A Connected-cluster of hydration around myoglobin: Correlation between molecular dynamics simulations and experiment

An analysis of a molecular dynamics simulation of metmyoglobin in an explicit solvent environment of 3,128 water molecules has been performed. Both statics and dynamics of the protein-solvent interface are addressed in a comparison with experiment. Three-dimensional density distributions, temperature factors, and occupancy weights are computed for the solvent by using the trajectory coordinates. Analysis of the hydration leads to the localization of more than 500 hydration sites distributed into multiple layers of solvation located between 2.6 and 6.8 Å from the atomic protein surface. After locating the local solvent density maxima or hydration sites we conclude that water molecules of hydration positions and hydration sites are distinct concepts. Both global and detailed properties of the hydration cluster around myoglobin are compared with recent neutron and X-ray data on myoglobin. Questions arising from differences between X-ray and neutron data concerning the locations of the protein-bound water are investigated. Analysis of water site differences found from X-ray and neutron experiments compared with our simulation shows that the simulation gives a way to unify the hydration picture given by the two experiments. © 1994 John Wiley & Sons, Inc.     

Orientational and rotational velocity correlation functions for water-hydrating B- and Z-DNA double helices

The molecular dynamics simulations reported earlier for the structure and dynamics of water molecules hydrating B- and Z-DNA double helices are analyzed for the orientational correlation functions and the proton rotational velocity autocorrelation functions. The spectra of the rotational velocity autocorrelation functions obtained from the simulation results are compared with the neutron inelastic scattering experiments on hydrated Na-DNA samples. The results predict a small frequency component associated with water molecules bound to the double helices that disappears for waters away from the double helix.     

SANS study of the distribution of water within starch granules

This study describes contrast variation small angle neutron scattering (SANS) experiments which focus on the role which the intra-granular room temperature distribution of water and carbohydrate plays in determining the native structure and subsequent functionality of starch. It is shown that variations in botanical origin and amylose content do not correlate with significant differences in room temperature composition of A-type starch granules. In turn, variations in the gelatinisation behaviour of A-type starches do not correlate with variations in room temperature water distribution. In contrast, the room temperature water content is found to differ significantly between granules of potato (B-type) and a range of A-type starch cultivars. A correlation is found between these compositional differences and variations in crystal structure, which has implications for biological growth conditions and gelatinisation behaviour.     

C-phycocyanin hydration water dynamics in the presence of trehalose: an incoherent elastic neutron scattering study at different energy resolutions

We present a study of C-phycocyanin hydration water dynamics in the presence of trehalose by incoherent elastic neutron scattering. By combining data from two backscattering spectrometers with a 10-fold difference in energy resolution we extract a scattering law S(Q,omega) from the Q-dependence of the elastic intensities without sampling the quasielastic range. The hydration water is described by two dynamically different populations--one diffusing inside a sphere and the other diffusing quasifreely--with a population ratio that depends on temperature. The scattering law derived describes the experimental data from both instruments excellently over a large temperature range (235-320 K). The effective diffusion coefficient extracted is reduced by a factor of 10-15 with respect to bulk water at corresponding temperatures. Our approach demonstrates the benefits and the efficiency of using different energy resolutions in incoherent elastic neutron scattering over a large angular range for the study of biological macromolecules and hydration water.     

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.     

The denaturation transition of DNA in mixed solvents

The helix-to-coil denaturation transition in DNA has been investigated in mixed solvents at high concentration using ultraviolet light absorption spectroscopy and small-angle neutron scattering. Two solvents have been used: water and ethylene glycol. The "melting" transition temperature was found to be 94 degrees C for 4% mass fraction DNA/d-water and 38 degrees C for 4% mass fraction DNA/d-ethylene glycol. The DNA melting transition temperature was found to vary linearly with the solvent fraction in the mixed solvents case. Deuterated solvents (d-water and d-ethylene glycol) were used to enhance the small-angle neutron scattering signal and 0.1M NaCl (or 0.0058 g/g mass fraction) salt concentration was added to screen charge interactions in all cases. DNA structural information was obtained by small-angle neutron scattering, including a correlation length characteristic of the inter-distance between the hydrogen-containing (desoxyribose sugar-amine base) groups. This correlation length was found to increase from 8.5 to 12.3 A across the melting transition. Ethylene glycol and water mixed solvents were found to mix randomly in the solvation region in the helix phase, but nonideal solvent mixing was found in the melted coil phase. In the coil phase, solvent mixtures are more effective solvating agents than either of the individual solvents. Once melted, DNA coils behave like swollen water-soluble synthetic polymer chains.     

