Wearne S J, Creighton T E
Medical Research Council, Laboratory of Molecular Biology, Cambridge, England.
Proteins. 1988;4(4):251-61. doi: 10.1002/prot.340040404.
Two very different mechanisms of folding have been proposed from experimental studies of disulfide formation in reduced ribonuclease A. (1) A pathway in which the rate-limiting step separates fully folded protein from all other disulfide intermediates and occurs solely in three-disulfide intermediates. (2) A multiple pathway mechanism with different rate-limiting steps for each pathway. The various rate-limiting steps involve disulfide breakage, formation, and rearrangement in intermediates with one, two, three, and four protein disulfides. To distinguish between these two mechanisms, we have carried out further studies of both unfolding and refolding. Refolding of reduced ribonuclease A requires three-disulfide intermediates to accumulate; negligible refolding occurs when only the nearly random one- and two-disulfide intermediate species are populated. Therefore, no rate-limiting steps of the type postulated in mechanism (2) occur in intermediates with one and two protein disulfides. Unfolding and disulfide reduction is an all-or-none process; no disulfide intermediates accumulate to detectable levels or precede the rate-limiting step. Mechanism (2) requires that such intermediates precede the rate-limiting step and accumulate to substantial levels. The different proposals were shown not to result from the use of different solution conditions or disulfide reagents; the two sets of data are not inconsistent. Instead, the inappropriate mechanism (2) resulted from an incorrect kinetic analysis and misinterpretation of the kinetics of disulfide formation and breakage.
通过对还原型核糖核酸酶A中二硫键形成的实验研究，人们提出了两种截然不同的折叠机制。(1) 一种途径，其中限速步骤将完全折叠的蛋白质与所有其他二硫键中间体分离，且仅在三硫键中间体中发生。(2) 一种多途径机制，每条途径具有不同的限速步骤。各种限速步骤涉及在具有一、二、三、四个蛋白质二硫键的中间体中二硫键的断裂、形成和重排。为了区分这两种机制，我们对解折叠和重折叠都进行了进一步研究。还原型核糖核酸酶A的重折叠需要三硫键中间体积累；当仅存在几乎随机的一硫键和二硫键中间体时，重折叠发生的量可以忽略不计。因此，在具有一、两个蛋白质二硫键的中间体中，不会发生机制(2)中假设的那种限速步骤。解折叠和二硫键还原是一个全或无的过程；没有二硫键中间体积累到可检测的水平，也不会先于限速步骤。机制(2)要求这些中间体先于限速步骤并积累到相当的水平。结果表明，不同的提议并非源于使用不同的溶液条件或二硫键试剂；这两组数据并非不一致。相反，不恰当的机制(2)是由于错误的动力学分析以及对二硫键形成和断裂动力学的错误解读。
