The unfolding pathway and conformational stability of potato carboxypeptidase inhibitor

The unfolding and denaturation curves of potato carboxypeptidase inhibitor (PCI) were investigated using the technique of disulfide scrambling. In the presence of denaturant and thiol initiator, the native PCI denatures by sh uffling its native disulfide bonds and converts to form a mixture of scramb led PCI that consists of 9 out of a possible 14 isomers. The denaturation c urve is determined by the fraction of native PCI converted to scrambled iso mers under increasing concentrations of denaturant. The concentration of gu anidine thiocyanate, guanidine hydrochloride, and urea required to denature 50% of the native PCI was found to be 0.7, 1.45, and 8 M, respectively. Th e PCI unfolding curve was constructed through the analysis of structures of scrambled isomers that were denatured under increasing concentrations of d enaturant. These results reveal the existence of structurally defined unfol ding intermediates and a progressive expansion of the polypeptide chain. Th e yield of the beads-form isomer (Cys(8)-Cys(12), Cys(18)-Cys(24), and Cys( 27)-Cys(34)) as a fraction of total denatured PCI was shown to be directly proportional to the strength of the denaturing condition. Furthermore, the PCI sequence was unable to fold quantitatively into a single native structu re, Under physiological conditions, the scrambled isomers of PCI that const itute about 4% of the protein were in equilibrium with native PCI.