Assessing the magnitude of folding forces along the oxidative folding pathway of multi-disulfide-containing proteins.

Oxidative folding involves the acquisition of the native set of disulfide bonds and native structure of a protein from its reduced, unfolded form. Two major factors that influence the distribution of disulfide bonds (over their entropically favored probability of formation) during the regeneration, eventually favoring and sustaining the native set of disulfide bonds, are enthalpic interactions that lead to conformational ordering among intermediates and conformational folding, resulting in native (or nativelike) structure which protects the native disulfide bonds from reduction and intramolecular thiol-disulfide exchange reactions. Here, we report a novel technique to provide a quantitative description of the enthalpic and conformational folding forces present at any stage of the oxidative folding process of multi-disulfide-containing proteins which is made feasible by assessing their ability to form "structures" that are able to protect disulfide bonds from reduction.