Native conformational tendencies in unfolded polypeptides: development of a novel method to assess native conformational tendencies in the reduced forms of multiple disulfide-bonded proteins.

Oxidative folding is the concomitant formation of the native disulfide bonds and the native tertiary structure from the reduced and unfolded polypeptide. Of interest is the inherent conformational tendency (bias) present in the reduced polypeptide to dictate the formation of the full set of native disulfide bonds. Here, by application of a novel tool, we have been able to assess this "native conformational tendency" present in reduced and unfolded bovine pancreatic ribonuclease A (RNase A). The essence of this method lies in the ability of the oxidant Pt(en)(2)Cl(2) (where "en" is ethylenediamine) to oxidize disulfide bonds under conditions in which both reduction and disulfide reshuffling, which are essential for rearranging non-native disulfide bonds, are extremely slow. When applied to RNase A, the method revealed little or no bias toward formation of the full native set of disulfide bonds in the fully reduced protein.