Conformational dynamics of cytochrome c: correlation to hydrogen exchange.

We study the dynamical fluctuations of horse heart cytochrome c by molecular dynamics (MD) simulations in aqueous solution, at four temperatures: 300 K, 360 K, 430 K, and 550 K. Each simulation covers a production time of at least 1.5 nanoseconds (ns). The conformational dynamics of the system is analyzed in terms of collective motions that involve the whole protein, and local motions that involve the formation and breaking of intramolecular hydrogen bonds. The character of the MD trajectories can be described within the framework of rugged energy landscape dynamics. The MD trajectories sample multiple conformational minima, with basins in protein conformational space being sampled for a few hundred picoseconds. The trajectories of the system in configurational space can be described in terms of diffusion of a particle in real space with a waiting time distribution due to partial trapping in shallow minima. As a consequence of the hierarchical nature of the dynamics, the mean square displacement autocorrelation function, <|x(t) - x(0)|2>, exhibits a power law dependence on time, with an exponent of around 0.5 for times shorter than 100 ps, and an exponent of 1.75 for longer times. This power law behavior indicates that the system exhibits suppressed diffusion (sub-diffusion) in sampling of configurational space at time scales shorter than 100 ps, and enhanced (super-diffusion) at longer time scales. The multi-basin feature of the trajectories is present at all temperatures simulated. Structural changes associated with inter-basin displacements correspond to collective motions of the Omega loops and coiled regions and relative motions of the alpha-helices as rigid bodies. Similar motions may be involved in experimentally observed amide hydrogen exchange. However, some groups showing large correlated motions do not expose the amino hydrogens to the solvent. We show that large fluctuations are not necessarily correlated to hydrogen exchange. For example, regions of the proteins forming alpha helices and turns show significant fluctuations, but as rigid bodies, and the hydrogen bonds involved in the formation of these structures do not break in proportion to these fluctuations. Proteins 1999;36:175-191. Published 1999 Wiley-Liss, Inc.     

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.     

Role of water on unfolding kinetics of helical peptides studied by molecular dynamics simulations

Molecular dynamics simulations have been carried out with four polypeptides, Ala13, Val(13), Ser13, and Ala4Gly5Ala4, in vacuo and with explicit hydration. The unfolding of the polypeptides, which are initially fully alpha-helix in conformation, has been monitored during trajectories of 0.3 ns at 350 K. A rank of Ala < Val < Ser < Gly is found in the order of increasing rate of unwinding. The unfolding of Ala13 and Val(13) is completed in hundreds of picoseconds, while that of Ser13 is about one order of magnitude faster. The helix content of the peptide containing glycine residues falls to zero within a few picoseconds. Ramachandran plots indicate quite distinct equilibrium distributions and time evolution of dihedral angles in water and in vacuum for each residue type. The unfolding of polyalanine and polyvaline helices is accelerated due to solvation. In contrast, polyserine is more stable in water compared to vacuum, because its side chains can form intramolecular hydrogen bonds with the backbone more readily in vacuum, which disrupts the helix. Distribution functions of the spatial and angular position of water molecules in the proximity of the polypeptide backbone polar groups reveal the stabilization of the coiled structures by hydration. The transition from helix to coil is characterized by the appearance of a new peak in the probability distribution at a specific location characteristic of hydrogen bond formation between water and backbone polar groups. No significant insertion of water molecules is observed at the precise onset of unwinding, while (i, i+3) hydrogen bond formation is frequently detected at the initiation of alpha-helix unwinding.     

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.     

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.     

Potassium and sodium binding to the outer mouth of the K+ channel.

Molecular dynamics simulations of the K+ channel from Streptomyces lividans (KcsA channel) were performed in a membrane-mimetic environment with Na+ and K+ in different initial locations. The structure of the channel remained stable and well preserved for simulations lasting up to 1.5 ns. Salt bridges between Asp80 and Arg89 of neighboring subunits, not detected in the X-ray structure, enhanced the stability of the tetrameric structure. Na+ or K+ ions located in the channel vestibule lost part of their hydration shell and diffused into the channel inner pore in less than a few hundred picoseconds. This powerful catalytic action was caused by strong electrostatic interactions with Asp80 and Glu71. The hydration state of the metal ions turned out to depend significantly on the conformational flexibility of the channel. Furthermore, Na+ entered the channel inner pore bound to more water molecules than K+. The different hydration state of the two ions may be a determinant factor in the ion selectivity of the channel.     

