This review of protein dynamics studied by neutron scattering focuses on data collected in the last 10 years. After an introduction to thermal neutron scattering and instrumental aspects, theoretical models that have been used to interpret the data are presented and discussed. Experiments are described according to sample type, protein powders, solutions and membranes. Neutron-scattering results are compared to those obtained from other techniques. The biological relevance of the experimental results is discussed. The major conclusion of the last decade concerns the strong dependence of internal dynamics on the macromolecular environment.

Mean macromolecular dynamics was quantified in vivo by neutron scattering in psychrophile, mesophile, thermophile and hyperthermophile bacteria. Root mean square atomic fluctuation amplitudes determining macromolecular flexibility were found to be similar for each organism at its physiological temperature (B1 Å in the 0.1 ns timescale). Effective force constants determining the mean macromolecular resilience were found to increase with physiological temperature from 0.2 N/m for the psychrophiles, which grow at 4 1C, to 0.6 N/m for the hyperthermophiles (85 1C), indicating that the increase in stabilization free energy is dominated by enthalpic rather than entropic terms. Larger resilience allows macromolecular stability at high temperatures, while maintaining flexibility within acceptable limits for biological activity.

Intracellular water dynamics in Haloarcula marismortui, an extremely halophilic organism originally isolated from the Dead Sea, was studied by neutron scattering. The water in centrifuged cell pellets was examined by means of two spectrometers, IN6 and IN16, sensitive to motions with time scales of 10 ps and 1 ns, respectively. From IN6 data, a translational diffusion constant of 1.3 ؋ 10 ؊5 cm 2 s ؊1 was determined at 285 K. This value is close to that found previously for other cells and close to that for bulk water, as well as that of the water in the 3.5 M NaCl solution bathing the cells. A very slow water component was discovered from the IN16 data. At 285 K the waterprotons of this component displays a residence time of 411 ps (compared with a few ps in bulk water). At 300 K, the residence time dropped to 243 ps and was associated with a translational diffusion of 9.3 ؋ 10 ؊8 cm 2 s ؊1 , or 250 times lower than that of bulk water. This slow water accounts for Ϸ76% of cell water in H. marismortui. No such water was found in Escherichia coli measured on BSS, a neutron spectrometer with properties similar to those of IN16. It is hypothesized that the slow mobility of a large part of H. marismortui cell water indicates a specific water structure responsible for the large amounts of K ؉ bound within these extremophile cells.Haloarcula marismortui ͉ water structure and dynamics ͉ extreme haplophile T he study of the specific properties of water in biological systems continues to yield fascinating surprises. Haloarcula marismortui, an archaeal extreme halophile isolated from the Dead Sea, attracted our attention some years ago because of its high selectivity for, despite a high membrane permeability (1). In distinction to other organisms, K ϩ was retained within the cell, even in the absence of metabolism, with a half-time of exchange with the outside medium of Ͼ24 h (2). At the time, the only systems known with a very high binding selectivity were antibiotics such as nonactin and valinomycin. The structure responsible for their specificity is a ''cage'' of six to eight carbonyl oxygen atoms arranged in space according to a definite pattern. If the same principle were to be responsible for K ϩ binding in H. marismortui, 24-32 moles oxygen per liter of cell water would be required, enormous amounts of oxygen atoms that cannot be supplied exclusively by the organic components of the cell. It is therefore suggested that the oxygen atoms required might be furnished by the ordering of water molecules forming a tertiary system of water, KCl, and cell proteins.Evidence in favor of more than a single phase of water in cell pellets of H. marismortui came initially from H-NMR measurements (3). The visible intensity of water in such pellets accounted for all of the total water present (101 Ϯ 7%). When the pellets were slowly cooled to below Ϫ19°C (the temperature at which the culture medium froze), 40% of the water signal remained visible. From analysis of the dynamic properties of the intracellular water, it was concluded ...

Protein thermal dynamics was evaluated by neutron scattering for halophilic malate dehydrogenase from Haloarcula marismortui (HmMalDH) and BSA under different solvent conditions. As a measure of thermal stability in each case, loss of secondary structure temperatures were determined by CD. HmMalDH requires molar salt and has different stability behavior in H 2 O, D 2 O, and in NaCl and KCl solvents. BSA remains soluble in molar NaCl. The neutron experiments provided values of mean-squared atomic fluctuations at the 0.1 ns time scale. Effective force constants, characterizing the mean resilience of the protein structure, were calculated from the variation of the mean-squared fluctuation with temperature. For HmMalDH, resilience increased progressively with increasing stability, from molar NaCl in H2O, via molar KCl in D 2O, to molar NaCl in D2O. Surprisingly, however, the opposite was observed for BSA; its resilience is higher in H 2O where it is less stable than in D 2O. These results confirmed the complexity of dynamics-stability relationships in different proteins. Softer dynamics for BSA in D 2O showed that the higher thermostability is associated with entropic fluctuations. In the halophilic protein, higher stability is associated with increased resilience showing the dominance of enthalpic terms arising from bonded interactions. From previous data, it is suggested that these are associated with hydrated ion binding stabilizing the protein in the high-salt solvent. S olvent interactions provide a complex contribution to protein structure stabilization through hydration, van der Waals interactions, hydrogen bonds, ion binding, and the hydrophobic effect. Because the same forces control thermal fluctuations, a relation among solvent interactions, protein stabilization, and dynamics is expected intuitively, in which a softer, more flexible protein structure would be less stable. Stability, however, need not necessarily be associated with lower flexibility. Neutronscattering experiments on ␣-amylase at room temperature have indicated larger amplitudes of motion for atoms in the thermophilic protein compared with the mesophilic homologue, suggesting that thermostability in this case is associated with entropic effects (1). Unfolding experiments on ␣-lytic protease have shown the existence of a partially unfolded state, I, which is favored entropically and has a lower free energy than the native state, N; under physiological conditions, N is not converted to I because of a very high activation-energy barrier (2). Where entropic terms are dominant, therefore, a more flexible protein could be more stable. Furthermore, measurements of flexibility and rigidity depend strongly on the experimental method used. They could relate to: thermal motions on very fast, ps to 100-ps time scales, measured by neutron scattering (1, 3-9); motions integrated up to the nanosecond or longer times, measured by NMR using isotope labeling (10); or slower conformational changes taking place in milliseconds, measured by hydrogen-exchange e...

