Circular dichroism spectroscopy of the intermediates that precede the rate-limiting step of the refolding pathway of bovine pancreatic trypsin inhibitor. Relationship of conformation and the refolding pathway

Abstract

Circular dichroism spectra of the partially folded trapped intermediates were measured to aid in the elucidation of the conformational forces which determine a nonrandom, nonsequential pathway of disulfide bond formation upon refolding of bovine pancreatic trypsin inhibitor. Whatever conformation was responsible for the kinetic rates of the intermediates should be stabilized by the presence of their trapped disulfide bonds. The near-UV spectra provide considerable information about the environments of the aromatic and disulfide side chains. The predominant single-disulfide intermediate has significant nonrandom conformation not present in the fully reduced protein, with aromatic rings and the disulfide bond in stabilized asymmetric environments. Forming either of the 2 nonnative, but kinetically important, second disulfides in this intermediate does not produce unequivocably different conformations. Forming a second native (but kinetically unproductive) disulfide produces a substantial decrease in randomness, which may hinder formation of the third disulfide. The largest conformational changes occur upon disulfide rearrangement to the stable, correctly refolded, 2- and 3-disulfide species. Interpretation of the far-UV spectra in terms of the secondary structure of the intermediates is uncertain due to the atypical spectra of the folded forms of the protein. Consequently, the secondary structure of the intermediates could not be determined unambiguously. However, all the spectra show that nonrandom conformations of the polypeptide chain gradually appear as disulfide bond formation progresses, as expected from the nonrandom pathway of the latter.