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.     

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.     

Amplitudes and time scales of picosecond-to-microsecond motion in proteins studied by solid-state NMR: a critical evaluation of experimental approaches and application to crystalline ubiquitin

Solid-state NMR provides insight into protein motion over time scales ranging from picoseconds to seconds. While in solution state the methodology to measure protein dynamics is well established, there is currently no such consensus protocol for measuring dynamics in solids. In this article, we perform a detailed investigation of measurement protocols for fast motions, i.e. motions ranging from picoseconds to a few microseconds, which is the range covered by dipolar coupling and relaxation experiments. We perform a detailed theoretical investigation how dipolar couplings and relaxation data can provide information about amplitudes and time scales of local motion. We show that the measurement of dipolar couplings is crucial for obtaining accurate motional parameters, while systematic errors are found when only relaxation data are used. Based on this realization, we investigate how the REDOR experiment can provide such data in a very accurate manner. We identify that with accurate rf calibration, and explicit consideration of rf field inhomogeneities, one can obtain highly accurate absolute order parameters. We then perform joint model-free analyses of 6 relaxation data sets and dipolar couplings, based on previously existing, as well as new data sets on microcrystalline ubiquitin. We show that nanosecond motion can be detected primarily in loop regions, and compare solid-state data to solution-state relaxation and RDC analyses. The protocols investigated here will serve as a useful basis towards the establishment of a routine protocol for the characterization of ps–μs motions in proteins by solid-state NMR.     

Hydration effect on low-frequency protein dynamics observed in simulated neutron scattering spectra

Hydration effects on protein dynamics were investigated by comparing the frequency dependence of the calculated neutron scattering spectra between full and minimal hydration states at temperatures between 100 and 300 K. The protein boson peak is observed in the frequency range 1-4 meV at 100 K in both states. The peak frequency in the minimal hydration state shifts to lower than that in the full hydration state. Protein motions with a frequency higher than 4 meV were shown to undergo almost harmonic motion in both states at all temperatures simulated, whereas those with a frequency lower than 1 meV dominate the total fluctuations above 220 K and contribute to the origin of the glass-like transition. At 300 K, the boson peak becomes buried in the quasielastic contributions in the full hydration state but is still observed in the minimal hydration state. The boson peak is observed when protein dynamics are trapped within a local minimum of its energy surface. Protein motions, which contribute to the boson peak, are distributed throughout the whole protein. The fine structure of the dynamics structure factor is expected to be detected by the experiment if a high resolution instrument (<∼20 μeV) is developed in the near future.     

The influence of hydration on the conformation of lysozyme studied by solid-state 13C-nmr spectroscopy

¹³C proton decoupled cross-polarization magic-angle spinning nmr spectra of lysozyme are reported as a function of hydration. Increases in hydration level enhance the resolution of the spectra, particularly in the aliphatic region, but has no significant effect on either the rotating frame proton spin–lattice relaxation time or the cross-relaxation time. The enhancement in spectral resolution with hydration is attributed to a decrease in the distribution of isotropic chemical shifts, which reflects a decrease in the distribution of conformational states sampled by the protein. Changes in the distribution of isotropic chemical shifts occur after the addition of water to the charged groups as coverage of the polar side chains and peptide groups takes place. The onset of this behavior occurs at a hydration level of about, 0.1–0.2 g water/g protein and is largely complete at about 0.3 g water/g protein, the same hydration range where changes in the heat capacity are observed. That hydrogen exchange of buried protons can occur at hydration levels significantly lower than those at which changes in the distribution of conformational states are first observed suggests that some motions that mediate exchange are already present in the dry protein. The preservation of efficient dipolar coupling indicates that the conformational rearrangements that do-occur on hydration are small and do not involve any significant overall expansion of free volume or weakening of interactions that would increase the reorientational freedom of protein groups. © 1993 John Wiley & Sons, Inc.     

