Distribution of Disulfide Bonds in the Two-Disulfide Intermediates in the Regeneration of Bovine Pancreatic Ribonuclease A: Further Insights into the Folding Process

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Abstract
The distribution of one-disulfide bonds in the two-disulfide intermediates in the oxidative refolding of bovine pancreatic ribonuclease A has been characterized. These two-disulfide intermediates were formed from the fully reduced 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 one-disulfide bonds in the two-disulfide ensemble was determined. Comparison with a statistical mechanical treatment of loop formation shows that the two-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 such specific long-range interactions in the two-disulfide species may elevate the concentration of kinetically important three-disulfide intermediates and thereby increase the folding rate. Bond [65−72] was found to make up ∼27% of the disulfide bonds of the two-disulfide species, significantly more than all other disulfides, because of stabilization by loop entropy factors and an energetically favorable β-turn. This turn may be one of several chain-folding initiation sites, accelerating folding by decreasing the dimensionality of the conformational space that has to be searched.