Distribution of disulfide bonds in the two-disulfide intermediates in the regeneration of bovine pancreatic ribonuclease A: Further insights into thefolding process

The distribution of one-disulfide bonds in the two-disulfide intermediates in the oxidative refolding of bovine pancreatic ribonuclease A has been cha racterized. These two-disulfide intermediates were formed from the fully re duced denatured protein by oxidation with dithiothreitol, then blocked with AEMTS, purified by cation-exchange chromatography, enzymatically digested, and analyzed by reversed-phase high-performance liquid chromatography and mass spectrometry. The relative concentration of each of the 28 possible on e-disulfide bonds in the two-disulfide ensemble was determined. Comparison with a statistical mechanical treatment of loop formation shows that the tw o-disulfide intermediates are probably compact. All 28 disulfide bonds were observed, demonstrating the absence of specific long-range interactions in these intermediates. Thermodynamic arguments suggest that the absence of s uch specific long-range interactions in the two-disulfide species may eleva te the concentration of kinetically important three-disulfide intermediates and thereby increase the folding rate. Bond [65-72] was found to make up s imilar to 27% of the disulfide bonds of the two-disulfide species, signific antly more than all other disulfides, because of stabilization by loop entr opy factors and an energetically favorable beta-turn. This turn may be one of several chain-folding initiation sites, accelerating folding by decreasi ng the dimensionality of the conformational space that has to be searched.