Controlling the protein dynamical transition with sugar-based bioprotectant matrices: a neutron scattering study

Through elastic neutron scattering we measured the mean-square displacements of the hydrogen atoms of lysozyme embedded in a glucose-water glassy matrix as a function of the temperature and at various water contents. The elastic intensity of all the samples has been interpreted in terms of the double-well model in the whole temperature range. The dry sample shows an onset of anharmonicity at approximately 100 K, which can be attributed to the activation of methyl group reorientations. Such a protein intrinsic dynamics is decoupled from the external environment on the whole investigated temperature range. In the hydrated samples an additional and larger anharmonic contribution is provided by the protein dynamical transition, which appears at a higher temperature Td. As hydration increases the coupling between the protein internal dynamics and the surrounding matrix relaxations becomes more effective. The behavior of Td that, as a function of the water content, diminishes by approximately 60 K, supports the picture of the protein dynamics as driven by solvent relaxations. A possible connection between the protein dynamical response versus T and the thermal stability in glucose-water bioprotectant matrices is proposed.     

Effects of soman inhibition and of structural differences on cholinesterase molecular dynamics: a neutron scattering study

Incoherent elastic neutron scattering experiments on members of the cholinesterase family were carried out to investigate how molecular dynamics is affected by covalent inhibitor binding and by differences in primary and quaternary structure. Tetrameric native and soman-inhibited human butyrylcholinesterase (HuBChE) as well as native dimeric Drosophila melanogaster acetylcholinesterase (DmAChE) hydrated protein powders were examined. Atomic mean-square displacements (MSDs) were found to be identical for native HuBChE and for DmAChE in the whole temperature range examined, leading to the conclusion that differences in activity and substrate specificity are not reflected by a global modification of subnanosecond molecular dynamics. MSDs of native and soman-inhibited HuBChE were identical below the thermal denaturation temperature of the native enzyme, indicating a common mean free-energy surface. Denaturation of the native enzyme is reflected by a relative increase of MSDs consistent with entropic stabilization of the unfolded state. The results suggest that the stabilization of HuBChE phosphorylated by soman is due to an increase in free energy of the unfolded state due to a decrease in entropy.     

Hydration affects both harmonic and anharmonic nature of protein dynamics

To understand the effect of hydration on protein dynamics, inelastic neutron-scattering experiments were performed on staphylococcal nuclease samples at differing hydration levels: dehydrated, partially hydrated, and hydrated. At cryogenic temperatures, hydration affected the collective motions with energies lower than 5 meV, whereas the high-energy localized motions were independent of hydration. The prominent change was a shift of boson peak toward higher energy by hydration, suggesting a hardening of harmonic potential at local minima on the energy landscape. The 240 K transition was observed only for the hydrated protein. Significant quasielastic scattering at 300 K was observed only for the hydrated sample, indicating that the origin of the transition is the motion activated by hydration water. The neutron-scattering profile of the partially hydrated sample was quite similar to that of the hydrated sample at 100 K and 200 K, whereas it was close to the dehydrated sample at 300 K, indicating that partial hydration is sufficient to affect the harmonic nature of protein dynamics, and that there is a threshold hydration level to activate anharmonic motions. Thus, hydration water controls both harmonic and anharmonic protein dynamics by differing means.     

Effect of conformational states on protein dynamical transition

In order to examine the properties specific to the folded protein, the effect of the conformational states on protein dynamical transition was studied by incoherent elastic neutron scattering for both wild type and a deletion mutant of staphylococcal nuclease. The deletion mutant of SNase which lacks C-terminal 13 residues takes a compact denatured structure, and can be regarded as a model of intrinsic unstructured protein. Incoherent elastic neutron scattering experiments were carried out at various temperature between 10 K and 300 K on IN10 and IN13 installed at ILL. Temperature dependence of mean-square displacements was obtained by the q-dependence of elastic scattering intensity. The measurements were performed on dried and hydrated powder samples. No significant differences were observed between wild type and the mutant for the hydrated samples, while significant differences were observed for the dried samples. A dynamical transition at ∼ 140 K observed for both dried and hydrated samples. The slopes of the temperature dependence of MSD before transition and after transition are different between wild type and the mutant, indicating the folding induces hardening. The hydration water activates a further transition at ∼ 240 K. The behavior of the temperature dependence of MSD is indistinguishable for wild type and the mutant, indicating that hydration water dynamics dominate the dynamical properties.     

