The temperature dependence of internal molecular motions in hydrated and dry alpha-amylase: the role of hydration water in the dynamical transition of proteins.

Internal molecular motions of proteins are strongly affected by environmental conditions, like temperature and hydration. As known from numerous studies, the dynamical behavior of hydrated proteins on the picosecond time scale is characterized by vibrational motions in the low-temperature regime and by an onset of stochastic large-amplitude fluctuations at a transition temperature of 180-230 K. The present study reports on the temperature dependence of internal molecular motions as measured with incoherent neutron scattering from the globular water-soluble protein alpha-amylase and from a protein-lipid complex of rhodopsin in disk membranes. Samples of alpha-amylase have been measured in a hydrated and dehydrated state. In contrast to the hydrated sample, which exhibits a pronounced dynamical transition near 200 K, the dehydrated alpha-amylase does not show an appreciable proportion of stochastic large-amplitude fluctuations and no dynamical transition in the measured temperature range of 140-300 K. The obtained results, which are compared to the dynamical behavior of protein-lipid complexes, are discussed with respect to the influence of hydration on the dynamical transition and in the framework of the glass transition.     

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.     

Influence of hydration on segmental chain librations and dynamical transition in lipid bilayers

Continuous wave electron paramagnetic resonance spectroscopy of chain-labeled phospholipids is used to investigate the effects of hydration on the librational oscillations and the dynamical transition of phospholipid membranes in the low-temperature range 120–270 K. Bilayers of dipalmitoylphostatidiycholine (DPPC) spin-labeled at the first acyl chain segments and at the methyl ends and prepared at full, low, and very low hydration are considered. The segmental mean-square angular amplitudes of librations, 〈α²〉, are larger in the bilayer interior than at the polar/apolar interface and larger in the fully and low hydrated than in the very low hydrated membranes. For chain segments at the beginning of the hydrocarbon region, 〈α²〉-values are markedly restricted and temperature independent in DPPC with the lowest water content, whereas they increase with temperature in the low and fully hydrated bilayers, particularly at the highest temperatures. For chain segments at the chain termini, the librational amplitudes increase progressively, first slowly and then more rapidly with temperature in bilayers at any level of hydration. From the temperature dependence of the mean-square librational amplitude, the dynamical transition is detected around 240 K at the polar/apolar interface in fully and low hydrated DPPC and at around 225 K at the inner hydrocarbon region for bilayers at any hydration condition. At the dynamical transition the bilayers cross low energy barriers of activation energy in the range 10–20 kJ/mol. The results highlight biophysical properties of DPPC bilayers at low-temperature and provide evidence of the effects of the hydration on the dynamical transition in bilayers.     

Translational hydration water dynamics drives the protein glass transition

Experimental and computer simulation studies have revealed the presence of a glass-like transition in the internal dynamics of hydrated proteins at approximately 200 K involving an increase of the amplitude of anharmonic dynamics. This increase in flexibility has been correlated with the onset of protein activity. Here, we determine the driving force behind the protein transition by performing molecular dynamics simulations of myoglobin surrounded by a shell of water. A dual heat bath method is used with which, in any given simulation, the protein and solvent are held at different temperatures, and sets of simulations are performed varying the temperature of the components. The results show that the protein transition is driven by a dynamical transition in the hydration water that induces increased fluctuations primarily in side chains in the external regions of the protein. The water transition involves activation of translational diffusion and occurs even in simulations where the protein atoms are held fixed.     

