The structure of aqueous solutions of hexafluoro-iso-propanol studied by neutron diffraction with hydrogen/deuterium isotope substitution and empirical potential structure refinement modeling.

Neutron diffraction measurements of H/D isotopic substitution are made at room temperature for seven H/D substituted hexafluoro-iso-propanol (HFIP; 1,1,1,3,3,3-hexafluoro-2-propanol)-water mixtures at 0.1, 0.2, and 0.4 HFIP mole fraction (xHFIP). The eight partial structure factors except for the H(CH)-H(CH) pair obtained are subjected to an empirical potential structure refinement (EPSR) method to derive all site-site pair correlation functions. It is found that with increasing HFIP concentration the ice-like network of water disappears between xHFIP = 0.1 and 0.2, followed by the formation of a chain-like water structure embedded in an intrinsic structure of HFIP evolved at xHFIP = 0.4. The hydroxyl group of HFIP forms hydrogen bonds with the surrounding water molecules at all HFIP mole fractions investigated. There is no evidence that the water structure is well defined around the CF3 groups of HFIP, but water molecules surround tangentially the CF3 groups of HFIP.

Ion interactions with non-polar and amphiphilic solutes in water.

Despite its extensive use in, for example, the concentration and crystallization of proteins, the salting out phenomenon remains poorly understood, with many--sometimes contradictory--explanations found in the literature. Following our earlier work using isotope substitution neutron scattering on an aqueous tertiary butyl alcohol (TBA) with added NaCl that examined in detail the molecular-level interactions in the system, and suggested that attempts to understand salting out through ion perturbation of the non-polar hydration shell may not be appropriate, we report here on two further sets of high resolution structural experiments that detail the structural interactions between ion, solvent and amphiphile in a wider range of relevant systems. First, a set of X-ray absorption spectroscopy experiments probed the effects on the hydrophobic hydration shell of Kr of adding a range of different salts (specifically Na2SO4, NaClO4, NaCl, NaNO3 and Mg(ClO4)2). The bottom line from these experiments is that the hydration shell is essentially unaffected by the added ions, underlining further the stability of the shell to such perturbations. The second set of experiments was a further isotope substitution neutron scattering study of the molecular-level solution structures of aqueous TBA, this time for a range of different added ions (CsF, NaBr, and NaCl at three concentrations), for which we have in addition extracted equilibrium constants to quantify the relative strengths of the various interactions. From this second set of results, we can conclude again that the solvent structure is essentially unperturbed by both the various ions and the amphiphile, and also identify a number of ion-specific effects. Comparing our results with those obtained from simulations that are not constrained by the experimental data underlines how sensitive structural conclusions are to the assumed potential functions, and that drawing conclusions from simulations not constrained to fit experimental data can lead to seriously erroneous conclusions.

The relationship between solution structure and crystal nucleation: a neutron scattering study of supersaturated methanolic solutions of benzoic acid.

In this contribution, neutron scattering experiments (with isotopic substitution) of concentrated and supersaturated methanolic benzoic acid solutions combined with empirical potential structure refinement (EPSR) were used to investigate the time-averaged atomistic details of this system. Through the determination of radial distribution functions, quantitative details emerge of the solution coordination, its relationship to the nature of the crystalline phase, and the response of the solution to imposed supersaturation.

Relaxation effects in low density amorphous ice: two distinct structural states observed by neutron diffraction.

Neutron diffraction with H/D isotopic substitution is used to investigate the structure of low density amorphous ice produced from (1) high density amorphous ice by isobaric warming and (2) very high density amorphous ice by isothermal decompression. Differences are found in the scattering patterns of the two low density amorphous ices that correlate with structural perturbations on intermediate length scales in the hydrogen bonded water network. Atomistic modeling suggests that the structural states of the two samples may relate to a competition between short range and intermediate range order and disorder. This structural difference in two low density amorphous (LDA) ices is also evident when comparing their compression behavior. In terms of the energy landscape formalism this finding implies that we have produced and characterized the structural difference of two different basins within the LDA-megabasin corresponding to identical macroscopic densities.

Relationship between solution structure and phase behavior: a neutron scattering study of concentrated aqueous hexamethylenetetramine solutions.

