KINETIC FOLDING AND CIS TRANS PROLYL ISOMERIZATION OF STAPHYLOCOCCAL NUCLEASE - A STUDY BY STOPPED-FLOW ABSORPTION, STOPPED-FLOW CIRCULAR-DICHROISM, AND MOLECULAR-DYNAMICS SIMULATIONS/

We Studied the urea-induced unfolding transition of staphylococcal nuc lease (SNase) and its five proline mutants (P47A, P47T, P117G, P47A/P1 17G, and P47T/P117G) by peptide and aromatic circular dichroism and ar omatic absorption spectroscopy at equilibrium and the refolding-unfold ing kinetics of the proteins by stopped-flow circular dichroism and st opped-flow absorption techniques. Recent studies have revealed that th e cis/trans isomerizations about the Pro47 and Pro117 peptide bonds of SNase occur not only in the unfolded state but also in the native sta te. The mutational effects on the stability and the refolding-unfoldin g kinetics of SNase were, however, remarkably different between the tw o sites. The substitution of Ala or Thr for Pro47 neither changed the stability nor affected the refolding-unfolding kinetics of SNase, wher eas the substitution of Gly for Pro117 increased the protein stability by 1.2 kcal/mol (pH 7.0 and 20 degrees C) 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 simulatio ns of native SNase. Under every condition studied, cooperative refoldi ng-unfolding kinetics of SNase were observed. Refolding of wild-type S Nase was represented by two urea concentration-dependent fast phases a nd a urea concentration-independent slow phase. The double mutant (P47 A/P117G) of SNase still showed multiphasic refolding kinetics that inv olved two urea concentration-independent slow phases, suggesting that isomerization of proline residues other than Pro47 and Pro 117 may occ ur in the unfolded state of the mutant. Two phases were observed in th e 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.