To explore protein adaptation to extremely high temperatures, two parameters related to macromolecular dynamics, the mean square atomic fluctuation and structural resilience, expressed as a mean force constant, were measured by neutron scattering for hyperthermophilic malate dehydrogenase from Methanococcus jannaschii and a mesophilic homologue, lactate dehydrogenase from Oryctolagus cunniculus (rabbit) muscle. The root mean square fluctuations, defining flexibility, were found to be similar for both enzymes (1.5 Å) at their optimal activity temperature. Resilience values, defining structural rigidity, are higher by an order of magnitude for the high temperature-adapted protein (0.15 Newtons/meter for O. cunniculus lactate dehydrogenase and 1.5 Newtons/meter for M. jannaschii malate dehydrogenase). Thermoadaptation appears to have been achieved by evolution through selection of appropriate structural rigidity in order to preserve specific protein structure while allowing the conformational flexibility required for activity.Hyperthermophilic organisms grow at temperatures above 80°C. Proteins from these organisms are themselves optimally active between 60 and 125°C and serve as paradigms for the characterization of factors responsible for protein fold stability and flexibility. Hyperthermophilic enzymes have also attracted considerable attention because of a range of biotechnological applications (1, 2).Sequence comparison studies and structural analyses carried out on hyperthermophilic proteins and their mesophilic homologues have shown that thermal stability is associated with multiple factors, including an increase in hydrogen bonding, complex salt bridge formation, and helix stabilization by acidic residues. The commonly accepted hypothesis is that increased thermal stability is due to enhanced conformational rigidity of the molecular structure (3). Hyperthermophilic enzymes are also characterized by a higher temperature of maximum activity (3, 4). The more rigid hyperthermophilic enzyme would then require higher temperatures to achieve the requisite conformational flexibility for activity.Experiments have shown that thermostable enzymes exhibit reduced structural flexibility at room temperature with respect to their mesophilic homologues (4, 5), whereas others, on ␣-amylase (6) and on rubredoxin (7,8), have shown the opposite effect, i.e. the thermostable homologues were found to be more flexible, suggesting stabilization through entropic effects. Relations between flexibility and stability are, therefore, complex. Atomic fluctuations only were measured in these experiments and interpreted in terms of flexibility.It is important to point out that reduced structural "flexibility" does not necessarily imply a more "rigid" structure. Atoms are maintained in a structure by forces that link them to their neighbors. In terms of a force field, the width of the potential well in which an atom moves is a measure of its flexibility in terms of a root mean square fluctuation amplitude ͑ ͌ Ͻu 2 Ͼ), whereas the detailed ...

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.

We present direct quasielastic neutron scattering measurements, in vivo, of macromolecular dynamics in Escherichia coli. The experiments were performed on a wide range of timescales to cover the large panel of internal and self-diffusion motions. Three major internal processes were extracted at physiological temperature: a fast picosecond process that corresponded to restricted jump diffusion motions and two slower processes that resulted from reorientational motions occurring in approximately 40 ps and 90 ps, respectively. The analysis of the fast process revealed that the cellular environment leads to an appreciable increase in internal molecular flexibility and diffusive motion rates compared with those evaluated in fully hydrated powders. The result showed that the amount of cell water plays a decisive role in internal molecular dynamics. Macromolecular interactions and confinement, however, attenuate slightly the lubricating effect of water, as revealed by the decrease of the in vivo parameters compared with those measured in solution. The study demonstrated that standard sample preparations do not mimic accurately the physiological environment and suggested that intracellular complexity participates in functional dynamics necessary for biological activity. Furthermore, the method allowed the extraction of the self-diffusion of E. coli macromolecules, which presented similar parameters as those extracted for hemoglobin in red blood cells.

Water constitutes the intracellular matrix in which biological molecules interact. Understanding its dynamic state is a main scientific challenge, which continues to provoke controversy after more than 50 years of study. We measured water dynamics in vivo in the cytoplasm of Escherichia coli by using neutron scattering and isotope labelling. Experimental timescales covered motions from pure water to interfacial water, on an atomic length scale. In contrast to the widespread opinion that water is 'tamed' by macromolecular confinement, the measurements established that water diffusion within the bacteria is similar to that of pure water at physiological temperature.