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 hydration water in deuterated purple membranes explored by neutron scattering

The function and dynamics of proteins depend on their direct environment, and much evidence has pointed to a strong coupling between water and protein motions. Recently however, neutron scattering measurements on deuterated and natural-abundance purple membrane (PM), hydrated in H(2)O and D(2)O, respectively, revealed that membrane and water motions on the ns-ps time scale are not directly coupled below 260 K (Wood et al. in Proc Natl Acad Sci USA 104:18049-18054, 2007). In the initial study, samples with a high level of hydration were measured. Here, we have measured the dynamics of PM and water separately, at a low-hydration level corresponding to the first layer of hydration water only. As in the case of the higher hydration samples previously studied, the dynamics of PM and water display different temperature dependencies, with a transition in the hydration water at 200 K not triggering a transition in the membrane at the same temperature. Furthermore, neutron diffraction experiments were carried out to monitor the lamellar spacing of a flash-cooled deuterated PM stack hydrated in H(2)O as a function of temperature. At 200 K, a sudden decrease in lamellar spacing indicated the onset of long-range translational water diffusion in the second hydration layer as has already been observed on flash-cooled natural-abundance PM stacks hydrated in D(2)O (Weik et al. in J Mol Biol 275:632-634, 2005), excluding thus a notable isotope effect. Our results reinforce the notion that membrane-protein dynamics may be less strongly coupled to hydration water motions than the dynamics of soluble proteins.     

The protein-solvent glass transition

The protein dynamical transition and its connection with the liquid-glass transition (GT) of hydration water and aqueous solvents are reviewed. The protein solvation shell exhibits a regular glass transition, characterized by steps in the specific heat and the thermal expansion coefficient at the calorimetric glass temperature T_G ≈ 170 K. It implies that the time scale of the structural α-relaxation has reached the experimental time window of 1–100 s. The protein dynamical transition, identified from elastic neutron scattering experiments by enhanced amplitudes of molecular motions exceeding the vibrational level [1], probes the α-process on a shorter time scale. The corresponding liquid-glass transition occurs at higher temperatures, typically 240 K. The GT is generally associated with diverging viscosities, the freezing of long-range translational diffusion in the supercooled liquid. Due to mutual hydrogen bonding, both, protein- and solvent relaxational degrees of freedom slow down in paralled near the GT. However, the freezing of protein motions, where surface-coupled rotational and librational degrees of freedom are arrested, is better characterized as a rubber-glass transition. In contrast, internal protein modes such as the rotation of side chains are not affected. Moreover, ligand binding experiments with myoglobin in various glass-forming solvents show, that only ligand entry and exit rates depend on the local viscosity near the protein surface, but protein-internal ligand migration is not coupled to the solvent. The GT leads to structural arrest on a macroscopic scale due to the microscopic cage effect on the scale of the intermolecular distance. Mode coupling theory provides a theoretical framework to understand the microcopic nature of the GT even in complex systems. The role of the α- and β-process in the dynamics of protein hydration water is evaluated. The protein-solvent GT is triggered by hydrogen bond fluctuations, which give rise to fast β-processes. High-frequency neutron scattering spectra indicate increasing hydrogen bond braking above T_G.     

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.     

Mean square fluctuations of hydrogen atoms and water-biopolymer interactions in hydrated saccharides.

We have used the elastic neutron scattering technique to investigate the dynamics of the two main saccharidic components of starch: amylose and amylopectin. The measurements were carried out in the temperature range of 20 to 320 K and at different hydration levels from the dry state up to 0.47 g saccharide/g D(2)O. In the dry samples, the atomic dynamics is harmonic up to approximately 300 K. In the hydrated samples a "glass-like" transition leading to an anharmonic dynamics is observed. The onset of the anharmonicity occurs at temperatures that increase from approximately 180 K to 260 K upon decreasing hydration from 0.5 to 0.1 g saccharide/g D(2)O. This behavior is qualitatively similar to that observed in hydrated globular proteins, but quantitative differences are present. Assuming a simple asymmetric double-well potential model, the temperature and hydration dependence of the transition have been described in terms of few physical parameters.     

