Study of folding mechanism of proline mutants of staphylococcal nuclease

利用多肽和芳香环二色性和芳香吸收技术研究了尿素诱导的葡萄球菌核酸酶（SNase）及其5个脯氨酸突变体（P47A、P47T、P117G、P47A/P117G和P47T/P117G）在平衡状态下的展开转变，并利用停流环二色性和停流吸收技术研究了蛋白质的折叠-展开动力学。近年来的研究表明，SNase的Pro47和Pro117肽键的顺/反异构化不仅发生在未折叠状态，也发生在天然状态。然而，突变对SNase稳定性和重新折叠-展开动力学的影响在两个位点之间有显著差异。以Ala或Thr取代Pro47既不改变SNase的稳定性，也不影响SNase的折叠展开动力学，而以Gly取代Pro117则增加了SNase的稳定性，并影响了SNase的折叠展开动力学。这些结果归因于Pro47周围环的高柔韧性，这已被天然SNase的分子动力学模拟所揭示。在每种条件下，都观察到SNase的协同折叠-展开动力学。野生型SNase的重折叠表现为两个与尿素浓度无关的快相和一个与尿素浓度无关的慢相。SNase的双突变体（P47A/P117G）仍然表现出多相重折叠动力学，涉及两个尿素浓度无关的慢相，这表明在突变体的未折叠状态下可能发生Pro47和Pro117以外的脯氨酸残基的异构化。在包含Pro117的野生型和突变型蛋白的展开过程中观察到两个阶段，一个快速阶段对应于反式异构体的展开，一个缓慢阶段对应于顺式异构体的展开。在这些结果的基础上，讨论了SNase的折叠方案。

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.