The water-hexamethylenetetramine system displays features of significant interest in the context of phase equilibria in molecular materials. First, it is possible to crystallize two solid phases depending on temperature, both hexahydrate and anhydrous forms. Second, saturated aqueous solutions in equilibrium with these forms exhibit a negative dependence of solubility (retrograde) on temperature. In this contribution, neutron scattering experiments (with isotopic substitution) of concentrated aqueous hexamethylenetetramine solutions combined with empirical potential structure refinement (EPSR) were used to investigate the time-averaged atomistic details of this system. Through the derivation of radial distribution functions, quantitative details emerge of the solution coordination, its relationship to the nature of the solid phases, and of the underlying cause of the solubility behavior of this molecule.

Association and dissociation of an aqueous amphiphile at elevated temperatures.

The hydrophobic interaction is often thought to increase with increasing temperature. Although there is good experimental evidence for decreased aqueous solubility and increased clustering of both nonpolar and amphiphilic molecules as temperature is increased, the detailed nature of the changes in intermolecular interactions with temperature remain unknown. By use of isotope substitution neutron scattering difference measurements on a 0.04 mole fraction solution of tert-butanol in water as the solute clustering passes through a temperature maximum, the changes in local intermolecular structures are examined. Although, as expected, the solute molecules cluster through increased contact between their nonpolar head groups with the exclusion of water, the detailed geometry of the mutual interactions changes as temperature increases. As the clustering breaks up with further temperature increase, the local structures formed do not mirror those that were found in the low-temperature dispersed system: the disassembly process is not the reverse of assembly. The clusters formed by the solute head groups are reminiscent of structures that are found in systems of spherical molecules, modulated by the additional constraint of near-maximal hydrogen bonding between the polar tails of the alcohol and the solvent water. Although the overall temperature behavior is qualitatively what would be expected of a hydrophobically driven system, the way the system resolves the competing interactions and their different temperature dependencies is complex, suggesting it could be misleading to think of the aggregation of aqueous amphiphiles solely in terms of a hydrophobic driving force.

The local and intermediate range structures of the five amorphous ices at 80 K and ambient pressure: a Faber-Ziman and Bhatia-Thornton analysis.

Using isotope substitution neutron scattering data, we present a detailed structural analysis of the short and intermediate range structures of the five known forms of amorphous ice. Two of the lower density forms--amorphous solid water and hyperquenched glassy water--have a structure very similar to each other and to low density amorphous ice, a structure which closely resembles a disordered, tetrahedrally coordinated, fully hydrogen bonded network. High density and very high density amorphous ices retain this tetrahedral organization at short range, but show significant differences beyond about 3.1 A from a typical water oxygen. The first diffraction peak in all structures is seen to be solely a function of the intermolecular organization. The short range connectivity in the two higher density forms is more homogeneous, while the hydrogen site disorder in these forms is greater. The low Q behavior of the structure factors indicates no significant density or concentration fluctuations over the length scale probed. We conclude that these three latter forms of ice are structurally distinct. Finally, the x-ray structure factors for all five amorphous systems are calculated for comparison with other studies.

The hydration of the neurotransmitter acetylcholine in aqueous solution.

Neutron diffraction augmented with hydrogen isotope substitution has been used to examine the water structure around the acetylcholine molecular ion in aqueous solution. It is shown that the nearest-neighbor water molecules in the region around the trimethylammonium headgroup are located either in a ring around the central nitrogen atom or between the carbon atoms, forming a sheath around the onium group. Moreover the water molecules in this cavity do not bond to the onium group but rather form hydrogen bonds with water molecules in the surrounding aqueous environment. Given that in the bound state the onium headgroup must be completely desolvated, the absence of bonding between the onium headgroup and the surrounding water solvent may be selectively favorable to acetylcholine-binding in the receptor site. Away from the headgroup, pronounced hydrogen-bonding of water to the carbonyl oxygen is observed, but not to the ether oxygen in the acetylcholine chain.

Dynamics of immobilized and native Escherichia coli dihydrofolate reductase by quasielastic neutron scattering.

The internal dynamics of native and immobilized Escherichia coli dihydrofolate reductase (DHFR) have been examined using incoherent quasielastic neutron scattering. These results reveal no difference between the high frequency vibration mean-square displacement of the native and the immobilized E. coli DHFR. However, length-scale-dependent, picosecond dynamical changes are found. On longer length scales, the dynamics are comparable for both DHFR samples. On shorter length scales, the dynamics is dominated by local jump motions over potential barriers. The residence time for the protons to stay in a potential well is tau = 7.95 +/- 1.02 ps for the native DHFR and tau = 20.36 +/- 1.80 ps for the immobilized DHFR. The average height of the potential barrier to the local motions is increased in the immobilized DHFR, and may increase the activation energy for the activity reaction, decreasing the rate as observed experimentally. These results suggest that the local motions on the picosecond timescale may act as a lubricant for those associated with DHFR activity occurring on a slower millisecond timescale. Experiments indicate a significantly slower catalytic reaction rate for the immobilized E. coli DHFR. However, the immobilization of the DHFR is on the exterior of the enzyme and essentially distal to the active site, thus this phenomenon has broad implications for the action of drugs distal to the active site.

