Study of folding mechanism of proline mutants of staphylococcal nuclease

我们用平衡状态下的肽和芳香族圆二色谱和芳香族吸收光谱研究了葡萄球菌核酸酶(SNase)及其5个突变体(P47A、P47T、P117G、P47A/P117G和P47T/P117G)在尿素诱导下的去折叠转变，并用停流圆二色谱和停流吸收技术研究了蛋白质的重折叠-去折叠动力学。最近的研究表明，SNase的Pro47和Pro117肽键的顺式/反式异构化不仅在未折叠状态下发生，而且在自然状态下也发生。然而，突变对SNase稳定性和复性-去折叠动力学的影响在这两个位点之间存在显著差异。Ala或Thr取代Pro47不改变SNase的稳定性，也不影响SNase的复性-去折叠动力学，而Gly取代Pro117提高了蛋白质的稳定性并影响了其动力学。这些结果归因于Pro47周围环的高度灵活性，这一点已经通过对天然SNase的分子动力学模拟得到了揭示。在所研究的各个条件下，观察了SNase的协同折叠-去折叠动力学。野生型SNase的复性表现为两个依赖于尿素浓度的快相和一个不依赖尿素浓度的慢相。SNase的双突变体(P47A/P117G)仍表现为多相折叠动力学，存在两个不依赖于尿素浓度的慢相，这表明在突变体的未折叠状态下，可能发生了Pro47和Pro117以外的Pro残基的异构化。在含有Pro117的野生型和突变型蛋白质的去折叠过程中观察到两个相，一个快相对应于反式异构体的去折叠，一个慢相对应于顺式异构体的去折叠。在此基础上，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.