Enzyme dynamics and activity: time-scale dependence of dynamical transitions in glutamate dehydrogenase solution.

We have examined the temperature dependence of motions in a cryosolution of the enzyme glutamate dehydrogenase (GDH) and compared these with activity. Dynamic neutron scattering was performed with two instruments of different energy resolution, permitting the separate determination of the average dynamical mean square displacements on the sub-approximately 100 ps and sub-approximately 5 ns time scales. The results demonstrate a marked dependence on the time scale of the temperature profile of the mean square displacement. The lowest temperature at which anharmonic motion is observed is heavily dependent on the time window of the instrument used to observe the dynamics. Several dynamical transitions (inflexions of the mean squared displacement) are observed in the slower dynamics. Comparison with the temperature profile of the activity of the enzyme in the same solvent reveals dynamical transitions that have no effect on GDH function.     

Temperature-dependent structural and functional features of a hyperthermostable enzyme using elastic neutron scattering

The dynamic behavior of an endoglucanase from the hyperthermophilic microorganism Pyrococcus furiosus was investigated using elastic neutron scattering. The temperature dependence of the atomic motions was correlated with conformational and functional characteristics of the enzyme. The onset of biological function at temperatures higher than approximately 25 degrees C (the hyperthermostable enzyme is essentially inactive at room temperature) was associated with a dynamical transition in the anharmonic motions domain. This transition from the nonactive to the enzymatically active conformation involved structurally similar conformational substates in the energy landscape. From the mean-square displacement of the protein atoms, the molecular flexibility and the effective force constants were calculated at different temperature zones. The results showed that the activity increases at higher temperatures where the intramolecular bonds are weakened and the overall rigidity of the protein is decreased. Further temperature increase resulted in significantly increased atomic fluctuations featuring heat denaturation of the protein.     

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.     

Dynamics of C-phycocyanin in various deuterated trehalose/water environments measured by quasielastic and elastic neutron scattering

The molecular understanding of protein stabilization by the disaccharide trehalose in extreme temperature or hydration conditions is still debated. In the present study, we investigated the role of trehalose on the dynamics of the protein C-phycocyanin (C-PC) by neutron scattering. To single out the motions of C-PC hydrogen (H) atoms in various trehalose/water environments, measurements were performed in deuterated trehalose and heavy water (D(2)O). We report that trehalose decreases the internal C-PC dynamics, as shown by a reduced diffusion coefficient of protein H atoms. By fitting the Elastic Incoherent Structure Factor-which gives access to the "geometry" of the internal proton motions-with the model of diffusion inside a sphere, we found that the presence of trehalose induces a significantly higher proportion of immobile C-PC hydrogens. We investigated, by elastic neutron scattering, the mean square displacements (MSDs) of deuterated trehalose/D(2)O-embedded C-PC as a function of temperature in the range of 40-318 K. Between 40 and approximately 225 K, harmonic MSDs of C-PC are slightly smaller in samples containing trehalose. Above a transition temperature of approximately 225 K, we observed anharmonic motions in all trehalose/water-coated C-PC samples. In the hydrated samples, MSDs are not significantly changed by addition of 15% trehalose but are slightly reduced by 30% trehalose. In opposition, no dynamical transition was detected in dry trehalose-embedded C-PC, whose hydrogen motions remain harmonic up to 318 K. These results suggest that a role of trehalose would be to stabilize proteins by inhibiting some fluctuations at the origin of protein unfolding and denaturation.     

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.     

Backbone motions in a crystalline protein from field-dependent 2H-NMR relaxation and line-shape analysis

