Publications - 2000

Using neutron diffraction with isotopic substitution, the structures of aqueous solutions of tertiary butanol have been studied as a function of concentration. As the behaviour of this system is thought to be driven by hydrophobic interactions, particular attention has been paid to the hydration of the non-polar headgroups and the nature of the intermolecular contacts. As concentration is increased from 0.06 to 0.16 mole fraction tertiary butanol, there is evidence for the growth of small clusters of the alcohol molecules, with butanol-butanol co-ordination numbers of 2 to 3 even at the lowest concentration. Orientational pair correlation functions show that the dominant intermolecular contacts between alcohol molecules are between the non-polar head groups, as would be expected for a system driven by hydrophobic interactions. As concentration increases, however, there is evidence of mixed polar - nonpolar contacts. The alcohol group's hydrogen bonding requirements appear to be fully met by polar contacts with water molecules: there is no evidence for significant butanol-butanol hydrogen bonding.

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:methoxyethanol: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 C. The least viscous of these, methanol:ethanediol [70%:10%], was miscible down to -130 C and showed no exo or endothermic transitions when examined using DSC. It is therefore potentially particularly suitable for very low temperature cryoenzymology.

The activity and dynamics of a simple, single submit enzyme, the xylanase from Thermotoga maritima strain Fj SS3B.1 have been measured under similar conditions, from -70 to +10C. The internal motions of the enzyme, as evidenced by neutron scattering, undergo a sharp transition within this temperature range; they show no evidence for picosecond-timescale anharmonic behaviour (e.g. local diffusive motions or jumps between alternative conformations) at temperatures below -50C, whereas these motions are strongly activated at higher temperatures. The activity follows Arrhenius behaviour over the whole of the temperature range investigated, -70 to +10C. The results indicate that a temperature range exists over which the enzyme rate-limiting step is independent of fast anharmonic dynamics.