Temperature dependence of dynamics of hydrated myoglobin. Comparison of force field calculations with neutron scattering data

Molecular dynamics is used to probe the atomic motions of the carboxy-myoglobin protein as a function of temperature. Simulations of 150 picoseconds in length are carried out on the protein at 20, 60, 100, 180, 220, 240, 260, 280, 300, 320 and 340 K. The simulations attempt to mimic neutron scattering experiments very closely by including a partial hydration shell around the protein. Theoretical elastic, quasielastic and inelastic neutron scattering data are derived from the trajectories and directly compared with experiment. Compared to experiment, the simulation-derived elastic scattering curves show a decrease in intensity as a function of the scattering wavevector, q2. The inelastic and quasielastic spectra show that the inelastic peak is shifted to lower frequency than the experimental value, while quasielastic behavior is in good agreement with experiment. This suggests that the theoretical model is too flexible in the harmonic limit (low temperature), but accurately reproduces high-temperature behavior. Time correlation functions of the intermediate scattering function are determined. At low temperature there is one fast decay process, and at high temperatures there is an additional slow relaxation process that is due to quasielastic scattering. The average atomic fluctuations show that the protein behaves harmonically at low temperatures. At approximately 210 K, a glass-like transition in atomic fluctuations is seen. Above the transition temperature, the atomic fluctuations exhibit both harmonic and anharmonic behavior. Comparison of protein mobility behavior with experiment indicate the fluctuations derived from simulations are larger in the harmonic region. However, the anharmonic region agrees very well with experiment. The anharmonicity is large at all temperatures, with a gradual monotonic increase from 0.5 at 20 K to greater than 0.7 at 340 K without a noticeable change at the glass transition temperature. Heavy-atom dihedral transitions are monitored as a function of temperature. Trends in the type of dihedral transitions that occur with temperature are clearly visible. Dihedral transitions involving backbone atoms occur only above the glass transition temperature. The overall protein behavior results suggest that at low temperatures there is purely vibrational motion with one fast decay process, and above the glass transition temperature there is more anharmonic motion with a fast and a slower relaxation process occurring simultaneously.(ABSTRACT TRUNCATED AT 400 WORDS)     

Picosecond Internal Dynamics of Lysozyme as Affected by Thermal Unfolding in Nonaqueous Environment

A neutron-scattering investigation of the internal picosecond dynamics of lysozyme solvated in glycerol as a function of temperature in the range 200–410 K has been undertaken. The inelastic contribution to the measured intensity is characterized by the presence of a bump generally known as “boson peak”, clearly distinguishable at low temperature. When the temperature is increased the quasielastic component of the spectrum becomes more and more intrusive and progressively overwhelms the vibrational bump. This happens especially for T > 345 K when the protein goes through an unfolding process, which leads to the complete denaturation. The quasielastic term is the superposition of two components whose intensities and linewidths have been studied as a function of temperature. The slower component describes motions with characteristic times of ~4 ps corresponding to reorientations of polypeptide side chains. Both the intensity and linewidth of this kind of relaxations show two distinct regimes with a crossover in the temperature range where the melting process occurs, thus suggesting the presence of a dynamical transition correlated to the protein unfolding. Conversely the faster component might be ascribed to the local dynamics of hydrogen atoms caged by the nearest neighbors with characteristic time of ~0.3 ps.     

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     

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.     

Temperature dependence of the low frequency dynamics of myoglobin. Measurement of the vibrational frequency distribution by inelastic neutron scattering.