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.     

How long does DNA keep the memory of its conformation? A time-dependent canonical correlation analysis of molecular dynamics simulation

The time dependence of the correlation between motions of different parts of DNA is analyzed from a 200 ps molecular dynamics simulation of the double-stranded self-complementary d(CTGATCAG) in the B form. Each nucleotide is decomposed into three subunits corresponding to the furanose ring (SU), the base (BA), and the backbone (SK). The motion of each subunit is considered as the superimposition of rigid body translation, rigid body rotation, and internal deformation. Canonical time-dependent correlation functions calculated with coordinates describing the different components of the subunits motion are defined and computed. This allows us to probe how long a particular type of motion of one subunit influences the other types of motions of other subunits (cross correlation functions) or how long a particular subunit keeps the memory of its own conformation or location (autocorrelation functions). From auto-correlation analysis it is found that deformation decorrelates within a few tenths of picoseconds, rotational correlation times are on the order of 8 ps, while translational motions are long-time correlated. The deformation of a subunit is not correlated to the deformation of another one (at the 200 ps time scale of our simulation), but influences slightly their translation and orientation as time increases. © 1996 John Wiley & Sons, Inc.     

Hydration dependence of backbone and side chain polylysine dynamics: a 13C solid-state NMR and IR spectroscopy study

The molecular dynamics of solid poly-L-lysine has been studied by the following natural abundance (13)C-NMR relaxation methods: measurements of the relaxation times T(1) at two resonance frequencies, off-resonance T(1rho) at two spin-lock frequencies, and proton-decoupled T(1rho). Experiments were performed at different temperatures and hydration levels (up to 17% H(2)O by weight). The natural abundance (13)C-CPMAS spectrum of polylysine provides spectral resolution of all types of backbone and side chain carbons and thus, dynamic parameters could be determined separately for each of them. At the same time, the conformational properties of polylysine were investigated by Fourier transform infrared spectroscopy. The data obtained from the different NMR experiments were simultaneously analyzed using the correlation function formalism and model-free approach. The results indicate that in dry polylysine both backbone and side chains take part in two low amplitude motions with correlation times of the order of 10(-4) s and 10(-9) s. Upon hydration, the dynamic parameters of the backbone remain almost constant except for the amplitude of the slower process that increases moderately. The side chain dynamics reveals a much stronger hydration response: the amplitudes of both slow and fast motions increase significantly and the correlation time of the slow motion shortens by about five orders of magnitude, and at hydration levels of more than 10% H(2)O fast and slow side chain motions are experimentally indistinguishable. These changes in the molecular dynamics cannot be ascribed to any hydration-dependent conformational transitions of polylysine because IR spectra reveal almost no hydration dependence in either backbone or side chain absorption domains. The physical nature of the fast and slow motions, their correlation time distributions, and hydration dependence of microdynamic parameters are discussed.     

Water penetration and escape in proteins

The kinetics of water penetration and escape in cytochrome c (cyt c) is studied by molecular dynamics (MD) simulations at various temperatures. Water molecules that penetrate the protein interior during the course of an MD simulation are identified by monitoring the number of water molecules in the first coordination shell (within 3.5 A) of each water molecule in the system. Water molecules in the interior of cyt c have 0-3 water molecules in their first hydration shell and this coordination number persists for extended periods of time. At T = 300 K we identify over 200 events in which water molecules penetrate the protein and reside inside for at least 5 picoseconds (ps) within a 1.5 nanoseconds (ns) time period. Twenty-seven (27) water molecules reside for at least 300 ps, 17 water molecules reside in the protein interior for times longer than 500 ps, and two interior water molecules do not escape; at T = 360 K one water molecule does not escape; at 430 K all water molecules exchange. Some of the internal water molecules show mean square displacements (MSD) of 1 A2 characteristic of structural waters. Others show MSD as large as 12 A2, suggesting that some of these water molecules occupy transient cavities and diffuse extensively within the protein. Motions of protein-bound water molecules are rotationally hindred, but show large librations. Analysis of the kinetics of water escape in terms of a survival time correlation function shows a power law behavior in time that can be interpreted in terms of a broad distribution of energy barriers, relative to kappa BT, for water exchange. At T = 300 K estimates of the roughness of the activation energy distribution is 4-10 kJ/mol (2-4 kappa BT). Activation enthalpies for water escape are 6-23 kJ/mol. The difference in activation entropies between fast exchanging (0.01 ns) and slow exchanging (0.1-1 ns) water molecules is -27 J/K/mol. Dunitz (Science 1997;264:670.) has estimated the maximum entropy loss of a water molecule due to binding to be 28 J/K/mol. Therefore, our results suggest that the entropy of interior water molecules is similar to entropy of bulk water.     

