Solvent-binding sites were compared in 10 different crystal forms of phage T4 lysozyme that were refined using data from 2.6 A to 1.7 A resolution. The sample included 18 crystallographically independent lysozyme molecules.Despite different crystallization conditions, variable crystal contacts, changes due to mutation, and varying attention to solvent during crystallographic refinement, 62% of the 20 most frequently occupied sites were conserved. Allowing for potential steric interference from neighboring molecules in the crystal lattice, this fraction increased to 79% of the sites. There was, however, no solvent-binding site that was occupied in all 18 lysozyme molecules. A buried double site was occupied in 17 instances and 2 other internal sites were occupied 15 times. Apart from these buried sites, the most frequently occupied sites were often at the amino-termini of a-helices. Solvent molecules at the most conserved sites tended to have crystallographic thermal factors lower than average, but atoms with low E-factors were not restricted to these sites.Although superficial inspection may suggest that only 50-60'70 (or less) of solvent-binding sites are conserved in different crystal forms of a protein, it appears that many sites appear to be empty either because of steric interference or because 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 that 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 conditions, it seems likely that binding of solvent to such sites will be maintained in solution under physiological conditions.

In a systematic attempt to identify residues important in the folding and stability of T4 lysozyme, five amino acids within a-helix 126-134 were substituted by alanine, either singly or in selected combinations. Together with three alanines already present in the wild-type structure this provided a set of mutant proteins with up to eight alanines in sequence. All the variants behaved normally, suggesting that the majority of residues in the a-helix are nonessential for the folding of T4 lysozyme. Of the five individual alanine substitutions it is inferred that four result in slightly increased protein stability and one, the replacement of a buried leucine with alanine, substantially decreased stability. The results support the idea that alanine is a residue of high helix propensity. The change in protein stability observed for each of the multiple mutants is approximately equal to the sum of the energies associated with each of the constituent substitutions.All of the variants could be crystallized isomorphously with wild-type lysozyme, and, with one trivial exception, their structures were determined at high resolution. Substitution of the largely solvent-exposed residues Asp 127, Glu 128, and Val 131 with alanine caused essentially no change in structure except at the immediate site of replacement. Substitutions of the partially buried Asn 132 and the buried Leu 133 with alanine were associated with modest ( 5 0 . 4 A) structural adjustments. The structural changes seen in the multiple mutants were essentially a combination of those seen in the constituent single replacements. The different replacements therefore act essentially independently not only so far as changes in energy are concerned but also in their effect on structure. The destabilizing replacement Leu 133 +Ala made a-helix 126-134 somewhat less regular. Incorporation of additional alanine replacements tended to make the helix more uniform. For the penta-alanine variant a distinct change occurred in a crystal-packing contact, and the "hinge-bending angle" between the amino-and carboxyterminal domains changed by 3.6". This tends to confirm that such hinge-bending in T4 lysozyme is a low-energy conformational change.Keywords: alanine; lysozyme; protein folding; protein structure; thermostability In a systematic attempt to identify residues important in the folding and stability of phage T4 lysozyme we previously substituted four alanines within the a-helix that includes residues 126-134 . The helix is amphipathic, located on the surface of the carboxy-terminal domain, and is remote from the active site ( Fig. 1; Kinemage 1). In wild-type lysozyme this a-helix already includes three alanines. It was found that substitution of three solvent-exposed residues, Glu 128, Val 131, and Asn