Study of folding mechanism of proline mutants of staphylococcal nuclease

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We studied the urea-induced unfolding transition of staphylococcal nuclease (SNase) and its five proline mutants (P47A,P47T,P117G,P47A/P117G,and P47T/P117G) by peptide and aromatic circular dichroism and aromatic absorption spectroscopy at equilibrium, and the refolding-unfolding kinetics of the proteins by stopped-flow circular dichroism and stopped-flow absorption techniques. Recent studies have revealed that the cis/trans isomerizations about the Pro47 and Pro117 peptide bonds of SNase occur not only in the unfolded state but also in the native state. The mutational effects on the stability and the refolding-unfolding kinetics of SNase were, however, remarkably different between the two sites. The substitution of Ala or Thr for Pro47 neither changed the stability nor affected the refolding-unfolding kinetics of SNase, whereas the substitution of Gly for Pro117 increased the protein stability and affected the kinetics. These results have been attributed to the high flexibility of the loop around Pro47, which has been revealed by molecular dynamics simulations of native SNase. Under every condition studied, cooperative refolding-unfolding kinetics of SNase were observed. Refolding of wild-type SNase was represented by two urea concentration-dependent fast phases and a urea concentration-independent slow phase. The double-mutant (P47A/P117G) of SNase still showed multiphasic refolding kinetics that involved two urea concentration-independent slow phases, suggesting that isomerization of proline residues other than Pro47 and Pro117 may occur in the unfolded state of the mutant. Two phases were observed in the unfolding of the wild-type and mutant proteins that contained Pro117, a fast phase corresponding to the unfolding of the trans isomer and a slow phase corresponding to that of the cis isomer. On the basis of these results, the folding scheme of SNase is discussed.