Segregation in aqueous methanol enhanced by cooling and compression.

Molecular segregation in methanol-water mixtures is studied across a wide concentration range as a function of temperature and pressure. Cluster distributions obtained from both neutron diffraction and molecular dynamics simulations point to significantly enhanced segregation as the mixtures are cooled or compressed. This evolution toward greater molecular heterogenity in the mixture accounts for the observed changes in the water-water radial distribution function and there are indications also of a change in the topology of the water clusters. The observed behavior is consistent with an approach to an upper critical solution point. Such a point would appear to be "hidden" below the freezing line, thereby precluding observation of the two-fluid region.

Methanol-water solutions: a bi-percolating liquid mixture.

An extensive series of neutron diffraction experiments and molecular dynamics simulations has shown that mixtures of methanol and water exhibit extended structures in solution despite the components being fully miscible in all proportions. Of particular interest is a concentration region (methanol mole fraction between 0.27 and 0.54) where both methanol and water appear to form separate, percolating networks. This is the concentration range where many transport properties and thermodynamic excess functions reach extremal values. The observed concentration dependence of several of these material properties of the solution may therefore have a structural origin.

Molecular and mesoscale structures in hydrophobically driven aqueous solutions.

Since Kauzmann's seminal 1959 paper, the hydrophobic interaction has dominated thinking on the forces that control protein folding and stability. Despite its wide importance in chemistry and biology, our understanding of this interaction at the molecular level remains poor, with little experimental evidence to support the idea of water ordering close to a non-polar group that is at the centre of the standard model for the source of the entropic driving force. Developments over recent years in neutron techniques now enable us to see directly how a non-polar group actually affects the molecular structure of the water in its immediate neighbourhood. On the basis of such work on aqueous solutions of small alcohols, the generally accepted standard model is found to be wanting, and alternative sources of the entropic driving force are suggested. Moreover, the fact that we can now follow changes in hydrogen bonding as the alcohol concentration is varied gives us the possibility of explaining the concentration dependence of the enthalpy of mixing. Complementary studies of solute association on the mesoscopic scale show a rich concentration and temperature behaviour, which reflects a complex balance of polar and non-polar interactions. Unravelling the detailed nature of this balance in simple aqueous amphiphiles may lead to a better understanding of the forces that control biomolecular structural stability and interactions.

The role of dynamics in enzyme activity.

Although protein function is thought to depend on flexibility, precisely how the dynamics of the molecule and its environment contribute to catalytic mechanisms is unclear. We review experimental and computational work relating to enzyme dynamics and function, including the role of solvent. The evidence suggests that fast motions on the 100 ps timescale, and any motions coupled to these, are not required for enzyme function. Proteins where the function is electron transfer, proton tunneling, or ligand binding may have different dynamical dependencies from those for enzymes, and enzymes with large turnover numbers may have different dynamical dependencies from those that turn over more slowly. The timescale differences between the fastest anharmonic fluctuations and the barrier-crossing rate point to the need to develop methods to resolve the range of motions present in enzymes on different time- and lengthscales.

Anion bridges drive salting out of a simple amphiphile from aqueous solution.

Neutron diffraction with isotope substitution has been used to determine the structural changes that occur on the addition of a simple salting-out agent to a dilute aqueous alcohol solution. The striking results obtained demonstrate a relatively simple process occurs in which interamphiphile anionic salt bridges are formed between the polar groups of the alcohol molecules. These ion bridges drive an increase in the exposure of the alcohol molecule nonpolar surface to the solvent water and hence point the way to their eventual salting out by the hydrophobic effect.

Structure of a new dense amorphous ice.

The detailed structure of a new dense amorphous ice, VHDA, is determined by isotope substitution neutron diffraction. Its structure is characterized by a doubled occupancy of the stabilizing interstitial location that was found in high density amorphous ice, HDA. As would be expected for a thermally activated unlocking of the stabilizing "interstitial," the transition from VHDA to LDA (low-density amorphous ice) is very sharp. Although its higher density makes VHDA a better candidate than HDA for a physical manifestation of the second putative liquid phase of water, as for the HDA case, the VHDA to LDA transition also appears to be kinetically controlled.

