VA-033

The Arc-MYL variant is hyperstable and has an earlier transition state than wild type as a consequence of replacing the wild-type salt-bridge triad formed by R31, E36 and R40 with hydrophobic interactions formed by M31, Y36 and L40. Amino acid substitution mutations were constructed at 16 positions in the Arc-MYL background and the equilibrium stabilities of the corresponding mutant proteins were determined. At three positions, mutations were found to be less destabilizing in MYL than in wild-type Arc, and, at one position, the opposite result was obtained. The kinetics of refolding and unfolding were determined for a subset of the Arc-MYL core mutants. Three mutations--VA18, LA19 and LA40--had their major energetic effects on the refolding rate. The interactions perturbed by these mutations appear to be substantially formed in the transition state. V18 and L19 are in the N-terminal turn of helix A and L40 is in the center of helix B. The remaining mutations--VA22, MA31, VA33, YA36, VA41, MA42 and FA45--had some effects on refolding but exerted their major effects on the unfolding rate. Approximately 30% of the energetic interactions mediated by these latter side chains seem to be present in the transition state of Arc-MYL.