Dynamics of tRNA at Different Levels of Hydration

The influence of hydration on the nanosecond timescale dynamics of tRNA is investigated using neutron scattering spectroscopy. Unlike protein dynamics, the dynamics of tRNA is not affected by methyl group rotation. This allows for a simpler analysis of the influence of hydration on the conformational motions in RNA. We find that hydration affects the dynamics of tRNA significantly more than that of lysozyme. Both the characteristic length scale and the timescale of the conformational motions in tRNA depend strongly on hydration. Even the characteristic temperature of the so-called “dynamical transition” appears to be hydration-dependent in tRNA. The amplitude of the conformational motions in fully hydrated tRNA is almost twice as large as in hydrated lysozyme. We ascribe these differences to a more open and flexible structure of hydrated RNA, and to a larger fraction and different nature of hydrophilic sites. The latter leads to a higher density of water that makes the biomolecule more flexible. All-atom molecular-dynamics simulations are used to show that the extent of hydration is greater in tRNA than in lysozyme. We propose that water acts as a “lubricant” in facilitating enhanced motion in solvated RNA molecules.     

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.     

Protein interaction with hydrated C60 fullerene in aqueous solutions

Physicochemical effects of hydrated C(60) fullerenes (HyFn) on serum albumin molecules were studied using ESR spin labeling and differential scanning microcalorimetry. Molecular-colloidal solution of hydrated C(60) fullerenes and their small spherical fractal clusters in water (C(60)FWS), was shown to stabilize protein hydration, and decrease specific surface energy in water-protein matrix in salt solutions. The mechanism of HyFn interaction with protein is discussed in terms of HyFn induced formation of protein clusters and phase transition of hydration water.     

Low-temperature glass transition in proteins

The viscoelastic properties of solid samples (crystals, amorphous films) of hen egg white lysozyme, bovine serum albumin, and sperm whale myoglobin were studied in the temperature range of 100–300 K at different hydration levels. Decreasing the temperature was shown to cause a steplike increase in the Young's modulus of highly hydrated protein samples (with water content exceeding 0.3 g/g dry weight of protein) in the temperature range of 237–251 K, followed by a large increase in the modulus in the broad temperature interval of 240–130 K, which we refer to as a mechanical glass transition. Soaking the samples in 50% glycerol solution completely removed the steplike transition without significantly affecting the glass transition. The apparent activation energy determined from the frequency dependence of the glass-transition temperature was found to be 18 kcal/mol for wet lysozyme crystals. Lowering the humidity causes both the change of the Young's modulus in response to the transition and the activation energy to decrease. The thermal expansion coefficient of amorphous protein films also indicates the glass transition at 150–170 K. The data presented suggest that the glass transition in hydrated samples is located in the surface layer of proteins and related to the immobilization of the protein groups and strongly bound water.     

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.     

DNA B to D transition can be explained in terms of hydration economy of the minor groove atoms

Adjacent phosphate oxygen atoms in A and Z-DNA are located much closer together than in the B form and can be hydrated more economically due to the formation of water bridges between them, whereas in the B form phosphates are hydrated individually. This principle of hydration economy of phosphate groups discovered by Saenger and colleagues could not be applied to the B-D transition, which, like the B-A and B-Z transitions, occurs in a situation of water deficiency, because the distances between adjacent phosphates of individual polynucleotide chains in the D form are not much different from B-DNA. It follows from our calculations of B and D-DNA accessibility to solvent performed by the method of Lee & Richards, and from a simulation of solvent structure near DNA, that there is an economy of hydration only for the minor groove atoms. This feature and some experimental data can explain why only a limited range of sequences consisting of A.T or I.C pairs undergo the transition to the D form. The conformational transition in DNAs with such sequences to a poly[d(A]).poly[d(T])-like conformation (Bh-DNA), which is accompanied by a narrowing of the minor groove, can be explained in the same way. Calculations suggest that in the D-form minor groove of different A-T or I-C DNAs there is a double-layer hydration spine similar to that observed by Drew & Dickerson in the A-T tract of the d(C-G-C-G-A-A-T-T-C-G-C-G) dodecamer. The B-D and B-Bh transitions in A + T-rich DNAs can have biological implications, e.g. they can facilitate DNA bending upon the interaction with proteins.     

