EFFECT OF PROLINE MUTATIONS ON THE STABILITY AND KINETICS OF FOLDING OF STAPHYLOCOCCAL NUCLEASE

The role of proline in the stability and kinetics of folding of wild-type staphylococcal nuclease and its P117G, P117T, and P31A mutants was examined as a function of guanidinium thiocyanate (Gdn.SCN) concentration. Replacement of Pro-117 with Gly or Thr caused small increases in stability, whereas substitution of Pro-31 by Ala led to a small decrease in stability. The slopes of the plots of DELTAG against denaturant concentration (m) for the mutant proteins are significantly smaller than for the wild-type, suggesting a decrease in the solvent-accessible surface area of the denatured state relative to that of the wild-type. The rates of unfolding and refolding were monitored using tryptophan fluorescence. The kinetic traces for refolding in the presence of Gdn.SCN were triphasic for the wild-type protein and P31A but biphasic for P117G and P117T mutants. The slower phases were typically 10% of the total amplitude except in the transition region. The rates of the fastest and medium phases of the wild-type were essentially unaffected by the mutations. Double-jump experiments in which the protein was unfolded in a high concentration of denaturant for a short time period and then refolded to final Gdn.SCN concentrations near the C(m) revealed a fast increase in fluorescence emission corresponding to formation of the native state, followed by a slower decrease with an amplitude that varied with the guanidine concentration and time of unfolding. The slower phase is ascribed to proline isomerization and was observed for wild-type and mutants at both proline positions, indicating the presence of a second, nonnative proline conformation in the unfolded protein. We conclude that the isomerization of Pro-117, which is in the less stable cis conformation in the native state, is responsible for the guanidine-independent slow-folding kinetic phase seen with the wild-type. An additional proline isomerization (or isomerizations), in which a significant amount of the nonnative (cis) conformation is formed in the unfolded state, is responsible for the intermediate kinetic phase seen with the wild-type and P31A mutant and the slow phase for the P117 mutants as well as the proline isomerization seen in the double-jump assays with the P117 mutants.