitfalls faced when dealing with non stationary and often noisy data will be stressed, and specific criteria for suspecting determinism in neuronal cells and/or assemblies will be described. We will finally address two fundamental questions, namely i) whether and how can one distinguish, deterministic patterns from stochastic ones, and, ii) what is the advantage of chaos over randomness: we will explain why and how the former can be controlled whereas, notoriously, the latter cannot be tamed. In the second paper of the series, results obtained at the level of single cells and their membrane conductances in real neuronal networks and in the study of higher brain functions, will be critically reviewed. It will be shown that the tools of nonlinear dynamics can be irreplaceable for revealing hidden mechanisms subserving, for example, neuronal synchronization and periodic oscillations. The benefits for the brain of adopting chaotic regimes with their wide range of potential behaviours and their aptitude to quickly react to changing conditions will also be considered.     

Analysis of red-fluorescent proteins provides insight into dark-state conversion and photodegradation

Fluorescent proteins (FPs) are powerful tools that permit real-time visualization of cellular processes. The utility of a given FP for a specific experiment depends strongly on its effective brightness and overall photostability. However, the brightness of FPs is limited by dark-state conversion (DSC) and irreversible photobleaching, which occur on different timescales. Here, we present in vivo ensemble assays for measuring DSC and irreversible photobleaching under continuous and pulsed illumination. An analysis of closely related red FPs reveals that DSC and irreversible photobleaching are not always connected by the same mechanistic pathway. DSC occurs out of the first-excited singlet state, and its magnitude depends predominantly on the kinetics for recovery out of the dark state. The experimental results can be replicated through kinetic simulations of a four-state model of the electronic states. The methodology presented here allows light-driven dynamics to be studied at the ensemble level over six orders of magnitude in time (microsecond to second timescales).     

Kinetics of unfolding and folding from amide hydrogen exchange in native ubiquitin

Amide hydrogen (NH) exchange is one of the few experimental techniques with the potential for determining the thermodynamics and kinetics of conformational motions at nearly every residue in native proteins. Quantitative interpretation of NH exchange in terms of molecular motions relies on a simple two-state kinetic model: at any given slowly exchanging NH, a closed or exchange-incompetent conformation is in equilibrium with an open or exchange-competent conformation. Previous studies have demonstrated the accuracy of this model in measuring conformational equilibria by comparing exchange data with the thermodynamics of protein unfolding. We report here a test of the accuracy of the model in determining the kinetics of conformational changes in native proteins. The kinetics of folding and unfolding for ubiquitin have been measured by conventional methods and compared with those derived from a comprehensive analysis of the pH dependence of exchange in native ubiquitin. Rate constants for folding and unfolding from these two very different types of experiments show good agreement. The simple model for NH exchange thus appears to be a robust framework for obtaining quantitative information about molecular motions in native proteins.     

Subunit exchange of polydisperse proteins: mass spectrometry reveals consequences of alphaA-crystallin truncation

The small heat shock protein, alpha-crystallin, plays a key role in maintaining lens transparency by chaperoning structurally compromised proteins. This is of particular importance in the human lens, where proteins are exposed to post-translational modifications over the life-time of an individual. Here, we examine the structural and functional consequences of one particular modification of alphaA-crystallin involving the truncation of 5 C-terminal residues (alphaA(1-168)). Using novel mass spectrometry approaches and established biophysical techniques, we show that alphaA(1-168) forms oligomeric assemblies with a lower average molecular mass than wild-type alphaA-crystallin (alphaA(WT)). Also apparent from the mass spectra of both alphaA(WT) and alphaA(1-168) assemblies is the predominance of oligomers containing even numbers of subunits; interestingly, this preference is more marked for alphaA(1-168). To examine the rate of exchange of subunits between assemblies, we mixed alphaB crystallin with either alphaA(WT) or alphaA(1-168) and monitored in a real-time mass spectrometry experiment the formation of heteroligomers. The results show that there is a significant decrease in the rate of exchange when alphaA(1-168) is involved. These reduced exchange kinetics, however, have no effect upon chaperone efficiency, which is found to be closely similar for both alphaA(WT) and alphaA(1-168). Overall, therefore, our results allow us to conclude that, in contrast to mechanisms established for analogous proteins from plants, yeast, and bacteria, the rate of subunit exchange is not the critical parameter in determining efficient chaperone behavior for mammalian alphaA-crystallin.     

EX1 hydrogen exchange and protein folding

Slow amide hydrogen exchange is an increasingly popular tool for investigating structure and function in proteins. The kinetic model for slow hydrogen exchange has two limits, called EX2 and EX1, wherein the thermodynamics and kinetics of protein motions, respectively, are reported by the exchange data. While many laboratories have demonstrated that EX2 exchange can indeed provide accurate results regarding the thermodynamics of protein stability, the potential of EX1 exchange to follow the kinetics of protein unfolding and folding is only beginning to be realized. EX1 hydrogen exchange has advantages over more traditional folding experiments: it provides single-residue resolution, as well as whole-molecule information, the latter of which can be interpreted in terms of the cooperativity of unfolding. However, key questions remain regarding the interpretation of EX1 hydrogen exchange.     