Protein flexibility from the dynamical transition: a force constant analysis

A standard analysis of the scattered neutron incoherent elastic intensity measured with very good energy resolution yields elastic scans, i.e., mean-square displacements of atomic motions (in a pico to nanosecond time scale) in a sample as a function of temperature. This provides a quick way for characterizing the dynamical behavior of biological macromolecules, such behavior being correlated with biological function and activity. Elastic scans of proteins exhibit a dynamical transition at approximately 200 K, marking a cross-over in molecular fluctuations between harmonic and nonharmonic dynamical regimes. This paper presents an approach allowing analysis of the elastic scan in terms of force constants and related parameters, such as the free energy barrier DeltaG at the transition. We find that the increased protein flexibility beyond the dynamical transition is associated with DeltaG approximately equals RT and effective force constants of the order of 0.1-3 N/m. The analysis provides a set of parameters for characterizing molecular resilience and exploring relations among dynamics, function, and activity in proteins.     

Differences in internal dynamics of actin under different structural states detected by neutron scattering

F-actin, a helical polymer formed by polymerization of the monomers (G-actin), plays crucial roles in various aspects of cell motility. Flexibility of F-actin has been suggested to be important for such a variety of functions. Understanding the flexibility of F-actin requires characterization of a hierarchy of dynamical properties, from internal dynamics of the actin monomers through domain motions within the monomers and relative motions between the monomers within F-actin to large-scale motions of F-actin as a whole. As a first step toward this ultimate purpose, we carried out elastic incoherent neutron scattering experiments on powders of F-actin and G-actin hydrated with D₂O and characterized the internal dynamics of F-actin and G-actin. Well established techniques and analysis enabled the extraction of mean-square displacements and their temperature dependence in F-actin and in G-actin. An effective force constant analysis with a model consisting of three energy states showed that two dynamical transitions occur at ∼150 K and ∼245 K, the former of which corresponds to the onset of anharmonic motions and the latter of which couples with the transition of hydration water. It is shown that behavior of the mean-square displacements is different between G-actin and F-actin, such that G-actin is “softer” than F-actin. The differences in the internal dynamics are detected for the first time between the different structural states (the monomeric state and the polymerized state). The different behavior observed is ascribed to the differences in dynamical heterogeneity between F-actin and G-actin. Based on structural data, the assignment of the differences observed in the two samples to dynamics of specific loop regions involved in the polymerization of G-actin into F-actin is proposed.     

Hydration dependent dynamics in sol–gel encapsulated myoglobin

In this work we study the effect of hydration on the dynamics of a protein in confined geometry, i.e. encapsulated in a porous silica matrix. Using elastic neutron scattering we investigate the temperature dependence of the mean square displacements of non-exchangeable hydrogen atoms of sol-gel encapsulated met-myoglobin. The study is extended to samples at 0.2, 0.3 and 0.5 g water/g protein fractions and comparison is made with met-myoglobin powders at the same average hydration and with a dry powder sample. Elastic data are analysed using a model of dynamical heterogeneity to take into account deviations of elastic intensity from gaussian behaviour in a large momentum transfer range and reveal a specific, model independent, effect of sol-gel confinement on protein dynamics, consisting mainly in a reduction of large-scale motions that are activated at temperatures larger than approximately 230 K. Surprisingly, the effect of confinement depends markedly on hydration and has a maximum at about 35% water/protein fraction corresponding to full first shell hydration. The presence of hydration-dependent MSD also in encapsulated met-Mb strongly supports the idea that the effect of sol-gel confinement on protein dynamics involves a modification of the structural/dynamical properties of the co-encapsulated solvent more than direct protein-matrix interactions.     