Molecular dynamics study of onset of water gelation around the collagen triple helix

The onset of water gelation around a collagen-like triple helix peptide was studied at ambient temperature and pressure by performing Molecular Dynamics simulations. The radial distribution functions of the oxygen and hydrogen atoms of water are distorted below 4 A from the peptide. The distortion is accompanied by the breakdown of the tetrahedral coordination of the hydrogen-bonded network of water molecules. The water shell around the peptide consists of alternating regions of higher and lower density. In agreement with experiments we find that the first hydration shell is kinetically labile, with a residence time in the order of picoseconds for a water molecule. From the computed diffusion coefficient, a key measure of the collective dynamics, we estimate the average diffusion speed decreases by a factor of 1.5 close to the peptide compared to the liquid. Our results give new insight in gel formation and structure on a molecular level.     

Effect of hydration on the molecular dynamic properties of human serum albumin at low temperatures

The effect of temperature and hydration on phosphorescence of chromatophores and on saturation curves of ESR spectra of spin labels covalently bound to human serum albumin was studied. It has been shown that at 90-260 degrees K albumin hydration results in intensification of motions of hydrophobic parts with low frequencies (vc less than or equal to 10(3) s-1) and does not affect the motions of hydrophobic and surfacial parts with high frequency.     

Hydration dependent dynamics in RNA

The essential role played by local and collective motions in RNA function has led to a growing interest in the characterization of RNA dynamics. Recent investigations have revealed that even relatively simple RNAs experience complex motions over multiple time scales covering the entire ms–ps motional range. In this work, we use deuterium solid-state NMR to systematically investigate motions in HIV-1 TAR RNA as a function of hydration. We probe dynamics at three uridine residues in different structural environments ranging from helical to completely unrestrained. We observe distinct and substantial changes in ²H solid-state relaxation times and lineshapes at each site as hydration levels increase. By comparing solid-state and solution state ¹³C relaxation measurements, we establish that ns–μs motions that may be indicative of collective dynamics suddenly arise in the RNA as hydration reaches a critical point coincident with the onset of bulk hydration. Beyond that point, we observe smaller changes in relaxation rates and lineshapes in these highly hydrated solid samples, compared to the dramatic activation of motion occurring at moderate hydration.     

Hydration-coupled dynamics in proteins studied by neutron scattering and NMR: the case of the typical EF-hand calcium-binding parvalbumin

The influence of hydration on the internal dynamics of a typical EF-hand calciprotein, parvalbumin, was investigated by incoherent quasi-elastic neutron scattering (IQNS) and solid-state 13C-NMR spectroscopy using the powdered protein at different hydration levels. Both approaches establish an increase in protein dynamics upon progressive hydration above a threshold that only corresponds to partial coverage of the protein surface by the water molecules. Selective motions are apparent by NMR in the 10-ns time scale at the level of the polar lysyl side chains (externally located), as well as of more internally located side chains (from Ala and Ile), whereas IQNS monitors diffusive motions of hydrogen atoms in the protein at time scales up to 20 ps. Hydration-induced dynamics at the level of the abundant lysyl residues mainly involve the ammonium extremity of the side chain, as shown by NMR. The combined results suggest that peripheral water-protein interactions influence the protein dynamics in a global manner. There is a progressive induction of mobility at increasing hydration from the periphery toward the protein interior. This study gives a microscopic view of the structural and dynamic events following the hydration of a globular protein.