Wedemeyer W J, Welker E, Narayan M, Scheraga H A
Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
Biochemistry. 2000 Apr 18;39(15):4207-16. doi: 10.1021/bi992922o.
The applications of disulfide-bond chemistry to studies of protein folding, structure, and stability are reviewed and illustrated with bovine pancreatic ribonuclease A (RNase A). After surveying the general properties and advantages of disulfide-bond studies, we illustrate the mechanism of reductive unfolding with RNase A, and discuss its application to probing structural fluctuations in folded proteins. The oxidative folding of RNase A is then described, focusing on the role of structure formation in the regeneration of the native disulfide bonds. The development of structure and conformational order in the disulfide intermediates during oxidative folding is characterized. Partially folded disulfide species are not observed, indicating that disulfide-coupled folding is highly cooperative. Contrary to the predictions of "rugged funnel" models of protein folding, misfolded disulfide species are also not observed despite the potentially stabilizing effect of many nonnative disulfide bonds. The mechanism of regenerating the native disulfide bonds suggests an analogous scenario for conformational folding. Finally, engineered covalent cross-links may be used to assay for the association of protein segments in the folding transition state, as illustrated with RNase A.
本文以牛胰核糖核酸酶A（RNase A）为例，综述并阐释了二硫键化学在蛋白质折叠、结构和稳定性研究中的应用。在概述二硫键研究的一般特性和优势后，我们阐述了RNase A的还原解折叠机制，并讨论了其在探测折叠蛋白质结构波动方面的应用。接着描述了RNase A的氧化折叠，重点关注结构形成在天然二硫键再生中的作用。对氧化折叠过程中二硫键中间体的结构和构象有序性发展进行了表征。未观察到部分折叠的二硫键物种，这表明二硫键偶联折叠具有高度协同性。与蛋白质折叠“崎岖漏斗”模型的预测相反，尽管许多非天然二硫键具有潜在的稳定作用，但也未观察到错误折叠的二硫键物种。天然二硫键再生机制提示了一种类似的构象折叠情况。最后，如RNase A所示，工程化的共价交联可用于检测折叠过渡态中蛋白质片段的缔合。
