Solvent behaviour in flash-cooled protein crystals at cryogenic temperatures

The solvent behaviour of flash-cooled protein crystals was studied in the r ange 100-180 K by X-ray diffraction. If the solvent is within large channel s it crystallizes at 155 K, as identified by a sharp change in the increase of unit-cell volume upon temperature increase. In contrast, if a similar a mount of solvent is confined to narrow channels and/or individual cavities it does not crystallize in the studied temperature range. It is concluded t hat the solvent in large channels behaves similarly to bulk water, whereas when confined to narrow channels it is mainly protein-associated. The analo gy with the behaviour of pure bulk water provides circumstantial evidence t hat only solvent in large channels undergoes a glass transition in the 100- 180 K temperature range. These studies reveal that flash-cooled protein cry stals are arrested in a metastable state up to at least 155 K, thus providi ng an upper temperature limit for their storage and handling. The results a re pertinent to the development of rational crystal annealing procedures an d to the study of temperature-dependent radiation damage to proteins. Furth ermore, they suggest an experimental paradigm for studying the correlation between solvent behaviour, protein dynamics and protein function.