Molecular dynamics decomposition of temperature-dependent elastic neutron scattering by a protein solution

Molecular dynamics simulations are performed of bovine pancreatic trypsin inhibitor in a cryosolution over a range of temperatures from 80 to 300 K and the origins identified of elastic dynamic neutron scattering from the solution. The elastic scattering and mean-square displacement calculated from the molecular dynamics trajectories are in reasonable agreement with experiments on a larger protein in the same solvent. The solvent and protein contributions to the scattering from the simulation model are determined. At lower temperatures (< approximately 200 K) or on shorter timescales ( approximately 10 ps) the scattering contributions are proportional to the isotopic nuclear scattering cross-sections of each component. However, for T > 200 K marked deviations from these cross-sections are seen due to differences in the dynamics of the components of the solution. Rapid activation of solvent diffusion leads to the variation with temperature of the total elastic intensity being determined largely by that of the solvent. At higher temperatures (>240 K) and longer times ( approximately 100 ps) the protein makes the only significant contribution to the scattering, the solvent scattering having moved out of the accessible time-space window. Decomposition of the protein mean-square displacement shows that the observed dynamical transition in the solution at 200-220 K involves activation of both internal motions and external whole-molecule rotational and translational diffusion. The proportion that the external dynamics contributes to the protein mean-square displacement increases to approximately 30 and 60% at 300 K on the 10- and 100-ps timescales, respectively.     

Protein dynamics and stability: the distribution of atomic fluctuations in thermophilic and mesophilic dihydrofolate reductase derived using elastic incoherent neutron scattering

The temperature dependence of the dynamics of mesophilic and thermophilic dihydrofolate reductase is examined using elastic incoherent neutron scattering. It is demonstrated that the distribution of atomic displacement amplitudes can be derived from the elastic scattering data by assuming a (Weibull) functional form that resembles distributions seen in molecular dynamics simulations. The thermophilic enzyme has a significantly broader distribution than its mesophilic counterpart. Furthermore, although the rate of increase with temperature of the atomic mean-square displacements extracted from the dynamic structure factor is found to be comparable for both enzymes, the amplitudes are found to be slightly larger for the thermophilic enzyme. Therefore, these results imply that the thermophilic enzyme is the more flexible of the two.     

Effect of the environment on the protein dynamical transition: a neutron scattering study

We performed an elastic neutron scattering investigation of the molecular dynamics of lysozyme solvated in glycerol, at different water contents h (grams of water/grams of lysozyme). The marked non-Gaussian behavior of the elastic intensity was studied in a wide experimental momentum transfer range, as a function of the temperature. The internal dynamics is well described in terms of the double-well jump model. At low temperature, the protein total mean square displacements exhibit an almost linear harmonic trend irrespective of the hydration level, whereas at the temperature T(d) a clear changeover toward an anharmonic regime marks a protein dynamical transition. The decrease of T(d) from approximately 238 K to approximately 195 K as a function of h is reminiscent of that found in the glass transition temperature of aqueous solutions of glycerol, thus suggesting that the protein internal dynamics as a whole is slave to the environment properties. Both T(d) and the total mean square displacements indicate that the protein flexibility strongly rises between 0.1 and 0.2h. This hydration-dependent dynamical activation, which is similar to that of hydrated lysozyme powders, is related to the specific interplay of the protein with the surrounding water and glycerol molecules.     

Incoherent neutron scattering of copper azurin: a comparison with molecular dynamics simulation results

The low-frequency dynamics of copper azurin has been studied at different temperatures for a dry and deuterium hydrated sample by incoherent neutron scattering and the experimental results have been compared with molecular dynamics (MD) simulations carried out in the same temperature range. Experimental Debye-Waller factors are consistent with a dynamical transition at approximately 200 K which appears partially suppressed in the dry sample. Inelastic and quasielastic scattering indicate that hydration water modulates both vibrational and diffusive motions. The low-temperature experimental dynamical structure factor of the hydrated protein shows an excess of inelastic scattering peaking at about 3 meV and whose position is slightly shifted downwards in the dry sample. Such an excess is reminiscent of the “boson peak” observed in glass-like materials. This vibrational peak is quite well reproduced by MD simulations, although at a lower energy. The experimental quasielastic scattering of the two samples at 300 K shows a two-step relaxation behaviour with similar characteristic times, while the corresponding intensities differ only by a scale factor. Also, MD simulations confirm the two-step diffusive trend, but the slow process seems to be characterized by a decay faster than the experimental one. Comparison with incoherent neutron scattering studies carried out on proteins having different structure indicates that globular proteins display common elastic, quasielastic and inelastic features, with an almost similar hydration dependence, irrespective of their secondary and tertiary structure. Received: 12 October 1998 / Revised version: 19 February 1999 / Accepted: 1 March 1999     

