Mutations in the N- and C-terminal tails of potato carboxypeptidase inhibitor influence its oxidative refolding process at the reshuffling stage

A comparative study of the oxidative refolding for nine selected potato car boxypeptidase inhibitor (PCI) mutants was carried out using the disulfide q uenching approach. The mutations were performed at the N- and C-terminal ta ils of PCI outside its disulfide stabilized central core. The differences b etween the refolding of wild type and mutant proteins were observed in the second phase of the refolding process, the reshuffling of disulfide bridges , although the first phase, nonspecific packing, was not greatly affected b y the mutations. Point mutations at the C-tail or deletion of up to three C -terminal residues of PCI resulted in a lower efficiency of the reshuffling process. In the case of the mutants lacking five N-terminal or four or fiv e C-terminal residues, no "native-like" form was observed after the refoldi ng process. On the other hand, the double mutant G35P/P36G did not attain a native-like form either, al though one slightly more stable species was ob served after being submitted to refolding, The disulfide pairing of this sp ecies is different from that of the wtPCI native form. The differences betw een the refolding process of wild type and mutant forms are interpreted in the light of the new view of protein folding. The results of the present st udy support the hypothesis that the refolding of this small disulfide-rich protein, and others, is driven by noncovalent interactions at the reshuffli ng stage. It is also shown that the interactions established between the N- and C-tail residues and the core of PCI are important for the proper refol ding of the protein.