Mutational studies of protein stability and folding of the hyperstable MYL Arc repressor variant Original Research Article

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.