1. Introduction 3282. Basic concepts of neutron scattering 3292.1 Introduction 3292.2 Neutron-scattering functions 3312.3 Coherent and incoherent neutron scattering. The particular role of hydrogen in incoherent scattering 3322.4 Total elastic scattering, EISF and mean square displacement (MSD) 3332.5 Quasielastic scattering and relaxation function 3342.6 Inelastic scattering and density of states 3353. Experimental aspects and instruments 3353.1 Energy and space resolution 3353.2 General sample aspects 3353.3 Potential effects of D₂O on dynamics 3363.4 Experimental ²H (deuterium) labelling 3364. Physics of protein dynamics 3364.1 Models 3364.2 The dynamical transition 3384.3 Effective force constants 3395. Dynamics of hydrated protein powders 3395.1 First experiments on myoglobin 3405.2 Dynamical transitions in other proteins 3405.3 The role of hydration water 3415.4 Influence of the solvent 3445.5 Diffusional motions within proteins by QENS 3465.6 Inelastic neutron scattering and vibrational spectra 3475.7 Conclusions 3516. Membranes 3526.1 Lipid bilayers 3536.2 BR and the purple membrane (PM) 3536.2.1 The dynamical transition in the PM 3536.2.2 QENS from oriented PM 3546.2.3 Hydration dependence of PM motions 3556.2.4 Local dynamics in PM studied by isotope labelling 3566.2.5 Dynamics of different BR conformations 3577. Protein solutions 3587.1 From powders to solutions 3587.2 Water dynamics and solvent dependence of the dynamical transition in proteins 3598. Comparing neutron scattering with other techniques 3599. Biological relevance 3609.1 Dynamics–activity relations 3609.2 Dynamics–stability relations (adaptation to extreme environments) 3609.3 Protein folding 36110. Acknowledgements 36411. References 364This 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 (∼1 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°C, to 0.6 N/m for the hyperthermophiles (85°C), 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.

Protein thermal dynamics was evaluated by neutron scattering for halophilic malate dehydrogenase from Haloarcula marismortui ( Hm MalDH) and BSA under different solvent conditions. As a measure of thermal stability in each case, loss of secondary structure temperatures were determined by CD. Hm MalDH 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 Hm MalDH, resilience increased progressively with increasing stability, from molar NaCl in H ₂ O, via molar KCl in D ₂ O, to molar NaCl in D ₂ O. Surprisingly, however, the opposite was observed for BSA; its resilience is higher in H ₂ O where it is less stable than in D ₂ O. These results confirmed the complexity of dynamics–stability relationships in different proteins. Softer dynamics for BSA in D ₂ O 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.

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 water-protons 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 ≈76% 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.

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.

An integrated picture of hydration shell dynamics and of its coupling to functional macromolecular motions is proposed from studies on a soluble protein, on a membrane protein in its natural lipid environment, and on the intracellular environment in bacteria and red blood cells. Water dynamics in multimolar salt solutions was also examined, in the context of the very slow water component previously discovered in the cytoplasm of extreme halophilic archaea. The data were obtained from neutron scattering by using deuterium labelling to focus on the dynamics of different parts of the complex systems examined.