Dynamics of Protein and its Hydration Water: Neutron Scattering Studies on Fully Deuterated GFP

We present a detailed analysis of the picosecond-to-nanosecond motions of green fluorescent protein (GFP) and its hydration water using neutron scattering spectroscopy and hydrogen/deuterium contrast. The analysis reveals that hydration water suppresses protein motions at lower temperatures (<∼200 K), and facilitates protein dynamics at high temperatures. Experimental data demonstrate that the hydration water is harmonic at temperatures <∼180–190 K and is not affected by the proteins’ methyl group rotations. The dynamics of the hydration water exhibits changes at ∼180–190 K that we ascribe to the glass transition in the hydrated protein. Our results confirm significant differences in the dynamics of protein and its hydration water at high temperatures: on the picosecond-to-nanosecond timescale, the hydration water exhibits diffusive dynamics, while the protein motions are localized to <∼3 Å. The diffusion of the GFP hydration water is similar to the behavior of hydration water previously observed for other proteins. Comparison with other globular proteins (e.g., lysozyme) reveals that on the timescale of 1 ns and at equivalent hydration level, GFP dynamics (mean-square displacements and quasielastic intensity) are of much smaller amplitude. Moreover, the suppression of the protein dynamics by the hydration water at low temperatures appears to be stronger in GFP than in other globular proteins. We ascribe this observation to the barrellike structure of GFP.     

Liquid-like water confined in stacks of biological membranes at 200 k and its relation to protein dynamics

Confined water is of considerable current interest owing to its biophysical importance and relevance to cryopreservation. It can be studied in its amorphous or supercooled state in the "no-man's land", i.e., in the temperature range between 150 and 235 K, in which bulk water is always crystalline. Amorphous deuterium oxide (D(2)O) was obtained in the intermembrane spaces of a stack of purple membranes from Halobacterium salinarum by flash cooling to 77 K. Neutron diffraction showed that upon heating to 200 K the intermembrane water space decreased sharply with an associated strengthening of ice diffraction, indicating that water beyond the first membrane hydration layer flowed out of the intermembrane space to form crystalline ice. It was concluded that the confined water undergoes a glass transition at or below 200 K to adopt an ultraviscous liquid state from which it crystallizes to form ice as soon as it finds itself in an unconfined, bulk-water environment. Our results provide model-free evidence for translational diffusion of confined water in the no-man's land. Potential effects of the confined-water glass transition on nanosecond membrane dynamics were investigated by incoherent elastic neutron scattering experiments. These revealed no differences between flash-cooled and slow-cooled samples (in the latter, the intermembrane space at temperatures <250 K is occupied only by the first membrane hydration layers), with dynamical transitions at 150 and 260 K, but not at 200 K, suggesting that nanosecond membrane dynamics are not sensitive to the state of the water beyond the first hydration shell at cryotemperatures.     

Direct Correlation between Molecular Dynamics and Enzymatic Stability: A Comparative Neutron Scattering Study of Native Human Butyrylcholinesterase and its “Aged” Soman Conjugate

An incoherent elastic neutron scattering study of the molecular dynamics of native human butyrylcholinesterase and its “aged” soman-inhibited conjugate revealed a significant change in molecular flexibility on an angstrom-nanosecond scale as a function of temperature. The results were related to the stability of each state as established previously by differential scanning calorimetry. A striking relationship was found between the denaturation behavior and the molecular flexibility of the native and inhibited enzymes as a function of temperature. This was reflected in a quantitative correlation between the atomic mean-square displacements on an angstrom-nanosecond scale determined by neutron spectroscopy and the calorimetric specific heat. By the application of a simple two-state model that describes the transition from a folded to a denatured state, the denaturation temperatures of the native and the inhibited enzyme were correctly extracted from the atomic mean-square displacements. Furthermore, the transition entropy and enthalpy extracted from the model fit of the neutron data were, within the experimental accuracy, compatible with the values determined by differential scanning calorimetry.     