Rheological behavior of living cells is timescale-dependent

The dynamic mechanical behavior of living cells has been proposed to result from timescale-invariant processes governed by the soft glass rheology theory derived from soft matter physics. But this theory is based on experimental measurements over timescales that are shorter than those most relevant for cell growth and function. Here we report results measured over a wider range of timescales which demonstrate that rheological behaviors of living cells are not timescale-invariant. These findings demonstrate that although soft glass rheology appears to accurately predict certain cell mechanical behaviors, it is not a unified model of cell rheology under biologically relevant conditions and thus, alternative mechanisms need to be considered.     

Mass spectrometric approaches using electrospray ionization charge states and hydrogen-deuterium exchange for determining protein structures and their conformational changes

Electrospray ionization (ESI) mass spectrometry (MS) is a powerful analytical tool for elucidating structural details of proteins in solution especially when coupled with amide hydrogen/deuterium (H/D) exchange analysis. ESI charge-state distributions and the envelopes of charges they form from proteins can provide an abundance of information on solution conformations that is not readily available through other biophysical techniques such as near ultraviolet circular dichroism (CD) and tryptophan fluorescence. The most compelling reason for the use of ESI-MS over nuclear magnetic resonance (NMR) for measuring H/D after exchange is that larger proteins and lesser amounts of samples can be studied. In addition, MS can provide structural details on transient or folding intermediates that may not be accessible by CD, fluorescence, and NMR because these techniques measure the average properties of large populations of proteins in solution. Correlations between measured H/D and calculated parameters that are often available from crystallographic data can be used to extend the range of structural details obtained on proteins. Molecular dynamics and energy minimization by simulation techniques such as assisted model building with energy refinement (AMBER) force field can be very useful in providing structural models of proteins that rationalize the experimental H/D exchange results. Charge-state envelopes and H/D exchange information from ESI-MS data used complementarily with NMR and CD data provides the most powerful approach available to understanding the structures and dynamics of proteins in solution.     

Constrained analysis of fluorescence anisotropy decay:application to experimental protein dynamics

Hydrodynamic properties as well as structural dynamics of proteins can be investigated by the well-established experimental method of fluorescence anisotropy decay. Successful use of this method depends on determination of the correct kinetic model, the extent of cross-correlation between parameters in the fitting function, and differences between the timescales of the depolarizing motions and the fluorophore's fluorescence lifetime. We have tested the utility of an independently measured steady-state anisotropy value as a constraint during data analysis to reduce parameter cross correlation and to increase the timescales over which anisotropy decay parameters can be recovered accurately for two calcium-binding proteins. Mutant rat F102W parvalbumin was used as a model system because its single tryptophan residue exhibits monoexponential fluorescence intensity and anisotropy decay kinetics. Cod parvalbumin, a protein with a single tryptophan residue that exhibits multiexponential fluorescence decay kinetics, was also examined as a more complex model. Anisotropy decays were measured for both proteins as a function of solution viscosity to vary hydrodynamic parameters. The use of the steady-state anisotropy as a constraint significantly improved the precision and accuracy of recovered parameters for both proteins, particularly for viscosities at which the protein's rotational correlation time was much longer than the fluorescence lifetime. Thus, basic hydrodynamic properties of larger biomolecules can now be determined with more precision and accuracy by fluorescence anisotropy decay.     

Directional persistence of EGF-induced cell migration is associated with stabilization of lamellipodial protrusions

Migrating cells can sustain a relatively constant direction of lamellipodial protrusion and locomotion over timescales ranging from minutes to hours. However, individual waves of lamellipodial extension occur over much shorter characteristic times. Little understanding exists regarding how cells might integrate biophysical processes across these disparate timescales to control the directional persistence of locomotion. We address this issue by examining the effects of epidermal growth factor (EGF) stimulation on long-timescale directional persistence and short-timescale lamellipodial dynamics of EGF receptor-transfected Chinese hamster ovary cells migrating on fibronectin-coated substrata. Addition of EGF increased persistence, with the magnitude of increase correlating with fibronectin coating concentration. Kymographic analysis of EGF-stimulated lamellipodial dynamics revealed that the temporal stability of lamellipodial protrusions similarly increased with fibronectin concentration. A soluble RGD peptide competitor reduced both the persistence of long-timescale cell paths and the stability of short-timescale membrane protrusions, indicating that cell-substratum adhesion concomitantly influences lamellipodial dynamics and directional persistence. These results reveal the importance of adhesion strength in regulating the directional motility of cells and suggest that the short-timescale kinetics of adhesion complex formation may play a key role in modulating directional persistence over much longer timescales.     

Characterizing and controlling the inherent dynamics of cyclophilin-A

With the recent advances in NMR relaxation techniques, protein motions on functionally important timescales can be studied at atomic resolution. Here, we have used NMR-based relaxation experiments at several temperatures and both 600 and 900 MHz to characterize the inherent dynamics of the enzyme cyclophilin-A (CypA). We have discovered multiple chemical exchange processes within the enzyme that form a “dynamic continuum” that spans 20–30 Å comprising active site residues and residues proximal to the active site. By combining mutagenesis with these NMR relaxation techniques, a simple method of counting the dynamically sampled conformations has been developed. Surprisingly, a combination of point mutations has allowed for the specific regulation of many of the exchange processes that occur within CypA, suggesting that the dynamics of an enzyme may be engineered.