Inelastic neutron scattering spectra of myoglobin hydrated to 0.33 g water (D2O)/g protein have been measured in the low frequency range (1-150 cm-1) at various temperatures between 100 and 350 K. The spectra at low temperatures show a well-resolved maximum in the incoherent dynamic structure factor Sinc(q, omega) at approximately 25 cm-1 and no elastic broadening. This maximum becomes gradually less distinct above 180 K due to the increasing amplitude of quasielastic scattering which extends out to 30 cm-1. The vibrational frequency distribution derived independently at 100 and 180 K are very similar, suggesting harmonic behavior at these temperatures. This result has been used to separate the vibrational motion from the quasielastic motion at temperatures above 180 K. The form of the density of states of myoglobin is discussed in relation to that of other amorphous systems, to theoretical calculations of low frequency modes in proteins, and to previous observations by electron-spin relaxation of fractal-like spectral properties of proteins. The onset of quasielastic scattering above 180 K is indicative of a dynamic transition of the system and correlates with an anomalous increase in the atomic mean-squared displacements observed by M?ssbauer spectroscopy (Parak, F., E. W. Knapp, and D. Kucheida. 1982. J. Mol. Biol. 161: 177-194.) and inelastic neutron scattering (Doster, W., S. Cusack, and W. Petry, 1989. Nature [Lond.]. 337: 754-756.) Similar behavior is observed for a hydrated powder of lysozyme suggesting that the low frequency dynamics of globular proteins have common features.     

C-phycocyanin hydration water dynamics in the presence of trehalose: an incoherent elastic neutron scattering study at different energy resolutions

We present a study of C-phycocyanin hydration water dynamics in the presence of trehalose by incoherent elastic neutron scattering. By combining data from two backscattering spectrometers with a 10-fold difference in energy resolution we extract a scattering law S(Q,omega) from the Q-dependence of the elastic intensities without sampling the quasielastic range. The hydration water is described by two dynamically different populations--one diffusing inside a sphere and the other diffusing quasifreely--with a population ratio that depends on temperature. The scattering law derived describes the experimental data from both instruments excellently over a large temperature range (235-320 K). The effective diffusion coefficient extracted is reduced by a factor of 10-15 with respect to bulk water at corresponding temperatures. Our approach demonstrates the benefits and the efficiency of using different energy resolutions in incoherent elastic neutron scattering over a large angular range for the study of biological macromolecules and hydration water.     

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.     

Probing the flexibility of the bacterial reaction center: the wild-type protein is more rigid than two site-specific mutants

Experimental and theoretical studies have stressed the importance of flexibility for protein function. However, more local studies of protein dynamics, using temperature factors from crystallographic data or elastic models of protein mechanics, suggest that active sites are among the most rigid parts of proteins. We have used quasielastic neutron scattering to study the native reaction center protein from the purple bacterium Rhodobacter sphaeroides, over a temperature range of 4-260 K, in parallel with two nonfunctional mutants both carrying the mutations L212Glu/L213Asp --> Ala/Ala (one mutant carrying, in addition, the M249Ala --> Tyr mutation). The so-called dynamical transition temperature, Td, remains the same for the three proteins around 230 K. Below Td the mean square displacement, u2, and the dynamical structure factor, S(Q,omega), as measured respectively by backscattering and time-of-flight techniques are identical. However, we report that above Td, where anharmonicity and diffusive motions take place, the native protein is more rigid than the two nonfunctional mutants. The higher flexibility of both mutant proteins is demonstrated by either their higher u2 values or the notable quasielastic broadening of S(Q,omega) that reveals the diffusive nature of the motions involved. Remarkably, we demonstrate here that in proteins, point genetic mutations may notably affect the overall protein dynamics, and this effect can be quantified by neutron scattering. Our results suggest a new direction of investigation for further understanding of the relationship between fast dynamics and activity in proteins. Brownian dynamics simulations we have carried out are consistent with the neutron experiments, suggesting that a rigid core within the native protein is specifically softened by distant point mutations. L212Glu, which is systematically conserved in all photosynthetic bacteria, seems to be one of the key residues that exerts a distant control over the rigidity of the core of the protein.     

