W0203

Evolution of Disorder in Protein Crystals During Dehydration. I. Dobrianov, C. Caylor, K. D. Finkelstein, and R. E. Thorne, Cornell University, Ithaca, NY 14853

Water is important to maintaining the native structure and activity of proteins in crystalline form. Studies of lysozyme structure as a function of crystal dehydration indicate that the unit cell shrinks and the protein molecules shift and rotate within the unit cell as water loss increases, and that there are conformation changes near the active site. We have employed X-ray topography combined with oscillation data collection to explore how structural changes associated with dehydration affect crystal quality. X-ray topography provides a powerful probe for observing defects, strains, and lattice orientation variations within the bulk of protein crystals. By dehydrating the crystals in situ, we are able to follow the time evolution of these features and correlate them with the evolution of the crystal lattice constant, diffraction resolution, and B-factor. For sufficiently large dehydrations, diffraction patterns degrade substantially and topographs develop extensive contrast, but the detailed behavior varies significantly from crystal to crystal. Some crystals fail catastrophically ñ often within a few hours or less ñ via formation of complex dislocation networks with concurrent degradation of the diffraction resolution. Other crystals develop patterns of strain and mosaicity consistent with the crystal symmetry but show no obvious dislocations. For these crystals, degradation of the diffraction resolution tends to be smaller and requires a day or more, an order of magnitude longer than the time scale for equilibration of the lattice constant. This suggests that dislocations facilitate the molecular-scale rearrangements that degrade the diffraction resolution, and that the large transient strains that can develop in their absence have much weaker effects. The difference between crystals that fail catastrophically and those that do not may be the presence of preexisting cracks or dislocations in the former that propagate and multiply under the influence of dehydration-related strains. Evolving lattice constants and inhomogeneous strains are commonly associated with post-growth protein crystal treatments including heavy atom soaking, ligand binding, and cryocooling. The present techniques and results may thus have broad applicability in understanding the degradation in diffraction properties that often accompany these treatments.