A MUTANT T4 LYSOZYME (VAL 131-]ALA) DESIGNED TO INCREASE THERMOSTABILITY BY THE REDUCTION OF STRAIN WITHIN AN ALPHA-HELIX

An attempt has been made to identify residues in T4 phage lyoszyme that may have strained conformations and, by appropriate site‐directed replacements, to reduce this strain and thus increase the thermostability of the protein. Valine 131, within α‐helix 126–134, was identified as a potential candidate. Its side‐chain rotational as a potential candidate. Its side‐chain rotation angle, χ1, differs by approximately 18° from the low‐energy trans configuration. In addition, it is largely solvent exposed, yet is held in a rigid conformation. The mutant protein with Val 131 replaced by alanine temperature 0.9°C higher than that of wildtype lyoszyme at pH 2.8. As a control, The mutant Val 131 → Thr was also constructed and its melting temperature was found to be marginally lower than wild type. Higher‐resolution crystal structure determination of the mutant lysozymes show that their structure are virtually identical with that of wild‐type lyoszyme, except for the Val → Ala or Val → Thr replacement. Analysis of the different structures suggests that the design of the Val→Ala substitution was, in principle, successful, although the apparent gain in stability caused by reduction in strain is modest and is somewhat offset by the loss of hydrophobic interactions and by entrophic effects. The results also help to provide a structural retionalization that alanine has a higher helix propensity than valine or theronine. Copyright © 1990 Wiley‐Liss, Inc.