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.     

Dynamics of apomyoglobin in the α-to-β transition and of partially unfolded aggregated protein

Changes of molecular dynamics in the α-to-β transition associated with amyloid fibril formation were explored on apomyoglobin (ApoMb) as a model system. Circular dichroism, neutron and X-ray scattering experiments were performed as a function of temperature on the protein, at different solvent conditions. A significant change in molecular dynamics was observed at the α-to-β transition at about 55°C, indicating a more resilient high temperature β structure phase. A similar effect at approximately the same temperature was observed in holo-myoglobin, associated with partial unfolding and protein aggregation. A study in a wide temperature range between 20 and 360 K revealed that a dynamical transition at about 200 K for motions in the 50 ps time scale exists also for a hydrated powder of heat-denatured aggregated ApoMb.     

The protein glass transition as measured by dielectric spectroscopy and differential scanning calorimetry

The glass transition and its related dynamics of myoglobin in water and in a water–glycerol mixture have been investigated by dielectric spectroscopy and differential scanning calorimetry (DSC). For all samples, the DSC measurements display a glass transition that extends over a large temperature range. Both the temperature of the transition and its broadness decrease rapidly with increasing amount of solvent in the system. The dielectric measurements show several dynamical processes, due to both protein and solvent relaxations, and in the case of pure water as solvent the main protein process (which most likely is due to conformational changes of the protein structure) exhibits a dynamic glass transition (i.e. reaches a relaxation time of 100 s) at about the same temperature as the calorimetric glass transition temperature T_g is found. This glass transition is most likely caused by the dynamic crossover and the associated vanishing of the α-relaxation of the main water relaxation, although it does not contribute to the calorimetric T_g. This is in contrast to myoglobin in water–glycerol, where the main solvent relaxation makes the strongest contribution to the calorimetric glass transition. For all samples it is clear that several proteins processes are involved in the calorimetric glass transition and the broadness of the transition depends on how much these different relaxations are separated in time.     

Multicomponent phase transitions of diacylphosphatidylethanolamine dispersions

The phase transition properties of aqueous suspensions of a series of nonhydrated (not heated above room temperature) and hydrated 1,2 diacylphosphatidylethanolamines (PE's) have been examined by high sensitivity differential scanning calorimetry at scan rates of 0.02-1.0 K min-1. At all scan rates nonhydrated PE's show a single asymmetric transition curve of excess heat capacity as a function of temperature. Multilamellar dispersions of hydrated PE's, however, exhibit transitions with fine structure, which can be fitted as the sum of three two-state component transitions, at scan rates of 0.02-0.1 K min-1, but give only a single asymmetric transition at 1.0 K min-1. At all scan rates the transition(s) of hydrated samples occur at lower temperatures than those of nonhydrated samples. One of the component transitions of hydrated PE's may be analogous to the pretransition that occurs in 1,2 diacylphosphatidylcholines.     

Conditioning action of the environment on the protein dynamics studied through elastic neutron scattering

The dynamics of lysozyme in the picosecond timescale has been studied when it is in dry and hydrated powder form and when it is embedded in glycerol, glycerol–water, glucose and glucose–water matrices. The investigation has been undertaken through elastic neutron scattering technique on the backscattering spectrometer IN13. The dynamics of dry powder and embedded-in-glucose lysozyme can be considered purely vibrational up to 100 K, where the onset of an anharmonic contribution takes place. This contribution can be attributed to the activation of methyl group reorientations and is described with an Arrhenius trend. An additional source of anharmonic dynamics appears at higher temperatures for lysozyme in hydrated powders and embedded in glycerol, glycerol–water and glucose–water matrices. This second process, also represented with an Arrhenius trend, corresponds to the so-called protein dynamical transition. Both the temperature where such a transition takes place and the magnitude of the protein mean square displacements depend on the environment. The dynamical response of the protein to temperature is put in relationship with its thermal stability.