Xu X, Scheraga H A
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
Biochemistry. 1998 May 19;37(20):7561-71. doi: 10.1021/bi980086x.
The oxidative refolding pathway of a three-disulfide mutant of bovine pancreatic ribonuclease A (RNase A) from the fully reduced unfolded form to the native state has been studied by using oxidized and reduced dithiothreitol as the redox reagents at pH 8.0 and 25 degrees C. This mutant was prepared by replacing Cys40 and Cys95 in RNase A with alanines while maintaining the other three native disulfide bonds to mimic one of the two major three-disulfide intermediates (des-[40-95]) observed in the regeneration of wild-type RNase A. The kinetics of refolding of this mutant were measured by quenching the regeneration reaction at various times with a rapid blocking reagent, 2-aminoethyl methanethiosulfonate (AEMTS), fractionating the disulfide intermediates by using cation-exchange HPLC, and analyzing the time course of each group of disulfide species. It was found that the disulfide intermediates formed during regeneration reach a steady-state distribution after a short period of preequilibration similar to that in the regeneration of wild-type RNase A. The experimental data acquired under different redox conditions were fit to a kinetic model with a steady-state treatment. The fitted results indicate that this mutant refolds through a rate-determining step which involves the oxidation of certain two-disulfide species to form a putative three-disulfide species which proceeds rapidly to the native protein. A rough estimation suggests that this pathway could constitute no more than 5% of the major pathway leading to the formation of des-[40-95] (the major three-disulfide intermediate formed) in the regeneration of wild-type RNase A. Several kinetic constants pertaining to the oxidation and reduction of various disulfide intermediates were compared with those obtained in the regeneration studies of wild-type RNase A to gain further understanding about the folding pathways of RNase A. Comparisons are also given for the oxidative refolding studies of several other three disulfide bond proteins, suggesting that the formation of a large number of disulfide-bonded intermediates during oxidative refolding is probably a common feature for most proteins.
在pH 8.0和25摄氏度条件下，以氧化型和还原型二硫苏糖醇作为氧化还原试剂，研究了牛胰核糖核酸酶A（RNase A）的一种三二硫键突变体从完全还原的未折叠形式到天然状态的氧化重折叠途径。该突变体通过将RNase A中的Cys40和Cys95替换为丙氨酸而制备，同时保留其他三个天然二硫键，以模拟在野生型RNase A再生过程中观察到的两种主要三二硫键中间体之一（去-[40-95]）。通过用快速阻断剂2-氨基乙基甲硫基磺酸盐（AEMTS）在不同时间淬灭再生反应、使用阳离子交换高效液相色谱法分离二硫键中间体以及分析每组二硫键物种的时间进程，来测量该突变体重折叠的动力学。结果发现，再生过程中形成的二硫键中间体在短时间的预平衡后达到稳态分布，这与野生型RNase A的再生情况相似。在不同氧化还原条件下获得的实验数据通过稳态处理拟合到一个动力学模型。拟合结果表明，该突变体通过一个速率决定步骤进行重折叠，该步骤涉及某些二二硫键物种的氧化以形成一种假定的三二硫键物种，然后该物种迅速转化为天然蛋白。粗略估计表明，该途径在野生型RNase A再生过程中导致去-[40-95]（形成的主要三二硫键中间体）形成的主要途径中所占比例不超过5%。将与各种二硫键中间体氧化和还原相关的几个动力学常数与在野生型RNase A再生研究中获得的常数进行比较，以进一步了解RNase A的折叠途径。还对其他几种三二硫键蛋白的氧化重折叠研究进行了比较，表明在氧化重折叠过程中形成大量二硫键结合中间体可能是大多数蛋白质的共同特征。
