CONSERVATION OF SOLVENT-BINDING SITES IN 10 CRYSTAL FORMS OF T4-LYSOZYME

Solvent-binding sites were compared in 10 different crystal forms of p hage T4 lysozyme that were refined using data from 2.6 Angstrom to 1.7 Angstrom resolution. The sample included 18 crystallographically inde pendent lysozyme molecules. Despite different crystallization conditio ns, variable crystal contacts, changes due to mutation, and varying at tention to solvent during crystallographic refinement, 62% of the 20 m ost frequently occupied sites were conserved. Allowing for potential s teric interference from neighboring molecules in the crystal lattice, this fraction increased to 79% of the sites. There was, however, no so lvent-binding site that was occupied in all 18 lysozyme molecules. A b uried double site was occupied in 17 instances and 2 other internal si tes were occupied 15 times. Apart from these buried sites, the most fr equently occupied sites were often at the amino-termini of oc-helices. Solvent molecules at the most conserved sites tended to have crystall ographic thermal factors lower than average, but atoms with low B-fact ors were not restricted to these sites. Although superficial inspectio n may suggest that only 50-60% (or less) of solvent-binding sites are conserved in different crystal forms of a protein, it appears that man y sites appear to be empty either because of steric interference or be cause the apparent occupancy of a given site can vary from crystal to crystal. The X-ray method of identifying sites is somewhat subjective and tends to result in specification only of those solvent molecules t hat are well ordered and bound with high occupancy, even though there is clear evidence for solvent bound at many additional sites. Because similar sites are occupied under a variety of crystallization conditio ns, it seems likely that binding of solvent to such sites will be main tained in solution under physiological conditions.