Structures of high and low density amorphous ice by neutron diffraction.

Neutron diffraction with isotope substitution is used to determine the structures of high (HDA) and low (LDA) density amorphous ice. Both "phases" are fully hydrogen bonded, tetrahedral networks, with local order similarities between LDA and ice Ih, and HDA and liquid water. Moving from HDA, through liquid water and LDA to ice Ih, the second shell radial order increases at the expense of spatial order. This is linked to a fifth first neighbor "interstitial" that restricts the orientations of first shell waters. This "lynch pin" molecule which keeps the HDA structure intact has implications for the nature of the HDA-LDA transition that bear on the current metastable water debate.

Molecular segregation observed in a concentrated alcohol-water solution.

When a simple alcohol such as methanol or ethanol is mixed with water, the entropy of the system increases far less than expected for an ideal solution of randomly mixed molecules. This well-known effect has been attributed to hydrophobic headgroups creating ice-like or clathrate-like structures in the surrounding water, although experimental support for this hypothesis is scarce. In fact, an increasing amount of experimental and theoretical work suggests that the hydrophobic headgroups of alcohol molecules in aqueous solution cluster together. However, a consistent description of the details of this self-association is lacking. Here we use neutron diffraction with isotope substitution to probe the molecular-scale structure of a concentrated alcohol water mixture (7:3 molar ratio). Our data indicate that most of the water molecules exist as small hydrogen-bonded strings and clusters in a 'fluid' of close-packed methyl groups, with water clusters bridging neighbouring methanol hydroxyl groups through hydrogen bonding. This behaviour suggests that the anomalous thermodynamics of water alcohol systems arises from incomplete mixing at the molecular level and from retention of remnants of the three-dimensional hydrogen-bonded network structure of bulk water.

Solvent dependence of dynamic transitions in protein solutions.

A transition as a function of increasing temperature from harmonic to anharmonic dynamics has been observed in globular proteins by using spectroscopic, scattering, and computer simulation techniques. We present here results of a dynamic neutron scattering analysis of the solvent dependence of the picosecond-time scale dynamic transition behavior of solutions of a simple single-subunit enzyme, xylanase. The protein is examined in powder form, in D(2)O, and in four two-component perdeuterated single-phase cryosolvents in which it is active and stable. The scattering profiles of the mixed solvent systems in the absence of protein are also determined. The general features of the dynamic transition behavior of the protein solutions follow those of the solvents. The dynamic transition in all of the mixed cryosolvent-protein systems is much more gradual than in pure D(2)O, consistent with a distribution of energy barriers. The differences between the dynamic behaviors of the various cryosolvent protein solutions themselves are remarkably small. The results are consistent with a picture in which the picosecond-time scale atomic dynamics respond strongly to melting of pure water solvent but are relatively invariant in cryosolvents of differing compositions and melting points.

Cryosolvents useful for protein and enzyme studies below -100 degrees C.

For the study of protein structure, dynamics, and function, at very low temperatures it is desirable to use cryosolvents that resist phase separation and crystallisation. We have examined these properties in a variety of cryosolvents. Using visual and X-ray diffraction criteria, methanol:ethanediol (70%:10%), methanol:glycerol (70%:10%), acetone:methoxy-ethanol:ethanediol (35%:35%:10%), dimethylformamide:ethanediol (70%:10%), dimethylformamide (80%), methoxyethanol (80%), and methoxyethanol:ethanediol (70%:10%) were all found to be free of phase-changes down to at least -160 degrees C. The least viscous of these, methanol:ethanediol (70%:10%), was miscible down to -125 degrees C and showed no exo or endothermic transitions when examined using DSC. It is therefore potentially particularly suitable for very low temperature cryoenzymology.

Enzyme dynamics and activity: time-scale dependence of dynamical transitions in glutamate dehydrogenase solution.

We have examined the temperature dependence of motions in a cryosolution of the enzyme glutamate dehydrogenase (GDH) and compared these with activity. Dynamic neutron scattering was performed with two instruments of different energy resolution, permitting the separate determination of the average dynamical mean square displacements on the sub-approximately 100 ps and sub-approximately 5 ns time scales. The results demonstrate a marked dependence on the time scale of the temperature profile of the mean square displacement. The lowest temperature at which anharmonic motion is observed is heavily dependent on the time window of the instrument used to observe the dynamics. Several dynamical transitions (inflexions of the mean squared displacement) are observed in the slower dynamics. Comparison with the temperature profile of the activity of the enzyme in the same solvent reveals dynamical transitions that have no effect on GDH function.