Talluri S, Rothwarf D M, Scheraga H A
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301.
Biochemistry. 1994 Aug 30;33(34):10437-49. doi: 10.1021/bi00200a027.
Earlier studies of the unfolding pathway of native bovine pancreatic ribonuclease A (using dithiothreitol as the reducing agent) revealed that the three-disulfide species lacking the disulfide bond between cysteine 65 and cysteine 72 is the most highly populated intermediate [Rothwarf & Scheraga (1991) J. Am. Chem. Soc. 113, 6293-6294]. This unfolding intermediate is referred to as des-[65-72]-RNase A. In order to determine the role of des-[65-72]-RNase A, i.e. of the 65-72 disulfide bond, in the structural folding/unfolding processes of RNase A, the stability and structure of this unfolding intermediate were determined by examining its thermal transition curve and by using two- and three-dimensional homonuclear 1H NMR spectroscopy. The midpoint of the thermal transition of des-[65-72]-RNase A was found to be 17.8 degrees C lower than that of native RNase A. A set of conformations that are consistent with the NMR-derived constraints was obtained by minimizing, first, a variable-target function and, then, the conformational energy. These conformations exhibit a well-defined structure that is very similar to that of native ribonuclease A in regions where the native protein has a regular backbone structure such as a beta-sheet or a helix. Some of the loop regions of the several computed structures exhibit large deviations from each other as well as from native ribonuclease A. However, these results indicate that des-[65-72]-RNase A has a close structural similarity to RNase A in all regions with the only major differences occurring in a loop region comprising residues 60-72. This led to the conclusion that, in reduction pathways that include des-[65-72]-RNase A (at 25 degrees C, pH 8.0), the rate-determining step corresponds to a partial unfolding event in one region of the protein and not to a global conformational unfolding process. The results further suggest that, in the regeneration pathways involving des-[65-72]-RNase A, the loop region from 60 to 72 is the last to fold.
早期对天然牛胰核糖核酸酶A展开途径的研究（使用二硫苏糖醇作为还原剂）表明，缺少半胱氨酸65和半胱氨酸72之间二硫键的三二硫键物种是含量最高的中间体[罗斯瓦尔夫和舍拉加（1991年）《美国化学会志》113，6293 - 6294]。这种展开中间体被称为去-[65 - 72]-核糖核酸酶A。为了确定去-[65 - 72]-核糖核酸酶A，即65 - 72二硫键，在核糖核酸酶A结构折叠/展开过程中的作用，通过检查其热转变曲线并使用二维和三维同核1H核磁共振光谱来确定这种展开中间体的稳定性和结构。发现去-[65 - 72]-核糖核酸酶A的热转变中点比天然核糖核酸酶A的低17.8摄氏度。通过首先最小化可变目标函数，然后最小化构象能量，获得了一组与核磁共振衍生约束一致的构象。这些构象呈现出明确的结构，在天然蛋白质具有规则主链结构（如β-折叠或螺旋）的区域，与天然核糖核酸酶A非常相似。几个计算结构的一些环区域彼此之间以及与天然核糖核酸酶A都有很大偏差。然而，这些结果表明，去-[65 - 72]-核糖核酸酶A在所有区域与核糖核酸酶A具有密切的结构相似性，唯一的主要差异出现在包含残基60 - 72的环区域。这导致得出结论，在包括去-[65 - 72]-核糖核酸酶A的还原途径中（在25摄氏度，pH 8.0），速率决定步骤对应于蛋白质一个区域的部分展开事件，而不是全局构象展开过程。结果进一步表明，在涉及去-[65 - 72]-核糖核酸酶A的再生途径中，60到72的环区域是最后折叠的。
