The disulfide folding pathway of potato carboxypeptidase inhibitor.
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.
Chang
J Y
JY
Pharmaceuticals Research Laboratories, Ciba-Geigy Ltd., Basel, Switzerland.
Canals
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Schindler
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Querol
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D002268:Carboxypeptidases / Q000037:antagonists & inhibitors*; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000378:metabolism*; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000378:metabolism*; D011480:Protease Inhibitors; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011198:Solanum tuberosum / Q000378:metabolism*
D004220:Disulfides; D010940:Plant Proteins; D011480:Protease Inhibitors; D002268:Carboxypeptidases