Coupling of protein and environment fluctuations

We review the concepts of protein dynamics developed over the last 35years and extend applications of the unified model of protein dynamics to heat flow and spatial fluctuations in hydrated myoglobin (Mb) powders. Differential scanning calorimetry (DSC) and incoherent neutron scattering (INS) data on hydration Mb powders are explained by the temperature-dependence of the hydration-shell β(h) process measured by dielectric relaxation spectroscopy (DRS). The unified model explains the temperature dependence of DSC and INS data as a kinetic effect due to a fixed experimental time window and a broad distribution of hydration-shell β(h) fluctuation rates. We review the slaving of large scale protein motions to the bulk solvent α process, and the metastability of Mb molecules in glass forming bulk solvent at low temperatures. This article is part of a Special Issue entitled: "Protein Dynamics: Experimental and Computational Approaches".     

A model for water motion in crystals of lysozyme based on an incoherent quasielastic neutron-scattering study

This paper reports an incoherent quasielastic neutron scattering study of the single particle, diffusive motions of water molecules surrounding a globular protein, the hen egg-white lysozyme. For the first time such an analysis has been done on protein crystals. It can thus be directly related and compared with a recent structural study of the same sample. The measurement temperature ranged from 100 to 300 K, but focus was on the room temperature analysis. The very good agreement between the structural and dynamical studies suggested a model for the dynamics of water in triclinic crystals of lysozyme in the time range approximately 330 ps and at 300 K. Herein, the dynamics of all water molecules is affected by the presence of the protein, and the water molecules can be divided into two populations. The first mainly corresponds to the first hydration shell, in which water molecules reorient themselves fivefold to 10-fold slower than in bulk solvent, and diffuse by jumps from hydration site to hydration site. The long-range diffusion coefficient is five to sixfold less than for bulk solvent. The second group corresponds to water molecules further away from the surface of the protein, in a second incomplete hydration layer, confined between hydrated macromolecules. Within the time scale probed they undergo a translational diffusion with a self-diffusion coefficient reduced approximately 50-fold compared with bulk solvent. As protein crystals have a highly crowded arrangement close to the packing of macromolecules in cells, our conclusion can be discussed with respect to solvent behavior in intracellular media: as the mobility is highest next to the surface, it suggests that under some crowding conditions, a two-dimensional motion for the transport of metabolites can be dominant.     

Atomic mean-square displacements in proteins by molecular dynamics: a case for analysis of variance

Information on protein internal motions is usually obtained through the analysis of atomic mean-square displacements, which are a measure of variability of the atomic positions distribution functions. We report a statistical approach to analyze molecular dynamics data on these displacements that is based on probability distribution functions. Using a technique inspired by the analysis of variance, we compute unbiased, reliable mean-square displacements of the atoms and analyze them statistically. We applied this procedure to characterize protein thermostability by comparing the results for a thermophilic enzyme and a mesophilic homolog. In agreement with previous experimental observations, our analysis suggests that the proteins surface regions can play a role in the different thermal behavior.     

