Effects of proline mutations on the folding of staphylococcal nuclease

Effects of proline isomerizations on the equilibrium unfolding and kinetic refolding of staphylococcal nuclease were studied by circular dichroism in the peptide region (225 nm) and fluorescence spectra of a tryptophan residu e. For this purpose, four single mutants (P11A, P31A, P42A, and P56A) and f our multiple mutants (P11A/P47T/P117G, P11A/P31A/P47T/P117G, P11A/P31A/P42A /P47T/P117G, and P11A/P31A/P42A/P47T/P56A/P117G) were constructed. These mu tants, together with the single and double mutants for Pro(47) and Pro(117) constructed in our previous study, cover all six proline sites of the nucl ease. The P11A, P31A, and P42A mutations did not change the stability of th e protein remarkably, while the P56A mutation increased protein stability t o a small extent by 0.5 kcal/mol. The refolding kinetics of the protein wer e, however, affected remarkably by three of the mutations, namely, P11A, P3 1A, and P56A. Most notably, the amplitude of the slow phase of the triphasi c refolding kinetics of the nuclease observed by stopped-flow circular dich roism decreased by increasing the number of the proline mutations; the slow phase disappeared completely in the proline-free mutant (P11A/P31A/P42A/P4 7T/P56A/P117G). The kinetic refolding reactions of the wild-type protein as sessed in the presence of Escherichia coli cyclophilin A showed that the sl ow phase was accelerated by cyclophilin, indicating that the slow phase was rate-limited by cis-trans isomerization of the proline residues. Although the fast and middle phases of the refolding kinetics were not affected by c yclophilin, the amplitude of the middle phase decreased when the number of the proline mutations increased; the percent amplitudes for the wild-type p rotein and the proline-free mutants were 43 and 13%, respectively. In addit ion to these three phases detected with stopped-flow circular dichroism, a very fast phase of refolding was observed with stopped-flow fluorescence, w hich had a shorter dead time (3.6 ms) than the stopped-flow circular dichro ism. The following conclusions were drawn. (1) The effects of the P11A, P31 A, and P56A mutations on the refolding kinetics indicate that the isomeriza tions of the three proline residues are rate-limiting, suggesting that the structures around these residues (Pro(11), Pro(31), and Pro(56)) may be org anized at an early stage of refolding. (2) The fast phase corresponds to th e refolding of the native proline isomer, and the middle phase whose amplit ude has decreased when the number of proline mutations was increased may co rrespond to the slow refolding of non-native proline isomers. The occurrenc e of the fast- and slow-refolding reactions together with the slow phase ra te-limited by the proline isomerization suggests that there are parallel fo lding pathways for the native and non-native proline isomers, (3) The middl e phase did not completely disappear in the proline-free mutant. This sugge sts that the slow-folding isomer is produced not only by the proline isomer izations but also by another conformational event that is not related to th e prolines. (4) The very fast phase detected with the fluorescent measureme nts suggests that there is an intermediate at a very early stage of kinetic refolding.