Low-temperature molecular dynamics simulations of horse heart cytochrome c and comparison with inelastic neutron scattering data

Molecular dynamics (MD) simulation combined with inelastic neutron scattering can provide information about the thermal dynamics of proteins, especially the low-frequency vibrational modes responsible for large movement of some parts of protein molecules. We performed several 30-ns MD simulations of cytochrome c (Cyt c) in a water box for temperatures ranging from 110 to 300 K and compared the results with those from experimental inelastic neutron scattering. The low-frequency vibrational modes were obtained via dynamic structure factors, S(Q, ω), obtained both from inelastic neutron scattering experiments and calculated from MD simulations for Cyt c in the same range of temperatures. The well known thermal transition in structural movements of Cyt c is clearly seen in MD simulations; it is, however, confined to unstructured fragments of loops Ω₁ and Ω₂; movement of structured loop Ω₃ and both helical ends of the protein is resistant to thermal disturbance. Calculated and experimental S(Q, ω) plots are in qualitative agreement for low temperatures whereas above 200 K a boson peak vanishes from the calculated plots. This may be a result of loss of crystal structure by the protein–water system compared with the protein crystal.     

Effect of conformational states on protein dynamical transition

In order to examine the properties specific to the folded protein, the effect of the conformational states on protein dynamical transition was studied by incoherent elastic neutron scattering for both wild type and a deletion mutant of staphylococcal nuclease. The deletion mutant of SNase which lacks C-terminal 13 residues takes a compact denatured structure, and can be regarded as a model of intrinsic unstructured protein. Incoherent elastic neutron scattering experiments were carried out at various temperature between 10 K and 300 K on IN10 and IN13 installed at ILL. Temperature dependence of mean-square displacements was obtained by the q-dependence of elastic scattering intensity. The measurements were performed on dried and hydrated powder samples. No significant differences were observed between wild type and the mutant for the hydrated samples, while significant differences were observed for the dried samples. A dynamical transition at ∼ 140 K observed for both dried and hydrated samples. The slopes of the temperature dependence of MSD before transition and after transition are different between wild type and the mutant, indicating the folding induces hardening. The hydration water activates a further transition at ∼ 240 K. The behavior of the temperature dependence of MSD is indistinguishable for wild type and the mutant, indicating that hydration water dynamics dominate the dynamical properties.     

Cation redistribution upon dehydration of Na58Y faujasite zeolite: a joint neutron diffraction and molecular simulation study

Using neutron scattering and Monte Carlo simulation, we investigate the distribution of cations in Na₅₈Y faujasite upon (de)hydration. We introduce a new method for the assignment of cations to specific sites in molecular simulations from their local environment. This allows us to bypass the need of the coordinates of crystallographic sites, which vary as water adsorption induces changes in the zeolite framework structure. Although the agreement between experiments and simulation is excellent at high temperature, some differences are observed below 150°C. We show that these differences are due to the presence of water and that temperature itself as well as adsorption-induced deformation of the framework play a less important role. We demonstrate the migration of sodium to sites III upon water adsorption, not observed for other Si:Al ratios.     

Hydration of NaDNA by neutron quasi-elastic scattering.

Preliminary results of neutron quasi-elastic scattering experiments are reported for hydrated paracrystals of sodium deoxyribonucleic acid (NaDNA). The samples were investigated at two water contents: 3.5 +/- 1.0 and 9.5 +/- 1.5 mol H2O per mole nucleotide. The results of the scattering experiments were almost independent of whether the NaDNA fibers were oriented parallel or perpendicular to the momentum transfer. The data indicate that at the lower hydration the water molecules do not diffuse appreciably on the time scale of the neutron measurements (approximately 3 X 10(-10) s). At the higher hydration the water molecules diffuse isotropically in a sphere of 9 A in diameter with a diffusion coefficient of (5 +/- 2) X 10(-6) cm2 s-1.     

Thickness measurements of single walled dimyristoyl phosphatidylcholine vesicles by neutron scattering

A method of deriving by neutron scattering thicknesses of lamellae in suspensions has been applied to single-walled vesicles of dimyristoyl phosphatidylcholine. The contrast variation method, based on data obtained for a range of isotope mixtures, has been used to extract a dimension Dw related to the lipid bilayer thickness and a measure alpha of the difference of density within the lamellae. Isotope mixtures for the lipid were used to optimize the information available. Dw is compared with results from multilayer stacks of lipid layers. The thickness for the low temperature L beta, structure has been observed to be higher than for the high temperature L alpha structure. Preliminary experiments on the kinetics of the mixing of the lipid isotope species are reported, and evidence is shown that the species are not segregated for lipids either above or below the transition temperature.     

