Immunochemical analysis of the conformational properties of intermediates trapped in the folding and unfolding of bovine pancreatic trypsin inhibitor.

Immunochemical methods have been used to examine the conformational properties of the entire polypeptide chain in the various trapped intermediate states which are kinetically important in the unfolding and refolding of pancreatic trypsin inhibitor. The interactions of each of the trapped intermediates, having their disulphide bonds frozen, with antibodies specific for either the native, folded or the reduced, unfolded states of the entire protein have been used to determine the probabilities of the various segments of the polypeptide chain adopting either conformation recognized by the antibodies.The results are considered with regard to the kinetic roles of the various species and to their conformational properties during folding and unfolding inferred from the observed propensities of each of the six cysteine residues to participate in disulphide bond formation, interchange, or breakage. It is concluded that no segment of the polypeptide chain adopts a stable native-like conformation until the entire polypeptide chain is able to do so simultaneously. The best correlation of conformation with the kinetic role in refolding of the intermediates is observed not with their propensity to adopt native-like conformation, but with their stability to full unfolding as measured by their interaction with antibodies against the reduced protein.