Circular dichroism, Raman spectroscopy, and gel filtration of trapped folding intermediates of ribonuclease

Abstract

The intermediates of [bovine pancreatic] RNase with 1-4 disulfide bonds trapped during refolding of the reduced protein were compared to the fully reduced and native forms of the protein by gel filtration, circular dichroism and Raman spectroscopy. Correctly refolded RNase is indistinguishable from native protein, while a 3 disulfide intermediate has a compact conformation which is very similar but not identical. All other intermediates with 1-4 disulfide bonds are qualitatively similar to fully reduced ribonuclease by their circular dichroism and Raman spectra, although the disulfide cross-links affect the dimensions of the polypeptide chain. The apparent absence of stable partially ordered structures in the initial disulfide intermediates implies that during refolding there are relatively few constraints on formation on disulfide bonds, although their formation is probably not entirely random. The stable conformation appears during refolding only when the 3 or 4 disulfide bonds capable of stabilizing a native-like conformation of the entire polypeptide chain occur simultaneously.