Effect of Irradiation on Microorganisms in a Nuclear Reactor

The effects of irradiation in the JRR-1 (Japan Research Reactor No. 1, a homogeneous light water nuclear reactor; max. power, 50 KW) on microorganisms such as bacterial and fungal spores and yeast cells were investigated in comparison with those of ⁶⁰Co gamma radiation. As far as the lethal effect was concerned the dose rate of radiation in the experimental hole No. 16 of the JRR-1 was equivalent to 3.0×l0⁶~3.4×l0⁶ r/hr with ⁶⁰Co gamma radiation, and a ratio of the neutron effect to the gamma radiation effect on microorganisms in this hole was estimated to be approximately 3~5.4. The results different from those with gamma radiation were obtained in experiments such as post-NaCl treatment and spore germination. The considerable contribution of fast neutrons to the total biological effect of neutrons, in comparison with the thermal neutron effect, could be presumed from the microbiological experiments with the help of physical and chemical data. Morphological changes in post-irradiation growth were observed by means of phase contrast microscopy. No specific aftereffect was found.     

Liquid-like water confined in stacks of biological membranes at 200 k and its relation to protein dynamics

Confined water is of considerable current interest owing to its biophysical importance and relevance to cryopreservation. It can be studied in its amorphous or supercooled state in the "no-man's land", i.e., in the temperature range between 150 and 235 K, in which bulk water is always crystalline. Amorphous deuterium oxide (D(2)O) was obtained in the intermembrane spaces of a stack of purple membranes from Halobacterium salinarum by flash cooling to 77 K. Neutron diffraction showed that upon heating to 200 K the intermembrane water space decreased sharply with an associated strengthening of ice diffraction, indicating that water beyond the first membrane hydration layer flowed out of the intermembrane space to form crystalline ice. It was concluded that the confined water undergoes a glass transition at or below 200 K to adopt an ultraviscous liquid state from which it crystallizes to form ice as soon as it finds itself in an unconfined, bulk-water environment. Our results provide model-free evidence for translational diffusion of confined water in the no-man's land. Potential effects of the confined-water glass transition on nanosecond membrane dynamics were investigated by incoherent elastic neutron scattering experiments. These revealed no differences between flash-cooled and slow-cooled samples (in the latter, the intermembrane space at temperatures <250 K is occupied only by the first membrane hydration layers), with dynamical transitions at 150 and 260 K, but not at 200 K, suggesting that nanosecond membrane dynamics are not sensitive to the state of the water beyond the first hydration shell at cryotemperatures.     

Nanostructures by self-assembling peptide amphiphile as potential selective drug carriers

The self-assembling behavior, at physiological pH, of the amphiphile peptide (C18)(2)L5CCK8 in nanostructures is reported. Stable aggregates presenting a critical micellar concentration of 2 x 10(-6) mol kg(-1), and characterized by water exposed CCK8 peptide in beta-sheet conformation, are obtained. Small angle neutron scattering experiments are indicative for a 3D structure with dimensions > or =100 nm. AFM images confirm the presence of nanostructures. Fluorescence experiments indicating the sequestration of pyrene, chosen as drug model, and the anticancer Doxorubicin within the nanostructures are reported.     

Experimental study of the dynamics of neutron emission from the GOL-3 multimirror trap

In experiments on the plasma heating and confinement in the GOL-3 multimirror trap, a deuterium plasma with a density of ～10¹⁵ cm^?3 and an ion temperature of 1–2 keV is confined for more than 1 ms. The plasma is heated by a relativistic electron beam. The ion temperature, which was measured by independent methods, reached 1.5–2 keV after the beginning of the beam injection. Since such a fast ion heating cannot be explained by the classical energy transfer from electrons to ions through binary collisions, a theoretical model of collective energy transfer was proposed. In order to verify this model, a new diagnostics was designed to study the dynamics of neutron emission from an individual mirror cell of the multimirror trap during electron beam injection. Intense neutron bursts predicted by this model were detected experimentally. Periodic neutron flux modulation caused by the macroscopic plasma flow along the solenoid was observed. The revealed mechanism of fast ion heating can be used to achieve fusion temperatures in the multimirror trap.     

Determination of bilayer thickness and lipid surface area in unilamellar dimyristoylphosphatidylcholine vesicles from small-angle neutron scattering curves: a comparison of evaluation methods

Small-angle neutron scattering (SANS) experiments were performed on unilamellar 1,2-dimyristoylphosphatidylcholine (DMPC) vesicles prepared in heavy water by extrusion through polycarbonate filters with 500 Å pores. The data obtained at 30±0.1 °C were evaluated using a five-strip function model of the bilayer coherent neutron scattering length density, three different approximate form factors describing scattering from vesicles, and different methods of evaluation of the experimental data. It is shown that the results obtained from the SANS data in the range of scattering vector values 0.0316 Å^–1<q<0.0775 Å^–1 are not sensitive to the vesicle form factor, nor to the evaluation method. Using the hollow sphere model of vesicles convoluted with the Gaussian distribution of their sizes, a constrained bilayer polar region thickness of 9 Å and a DMPC headgroup volume of 325.5 ų, it was possible to obtain from the experimental data the DMPC surface area as 58.9±0.8 Ų, the bilayer thickness as 44.5±0.3 Å and the number of water molecules as 6.8±0.2 per DMPC located in the bilayer polar region.     