The inverse relationship between protein dynamics and thermal stability

Protein powders that are dehydrated or mixed with a glassy compound are known to have improved thermal stability. We present elastic and quasielastic neutron scattering measurements of the global dynamics of lysozyme and ribonuclease A powders. In the absence of solvation water, both protein powders exhibit largely harmonic motions on the timescale of the measurements. Upon partial hydration, quasielastic scattering indicative of relaxational processes appears at sufficiently high temperature. When the scattering spectrum are analyzed with the Kohlrausch-Williams-Watts formalism, the exponent beta decreases with increasing temperature, suggesting that multiple relaxation modes are emerging. When lysozyme was mixed with glycerol, its beta values were higher than the hydrated sample at comparable temperatures, reflecting the viscosity and stabilizing effects of glycerol.     

Vibrational modes of hemoglobin in red blood cells.

Equine red blood cells were washed in saline heavy water (2H2O) to exchange the hydrogen atoms of the non-hemoglobin components with deuterons. This led to novel neutron scattering measurements of protein vibrations within a cellular system and permitted a comparison with inelastic neutron scattering measurements on purified horse hemoglobin, either dry or wetted with 2H2O. As a function of wavevector transfer Q and the frequency transfer v the neutron response typified by the dynamic structure factor S(Q, v) was found to be similar for extracted and cellular hemoglobin at low and high temperatures. At 77 K, in the cells, a peak in S(Q, v) due to the protein was found near 0.7 THz, approximately half the frequency of a strong peak in the aqueous medium. Measurements at higher temperatures (170 and 230 K) indicated similar small shifts downwards in the peak frequencies of both components. At 260 K the low frequency component became predominantly quasielastic, but a significant inelastic component could still be ascribed to the aqueous scattering. Near 295 K the frequency responses of both components were similar and centered near zero. When scattering due to water is taken into account it appears that the protein neutron response in, or out of, red blood cells is little affected by hydration in the low frequency regime where Van der Waals forces are thought to be effective.     

Enzyme activity and flexibility at very low hydration

Recent measurements have demonstrated enzyme activity at hydrations as low as 3%. This raises the question of whether hydration-induced enzyme flexibility is important for activity. Here, to address this, picosecond dynamic neutron scattering experiments are performed on pig liver esterase powders at 0%, 3%, 12%, and 50% hydration by weight and at temperatures ranging from 120 to 300 K. At all temperatures and hydrations, significant quasielastic scattering intensity is found in the protein, indicating the presence of anharmonic, diffusive motion. As the hydration increases, a temperature-dependent dynamical transition appears and strengthens involving additional diffusive motion. The implication of these results is that, although the additional hydration-induced diffusive motion in the protein detected here may be related to increased activity, it is not required for the enzyme to function.     

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.     

From Powder to Solution: Hydration Dependence of Human Hemoglobin Dynamics Correlated to Body Temperature

A transition in hemoglobin (Hb), involving partial unfolding and aggregation, has been shown previously by various biophysical methods. The correlation between the transition temperature and body temperature for Hb from different species, suggested that it might be significant for biological function. To focus on such biologically relevant human Hb dynamics, we studied the protein internal picosecond motions as a response to hydration, by elastic and quasielastic neutron scattering. Rates of fast diffusive motions were found to be significantly enhanced with increasing hydration from fully hydrated powder to concentrated Hb solution. In concentrated protein solution, the data showed that amino acid side chains can explore larger volumes above body temperature than expected from normal temperature dependence. The body temperature transition in protein dynamics was absent in fully hydrated powder, indicating that picosecond protein dynamics responsible for the transition is activated only at a sufficient level of hydration. A collateral result from the study is that fully hydrated protein powder samples do not accurately describe all aspects of protein picosecond dynamics that might be necessary for biological function.     