We have used 2H-nmr to study backbone dynamics of the 2H-labeled, slowly exchanging amide sites of fully hydrated, crystalline hen egg white lysozyme. Order parameters are determined from the residual quadrupole coupling and values increase from S2 = 0.85 at 290 K to S2 = 0.94 at 200 K. Dynamical rates are determined from spin-lattice relaxation at three nmr frequencies (38.8, 61.5, and 76.7 MHz). The approach used here is thus distinct from solution nmr studies where dynamical amplitudes and rates are both determined from relaxation measurements. At temperatures below 250 K, relaxation is independent of the nmr frequency indicating that backbone motions are fast compared to the nmr frequencies. However, as the temperature is increased above 250 K, relaxation is significantly more efficient at the lowest frequency, which shows, in addition, the presence of motions that are slow compared to the nmr frequencies. Using the values of S2 determined from the residual quadrupole coupling and a model-free relaxation formalism that allows for fast and slow internal motions, we conclude that these slow motions have correlation times in the range of 0.1 to 1.0 microsecond and are effectively frozen out at 250 K where fast motions of the amide planes with approximately 15 ps effective correlation times and 9 degrees rms amplitudes dominate relaxation. The fast internal motions increase slightly in amplitude as the temperature rises toward 290 K, but the correlation time, as is also observed in solution nmr studies of RNase H, is approximately constant. These findings are consistent with hypotheses of dynamic glass transitions in hydrated proteins arising from temperature-dependent damping of harmonic modes of motion above the transition point.     

On the relationship between thermal stability and catalytic power of enzymes

The possible relationship between the thermal stability and the catalytic power of enzymes is of great current interest. In particular, it has been suggested that thermophilic or hyperthermophilic (Tm) enzymes have lower catalytic power at a given temperature than the corresponding mesophilic (Ms) enzymes, because the thermophilic enzymes are less flexible (assuming that flexibility and catalysis are directly correlated). These suggestions presume that the reduced dynamics of the thermophilic enzymes is the reason for their reduced catalytic power. The present paper takes the specific case of dihydrofolate reductase (DHFR) and explores the validity of the above argument by simulation approaches. It is found that the Tm enzymes have restricted motions in the direction of the folding coordinate, but this is not relevant to the chemical process, since the motions along the reaction coordinate are perpendicular to the folding motions. Moreover, it is shown that the rate of the chemical reaction is determined by the activation barrier and the corresponding reorganization energy, rather than by dynamics or flexibility in the ground state. In fact, as far as flexibility is concerned, we conclude that the displacement along the reaction coordinate is larger in the Tm enzyme than in the Ms enzyme and that the general trend in enzyme catalysis is that the best catalyst involves less motion during the reaction than the less optimal catalyst. The relationship between thermal stability and catalysis appears to reflect the fact that to obtain small electrostatic reorganization energy it is necessary to invest some folding energy in the overall preorganization process. Thus, the optimized catalysts are less stable. This trend is clearly observed in the DHFR case.     

Radially softening diffusive motions in a globular protein

Molecular dynamics simulation, quasielastic neutron scattering and analytical theory are combined to characterize diffusive motions in a hydrated protein, C-phycocyanin. The simulation-derived scattering function is in approximate agreement with experiment and is decomposed to determine the essential contributions. It is found that the geometry of the atomic motions can be modeled as diffusion in spheres with a distribution of radii. The time dependence of the dynamics follows stretched exponential behavior, reflecting a distribution of relaxation times. The average side chain and backbone dynamics are quantified and compared. The dynamical parameters are shown to present a smooth variation with distance from the core of the protein. Moving outward from the center of the protein there is a progressive increase of the mean sphere size, accompanied by a narrowing and shifting to shorter times of the relaxation time distribution. This smooth, "radially softening" dynamics may have important consequences for protein function. It also raises the possibility that the dynamical or "glass" transition with temperature observed experimentally in proteins might be depth dependent, involving, as the temperature decreases, progressive freezing out of the anharmonic dynamics with increasing distance from the center of the protein.     

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.     

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.     

Activity and dynamics of an enzyme, pig liver esterase, in near-anhydrous conditions

Water is widely assumed to be essential for life, although the exact molecular basis of this requirement is unclear. Water facilitates protein motions, and although enzyme activity has been demonstrated at low hydrations in organic solvents, such nonaqueous solvents may allow the necessary motions for catalysis. To examine enzyme function in the absence of solvation and bypass diffusional constraints we have tested the ability of an enzyme, pig liver esterase, to catalyze alcoholysis as an anhydrous powder, in a reaction system of defined water content and where the substrates and products are gaseous. At hydrations of 3 (±2) molecules of water per molecule of enzyme, activity is several orders-of-magnitude greater than nonenzymatic catalysis. Neutron spectroscopy indicates that the fast (≤nanosecond) global anharmonic dynamics of the anhydrous functional enzyme are suppressed. This indicates that neither hydration water nor fast anharmonic dynamics are required for catalysis by this enzyme, implying that one of the biological requirements of water may lie with its role as a diffusion medium rather than any of its more specific properties.     

