Chang J Y, Canals F, Schindler P, Querol E, Avilés F X
Pharmaceuticals Research Laboratories, Ciba-Geigy Ltd., Basel, Switzerland.
J Biol Chem. 1994 Sep 2;269(35):22087-94.
Potato carboxypeptidase inhibitor (PCI) contains 39 amino acids and three disulfides. Reduced and denatured PCI refolds spontaneously in vitro to regain its native structure. The folding pathway of a recombinant form of this protein has been elucidated by structural analysis and stop/go folding experiments of both acid and iodoacetate-trapped intermediates. The results reveal that folding of PCI proceeds through an initial stage of nonspecific disulfide formation (packing), followed by disulfide reshuffling (consolidation) of partially packed intermediates to acquire the native structure. The process of nonspecific packing involves a sequential flow of fully reduced PCI through 1- and 2-disulfide intermediates and leads to the formation of scrambled 3-disulfide species. All three classes of intermediates are highly heterogeneous. Disulfide reshuffling occurs at the final stage which refines and consolidates the scrambled species to acquire the native conformation. The efficiencies of disulfide formation and disulfide reshuffling can be selectively regulated by redox potential. Disulfide formation is promoted by cystine or oxidized glutathione, whereas disulfide reshuffling requires free thiols, such as cysteine, reduced glutathione, or beta-mercaptoethanol. Consolidation of scrambled species to form the native PCI represents the major rate-limiting step. When folding of PCI was carried out in the presence of cystine (2 mM) alone, more than 98% of the intermediates accumulate as the scrambled species after 1 min of folding. Furthermore, denaturant (5 M GdmCl or 8 M urea) mainly disrupts the final stage of PCI folding and exerts no apparent influence on the early stage of nonspecific packing. The compositions of 1- and 2-disulfide intermediates, according to their high performance liquid chromatography patterns, remain indistinguishable regardless of whether folding is performed in the absence or presence of denaturant.
马铃薯羧肽酶抑制剂（PCI）含有39个氨基酸和三个二硫键。还原和变性的PCI在体外可自发重折叠以恢复其天然结构。通过对该蛋白质重组形式的结构分析以及对酸和碘乙酸捕获的中间体进行的停止/继续折叠实验，阐明了其折叠途径。结果表明，PCI的折叠过程首先是通过非特异性二硫键形成（堆积）的初始阶段，然后是部分堆积中间体的二硫键重排（巩固）以获得天然结构。非特异性堆积过程涉及完全还原的PCI依次通过1-和2-二硫键中间体，并导致形成混乱的3-二硫键物种。所有这三类中间体都具有高度的异质性。二硫键重排在最后阶段发生，该阶段对混乱的物种进行细化和巩固以获得天然构象。二硫键形成和二硫键重排的效率可以通过氧化还原电位进行选择性调节。胱氨酸或氧化型谷胱甘肽促进二硫键形成，而二硫键重排需要游离巯基，如半胱氨酸、还原型谷胱甘肽或β-巯基乙醇。混乱物种的巩固以形成天然PCI是主要的限速步骤。当仅在胱氨酸（2 mM）存在下进行PCI折叠时，折叠1分钟后，超过98%的中间体以混乱物种的形式积累。此外，变性剂（5 M盐酸胍或8 M尿素）主要破坏PCI折叠的最后阶段，对非特异性堆积的早期阶段没有明显影响。根据其高效液相色谱图谱，无论折叠是在不存在还是存在变性剂的情况下进行，1-和2-二硫键中间体的组成都保持无法区分。
