Structural fluctuations in proteins on the picosecond timescale have been studied in considerable detail by theoretical methods such as molecular dynamics simulation, but there exist very few experimental data with which to test the conclusions. We have used the technique of inelastic neutron scattering to investigate atomic motion in hydrated myoglobin over the temperature range 4-350 K and on the molecular dynamics timescale 0.1-100 ps. At temperatures below 180 K myoglobin behaves as a harmonic solid, with essentially only vibrational motion. Above 180 K there is a striking dynamic transition arising from the excitation of nonvibrational motion, which we interpret as corresponding to torsional jumps between states of different energy, with a mean energy asymmetry of 12 kJ mol-1. This extra mobility is reflected in a strong temperature dependence of the mean-square atomic displacements, a phenomenon previously observed specifically for the heme iron by Mössbauer spectroscopy, but on a much slower timescale (10(-7) s). It also correlates with a glass-like transition in the hydration shell of myoglobin and with the temperature-dependence of ligand-binding rates at the heme iron, as monitored by flash photolysis. In contrast, the crystal structure of myoglobin determined down to 80 K shows no significant structural transition. The dynamical behaviour we find for myoglobin (and other globular proteins) suggests a coupling of fast local motions to slower collective motions, which is a characteristic feature of other dense glass-forming systems.

No abstract

No abstract

No abstract

Thin layers of water on biomolecular and other nanostructured surfaces can be supercooled to temperatures not accessible with bulk water. Chen et al. [PNAS 103, 9012 (2006)] suggested that anomalies near 220 K observed by quasi-elastic neutron scattering can be explained by a hidden critical point of bulk water. Based on more sensitive measurements of water on perdeuterated phycocyanin, using the new neutron backscattering spectrometer SPHERES, and an improved data analysis, we present results that show no sign of such a fragile-to-strong transition. The inflection of the elastic intensity at 220 K has a dynamic origin that is compatible with a calorimetric glass transition at 170 K. The temperature dependence of the relaxation times is highly sensitive to data evaluation; it can be brought into perfect agreement with the results of other techniques, without any anomaly. In contrast to bulk water, protein hydration water can be supercooled down to a glass transition at T g ≃ 170 K. Near T g translational degrees of freedom arrest, which induces discontinuities in the specific heat and the thermal expansion coefficient of the hydration water [1][2][3][4]. Due to the dynamic nature of the glass transition, freezing of microscopic degrees of freedom can already be observed far above T g .The protein dynamic transition is an abrupt onset of atomic displacements on the microscopic length and time scale probed by quasielastic neutron scattering (QENS). First observed twenty years ago in hydrated myoglobin and lysozyme at T ∆ ≃ 240 K [5], it is now known to be a generic property of hydrated proteins, while it is absent in dehydrated systems. It is therefore related to the dynamics of the hydration shell.In QENS, mean squared dispacements δx 2 are deduced from the elastic scattering intensity S(q, 0). Due to the finite spectrometer resolution (fwhm 2∆ω), one actually measures S(q, |ω| ∆ω). Full spectral measurements show that the anomalous decrease of the central peak is compensated by increasing inelastic wings [5,6]. These effects have been interpreted as precursors of the glass transition. Since QENS probes structural relaxation at ∆ω −1 ≃ 100 ps, while the calorimetric T g refers to a time scale of 100 s, it is natural that T ∆ is located far above T g : the protein dynamic transition is the microscopic manifestation of the glass transition in the hydration shell. The time-scale dependence of T ∆ also explains its variation with viscosity [7][8][9]. * Electronic address: wdoster@ph.tum.de Recently, these views have been challenged by the suggestion that there might be a time-scale independent transition. Support came mainly from QENS experiments on the backscattering spectrometer HFBS at NIST. In hydrated lysozyme, DNA and RNA, a kink was found not only in S(q, |ω| ∆ω), but also in the α relaxation time τ deduced from full spectra S(q, ω) [10][11][12][13]. This change of τ (T ) from high-T super-Arrhenius to low-T Arrhenius behavior at T L ≃ 220 K has been interpreted as a fragile-to-strong-transition (FST) from the...

The dynamics of hydrated proteins and of protein crystals can be studied within a wide temperature range, since the water of hydration does not crystallize at low temperature. Instead it turns into an amorphous glassy state below 200 K. Extending the temperature range facilitates the spectral separation of different molecular processes. The conformational motions of proteins show an abrupt enhancement near 180 K, which has been called a "dynamical transition". In this contribution various aspects of the transition are critically reviewed: the role of the instrumental resolution function in extracting displacements from neutron elastic scattering data and the question of the appropriate dynamic model, discrete transitions between states of different energy versus continuous diffusion inside a harmonic well, are discussed. A decomposition of the transition involving two motional components is performed: rotational transitions of methyl groups and small scale librations of side-chains, induced by water at the protein surface. Both processes create an enhancement of the observed amplitude. The onset occurs, when their time scale becomes compatible with the resolution of the spectrometer. The reorientational rate of hydration water follows a super-Arrhenius temperature dependence, a characteristic feature of a dynamical transition. It occurs only with hydrated proteins, while the torsional motion of methyl groups takes place also in the dehydrated or solvent-vitrified system. Finally, the role of fast hydrogen bond fluctuations contributing to the amplitude enhancement is discussed.

The water of hydration in myoglobin crystals and solutions was studied at subzero temperatures by calorimetry and infrared spectroscopy (ir). For comparison we also investigated glycine, DL-alanine and DL-valine solutions. The hydration water remains amorphorus at low temperatures. We find a broad glass transition between 180 and 270 K depending on the degree of hydration. The ice component shows a noncolligative melting point depression that is attributed to a finite conformational flexibility. The ir spectrum and the specific heat of water in myoglobin crystals was determined for the first time between 180 and 290 K. The glass transition in crystals is qualitatively similar to what is found in amorphous samples at the same water content. These data are compared with Mossbauer experiments and dielectric relaxation of water in myoglobin crystals. The similar temperature dependencies suggest a cross correlation between structural fluctuations and the thermal motion of crystal water. A hydrogen bond network model is proposed to explain these features. The essential ingredients are cooperativity and a distribution of hydrogen-bonded clusters.

Conformational changes of proteins often involve the relative motion of rigid structural domains. Normal mode analysis and molecular dynamics simulations of small globular proteins predict delocalized vibrations with frequencies below 20 cm(-1), which may be overdamped in solution due to solvent friction. In search of these modes, we have studied deuterium-exchanged myoglobin and lysozyme using inelastic neutron scattering in the low-frequency range at full and low hydration to modify the degree of damping. At room temperature, the hydrated samples exhibit a more pronounced quasielastic spectrum due to diffusive motions than the dehydrated samples. The analysis of the corresponding lineshapes suggests that water modifies mainly the amplitude, but not the characteristic time of fast protein motions. At low temperatures, in contrast, the dehydrated samples exhibit larger motional amplitudes than the hydrated ones. The excess scattering, culminating at 16 cm(-1), is suggested to reflect water-coupled librations of polar side chains that are depressed in the hydrated system by strong intermolecular hydrogen bonding. Both myoglobin and lysozyme exhibit ultra-low-frequency modes below 10 cm(-1) in the dry state, possibly related to the breathing modes predicted by harmonic analysis.