Local dynamics of DNA probed with optical absorption spectroscopy of bound ethidium bromide.

We have studied the local dynamics of calf thymus double-helical DNA by means of an "optical labeling" technique. The study has been performed by measuring the visible absorption band of the cationic dye ethidium bromide, both free in solution and bound to DNA, in the temperature interval 360-30 K and in two different solvent conditions. The temperature dependence of the absorption line shape has been analyzed within the framework of the vibronic coupling theory, to extract information on the dynamic properties of the system; comparison of the thermal behavior of the absorption band of free and DNA-bound ethidium bromide gave information on the local dynamics of the double helix in the proximity of the chromophore. For the dye free in solution, large spectral heterogeneity and coupling to a "bath" of low-frequency (soft) modes is observed; moreover, anharmonic motions become evident at suitably high temperatures. The average frequency of the soft modes and the amplitude of anharmonic motions depend upon solvent composition. For the DNA-bound dye, at low temperatures, heterogeneity is decreased, the average frequency of the soft modes is increased, and anharmonic motions are hindered. However, a new dynamic regime characterized by a large increase in anharmonic motions is observed at temperatures higher than approximately 280 K. The DNA double helix therefore appears to provide, at low temperatures, a rather rigid environment for the bound chromophore, in which conformational heterogeneity is reduced and low-frequency motions (both harmonic vibrations and anharmonic contributions) are hindered. The system becomes anharmonic at approximately 180 K; however, above approximately 280 K, anharmonicity starts to increase much more rapidly than for the dye free in solution; this can be attributed to the onset of wobbling of the dye in its intercalation site, which is likely connected with the onset of (functionally relevant) DNA motions, involving local opening/unwinding of the double helix. As shown by parallel measurements of the melting curves, these motions precede the melting of the double helix and depend upon solvent composition much more than does the melting itself.     

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.     

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.     

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)     

Nanosecond Motions in Proteins Impose Bounds on the Timescale Distributions of Local Dynamics

We elucidate the physics of protein dynamical transition via 10-100-ns molecular dynamics simulations at temperatures spanning 160-300 K. By tracking the energy fluctuations, we show that the protein dynamical transition is marked by a crossover from nonstationary to stationary processes that underlie the dynamics of protein motions. A two-timescale function captures the nonexponential character of backbone structural relaxations. One timescale is attributed to the collective segmental motions and the other to local relaxations. The former is well defined by a single-exponential, nanosecond decay, operative at all temperatures. The latter is described by a set of processes that display a distribution of timescales. Although their average remains on the picosecond timescale, the distribution is markedly contracted at the onset of the transition. It is shown that the collective motions impose bounds on timescales spanned by local dynamical processes. The nonstationary character below the transition implicates the presence of a collection of substates whose interactions are restricted. At these temperatures, a wide distribution of local-motion timescales, extending beyond that of nanoseconds, is observed. At physiological temperatures, local motions are confined to timescales faster than nanoseconds. This relatively narrow window makes possible the appearance of multiple channels for the backbone dynamics to operate.     

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.     

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.     

Temperature derivative fluorescence spectroscopy as a tool to study dynamical changes in protein crystals

Motions through the energy landscape of proteins lead to biological function. At temperatures below a dynamical transition (150-250 K), some of these motions are arrested and the activity of some proteins ceases. Here, we introduce the technique of temperature-derivative fluorescence microspectrophotometry to investigate the dynamical behavior of single protein crystals. The observation of glass transitions in thin films of water/glycerol mixtures allowed us to demonstrate the potential of the technique. Then, protein crystals were investigated, after soaking the samples in a small amount of fluorescein. If the fluorophore resides within the crystal channels, temperature-dependent changes in solvent dynamics can be monitored. Alternatively, if the fluorophore binds to the protein, local dynamical transitions within the biomolecule can be probed directly. A clear dynamical transition was observed at 175 K in the active site of crystalline human butyrylcholinesterase. The results suggest that the dynamics of crystalline proteins is strongly dependent on solvent composition and confinement in the crystal channels. Beyond applications in the field of kinetic crystallography, the highly sensitive temperature-derivative fluorescence microspectrophotometry technique opens the way to many studies on the dynamics of biological nanosamples.     

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.