Neutron frequency windows and the protein dynamical transition

Proteins undergo an apparent dynamical transition on temperature variation that has been correlated with the onset of function. The transition in the mean-square displacement, , that is observed using a spectrometer or computer simulation, depends on the relationship between the timescales of the relaxation processes activated and the timescale accessible to the instrument or simulation. Models are described of two extreme situations---an "equilibrium" model, in which the long-time dynamics changes with temperature and all motions are resolved by the instrument used; and a "frequency window" model, in which there is no change in the long-time dynamics but as the temperature increases, the relaxation frequencies move into the instrumental range. Here we demonstrate that the latter, frequency-window model can describe the temperature and timescale dependences of both the intermediate neutron scattering function and derived from molecular dynamics simulations of a small protein in a cryosolution. The frequency-window model also describes the energy-resolution and temperature-dependences of obtained from experimental neutron scattering on glutamate dehydrogenase in the same solvent. Although equilibrium effects should also contribute to dynamical transitions in proteins, the present results suggests that frequency-window effects can play a role in the simulations and experiments examined. Finally, misquotations of previous findings are discussed in the context of solvent activation of protein dynamics and the possible relationship of this to activity.     

Biophysical aspects of neutron scattering from vibrational modes of proteins

This review describes a major portion of the published work on neutron scattering experiments aimed at measuring large scale motions in proteins. The importance of these motions for enzyme function and oxygen transport is indicated. The theory applicable to each type of neutron scattering measurement is given and results are discussed with a view to biological relevance. New experiments are suggested and a comparison of neutron scattering data is made with results from other techniques such as raman scattering, infrared absorption, photolysis and molecular dynamics simulations.     

Vibrational neutron spectroscopy of collagen and model polypeptides.

A pulsed source neutron spectrometer has been used to measure vibrational spectra (20-4000 cm-1) of dry and hydrated type I collagen fibers, and of two model polypeptides, polyproline II and (prolyl-prolyl-glycine)10, at temperatures of 30 and 120 K. the collagen spectra provide the first high resolution neutron views of the proton-dominated modes of a protein over a wide energy range from the low frequency phonon region to the rich spectrum of localized high frequency modes. Several bands show a level of fine structure approaching that of optical data. The principal features of the spectra are assigned. A difference spectrum is obtained for protein associated water, which displays an acoustic peak similar to pure ice and a librational band shifted to lower frequency by the influence of the protein. Hydrogen-weighted densities of states are extracted for collagen and the model polypeptides, and compared with published calculations. Proton mean-square displacements are calculated from Debye-Waller factors measured in parallel quasi-elastic neutron-scattering experiments. Combined with the collagen density of states function, these yield an effective mass of 14.5 a.m.u. for the low frequency harmonic oscillators, indicating that the extended atom approximation, which simplifies analyses of low frequency protein dynamics, is appropriate.     

Solvent structure in crystals of trypsin determined by X-ray and neutron diffraction.

The solvent structure in orthorhombic crystals of bovine trypsin has been independently determined by X-ray diffraction to 1.35 A resolution and by neutron diffraction to 2.1 A resolution. A consensus model of the water molecule positions was obtained using oxygen positions identified in the electron density map determined by X-ray diffraction, which were verified by comparison to D2O-H2O difference neutron scattering density. Six of 184 water molecules in the X-ray structure, all with B-factors greater than 50 A2, were found to be spurious after comparison with neutron results. Roughly two-thirds of the water of hydration expected from thermodynamic data for proteins was localized by neutron diffraction; approximately one-half of the water of hydration was located by X-ray diffraction. Polar regions of the protein are well hydrated, and significant D2O-H2O difference density is seen for a small number of water molecules in a second shell of hydration. Hydrogen bond lengths and angles calculated from unconstrained refinement of water positions are distributed about values typically seen in small molecule structures. Solvent models found in seven other bovine trypsin and trypsinogen and rat trypsin structures determined by X-ray diffraction were compared. Internal water molecules are well conserved in all trypsin structures including anionic rat trypsin, which is 65% homologous to bovine trypsin. Of the 22 conserved waters in trypsin, 19 were also found in trypsinogen, suggesting that they are located in regions of the apoprotein that are structurally conserved in the transition to the mature protein. Seven waters were displaced upon activation of trypsinogen. Water structure at crystal contacts is not generally conserved in different crystal forms. Three groups of integral structural water molecules are highly conserved in all solvent structures, including a spline of water molecules inserted between two beta-strands, which may resemble an intermediate in the formation of beta sheets during the folding of a protein.