Temperature- and hydration-dependent protein dynamics in photosystem II of green plants studied by quasielastic neutron scattering

Protein dynamics in hydrated and vacuum-dried photosystem II (PS II) membrane fragments from spinach has been investigated by quasielastic neutron scattering (QENS) in the temperature range between 5 and 300 K. Three distinct temperature ranges can be clearly distinguished by active type(s) of protein dynamics: (A) At low temperatures (T < 120 K), the protein dynamics of both dry and hydrated PS II is characterized by harmonic vibrational motions. (B) In the intermediate temperature range (120 < T < 240 K), the total mean square displacement total slightly deviates from the predicted linear behavior. The QENS data indicate that this deviation, which is virtually independent of the extent of hydration, is due to a partial onset of diffusive protein motions. (C) At temperatures above 240 K, the protein flexibility drastically changes because of the onset of diffusive (large-amplitude) protein motions. This dynamical transition is clearly hydration-dependent since it is strongly suppressed in dry PS II. The thermally activated onset of protein flexibility as monitored by QENS is found to be strictly correlated with the temperature-dependent increase of the electron transport efficiency from Q(A)(-) to QB (Garbers et al. (1998) Biochemistry 37, 11399-11404). Analogously, the freezing of protein mobility by dehydration in dry PS II appears to be responsible for the blockage of Q(A)(-) reoxidation by Q(B) at hydration values lower than 45% r.h. (Kaminskaya et al. (2003) Biochemistry 42, 8119-8132). Similar effects were observed for reactions of the water-oxidizing complex as outlined in the Discussion section.     

Measurement of coherent Debye-Waller factor in in vivo deuterated C-phycocyanin by inelastic neutron scattering.

Quasielastic neutron scattering measurements of dry and 35% D2O hydrated amorphous protein powder of C-phycocyanin were made as a function of temperature ranging from 313K down to 100K. The protein is grown from blue-green algae cultured in D2O and is deuterated up to 99%. The scattering is thus dominated by coherent scattering. Within the best energy resolution of the time-of-flight instrument, which is 28 mueV FWHM, the scattering appears entirely elastic. For this reason we are able to extract a coherent Debye-Waller factor by making an independent measurement of the static structure factor. We observe a considerable difference in the q dependence of the Debye-Waller factor between the dry and hydrated proteins; furthermore, there is an interesting temperature dependence of the Debye-Waller factor that is quite different from that predicted for dense hard-sphere liquids.     

Evolution of the internal dynamics of two globular proteins from dry powder to solution.

Myoglobin and lysozyme picosecond internal dynamics in solution is compared to that in hydrated powders by quasielastic incoherent neutron scattering. This technique is sensitive to the motions of the nonexchangeable hydrogen atoms in a sample. Because these are homogeneously distributed throughout the protein structure, the average dynamics of the protein is described. We first propose an original data treatment to deal with the protein global motions in the case of solution samples. The validity of this treatment is checked by comparison with classical measurements of the diffusion constants. The evolution with the scattering vector of the width and relative contribution of the quasielastic component was then used to derive information on the amount of local diffusive motions and their characteristic average relaxation time. From dry powder to coverage by one water layer, the surface side chains progressively acquire the possibility to diffuse locally. On subsequent hydration, the main effect of water is to improve the rate of these diffusive motions. Motions with higher average amplitude occur in solution, about three times more than for a hydrated powder at complete coverage, with a shorter average relaxation time, approximately 4.5 ps compared to 9.4 ps for one water monolayer.     

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.     

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.     

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.     

Hydration effect on low-frequency protein dynamics observed in simulated neutron scattering spectra

Hydration effects on protein dynamics were investigated by comparing the frequency dependence of the calculated neutron scattering spectra between full and minimal hydration states at temperatures between 100 and 300 K. The protein boson peak is observed in the frequency range 1-4 meV at 100 K in both states. The peak frequency in the minimal hydration state shifts to lower than that in the full hydration state. Protein motions with a frequency higher than 4 meV were shown to undergo almost harmonic motion in both states at all temperatures simulated, whereas those with a frequency lower than 1 meV dominate the total fluctuations above 220 K and contribute to the origin of the glass-like transition. At 300 K, the boson peak becomes buried in the quasielastic contributions in the full hydration state but is still observed in the minimal hydration state. The boson peak is observed when protein dynamics are trapped within a local minimum of its energy surface. Protein motions, which contribute to the boson peak, are distributed throughout the whole protein. The fine structure of the dynamics structure factor is expected to be detected by the experiment if a high resolution instrument (<∼20 μeV) is developed in the near future.