STRUCTURES OF RANDOMLY GENERATED MUTANTS OF T4-LYSOZYME SHOW THAT PROTEIN STABILITY CAN BE ENHANCED BY RELAXATION OF STRAIN AND BY IMPROVEDHYDROGEN-BONDING VIA BOUND SOLVENT

The structures of three mutants of bacteriophage T4 lysozyme selected using a screen designed to identify thermostable variants are describe d. Each of the mutants has a substitution involving threonine. Two of the variants, Thr 26 --> Ser (T26S) and Thr 151 --> Ser (T151S), have increased reversible melting temperatures with respect to the wild-typ e protein. The third, Ala 93 --> Thr (A93T), has essentially the same stability as wild type. Thr 26 is in the wall of the active-site cleft . Its replacement with serine results in the rearrangement of nearby r esidues, most notably Tyr 18, suggesting that the increase in stabilit y may result from the removal of strain. Thr 151 in the wild-type stru cture is far from the active site and appears to sterically prevent th e access of solvent to a preformed binding site. In the mutant, the re moval of the methyl group allows access to the solvent binding site an d, in addition, the Ser 151 hydroxyl rotates to a new position so that it also contributes to solvent binding. Residue 93 is in a highly exp osed site on the surface of the molecule, and presumably is equally so lvent exposed in the unfolded protein. It is, therefore, not surprisin g that the substitution Ala 93 --> Thr does not change stability. The mutant structures show how chemically similar mutations can have diffe rent effects on both the structure and stability of the protein, depen ding on the structural context. The results also illustrate the power of random mutagenesis in obtaining variants with a desired phenotype. Although knowledge of the mutant structures makes it possible to ratio nalize their behavior, it would have been very difficult to predict in advance that these mutants would be stabilizing.