Photoactivation Reduces Side-Chain Dynamics of a LOV Photoreceptor

We used neutron-scattering experiments to probe the conformational dynamics of the light, oxygen, voltage (LOV) photoreceptor PpSB1-LOV from Pseudomonas putida in both the dark and light states. Global protein diffusion and internal macromolecular dynamics were measured using incoherent neutron time-of-flight and backscattering spectroscopy on the picosecond to nanosecond timescales. Global protein diffusion of PpSB1-LOV is not influenced by photoactivation. Observation-time-dependent global diffusion coefficients were found, which converge on the nanosecond timescale toward diffusion coefficients determined by dynamic light scattering. Mean-square displacements of localized internal motions and effective force constants, <k′>, describing the resilience of the proteins were determined on the respective timescales. Photoactivation significantly modifies the flexibility and the resilience of PpSB1-LOV. On the fast, picosecond timescale, small changes in the mean-square displacement and <k′> are observed, which are enhanced on the slower, nanosecond timescale. Photoactivation results in a slightly larger resilience of the photoreceptor on the fast, picosecond timescale, whereas in the nanosecond range, a significantly less resilient structure of the light-state protein is observed. For a residue-resolved interpretation of the experimental neutron-scattering data, we analyzed molecular dynamics simulations of the PpSB1-LOV X-ray structure. Based on these data, it is tempting to speculate that light-induced changes in the protein result in altered side-chain mobility mostly for residues on the protruding Jα helix and on the LOV-LOV dimer interface. Our results provide strong experimental evidence that side-chain dynamics play a crucial role in photoactivation and signaling of PpSB1-LOV via modulation of conformational entropy.     

Incoherent elastic and quasi-elastic neutron scattering investigation of hemoglobin dynamics

In this work we investigate the dynamic properties of hemoglobin in glycerolD(8)/D(2)O solution using incoherent elastic (ENS) and quasi-elastic (QENS) neutron scattering. Taking advantage of complementary energy resolutions of backscattering spectrometers at ILL (Grenoble), we explore motions in a large space-time window, up to 1 ns and 14 A; moreover, in order to cover the harmonic and anharmonic protein dynamics regimes, the elastic experiments have been performed over the wide temperature interval of 20-300 K. To study the dependence of the measured dynamics upon the protein quaternary structure, both deoxyhemoglobin (in T quaternary conformation) and carbonmonoxyhemoglobin (in R quaternary conformation) have been investigated. From the ENS data the mean square displacements of the non-exchangeable hydrogen atoms of the protein and their temperature dependence are obtained. In agreement with previous results on hydrated powders, a dynamical transition at about 220 K is detected. The results show interesting differences between the two hemoglobin quaternary conformations, the T-state protein appearing more rigid and performing faster motions than the R-state one; however, these differences involve motions occurring in the nanosecond time scale and are not detected when only faster atomic motions in the time scale up to 100 ps are investigated. The QENS results put in evidence a relevant Lorentzian quasi-elastic contribution. Analysis of the dependence of the Elastic Incoherent Structure Factor (EISF) and of the Lorentzian halfwidth upon the momentum transfer suggests that the above quasi-elastic contribution arises from the diffusion inside a confined space, values of confinement radius and local diffusion coefficient being compatible with motions of hydrogen atoms of the amino acid side chains. When averaged over the whole range of momentum transfer the QENS data put in evidence differences between deoxy and carbonmonoxy hemoglobin and confirm the quaternary structure dependence of the protein dynamics in the nanosecond time scale.     

The dynamics of protein hydration water: a quantitative comparison of molecular dynamics simulations and neutron-scattering experiments

We present results from an extensive molecular dynamics simulation study of water hydrating the protein Ribonuclease A, at a series of temperatures in cluster, crystal, and powder environments. The dynamics of protein hydration water appear to be very similar in crystal and powder environments at moderate to high hydration levels. Thus, we contend that experiments performed on powder samples are appropriate for discussing hydration water dynamics in native protein environments. Our analysis reveals that simulations performed on cluster models consisting of proteins surrounded by a finite water shell with free boundaries are not appropriate for the study of the solvent dynamics. Detailed comparison to available x-ray diffraction and inelastic neutron-scattering data shows that current generation force fields are capable of accurately reproducing the structural and dynamical observables. On the time scale of tens of picoseconds, at room temperature and high hydration, significant water translational diffusion and rotational motion occur. At low hydration, the water molecules are translationally confined but display appreciable rotational motion. Below the protein dynamical transition temperature, both translational and rotational motions of the water molecules are essentially arrested. Taken together, these results suggest that water translational motion is necessary for the structural relaxation that permits anharmonic and diffusive motions in proteins. Furthermore, it appears that the exchange of protein-water hydrogen bonds by water rotational/librational motion is not sufficient to permit protein structural relaxation. Rather, the complete exchange of protein-bound water molecules by translational displacement seems to be required.