Dissimilarity in the oxidative folding of onconase and ribonuclease A, two structural homologues

Abstract

The oxidative folding of frog onconase (ONC), a member of the ribonuclease A family, was examined and shows markedly different behavior compared to its structural homologue bovine pancreatic ribonuclease A (RNase A) under similar conditions. Application of a reduction pulse (using a small amount of reduced dithiothreitol) during the oxidative regeneration of ONC indicated the survival of the native protein along with three other (structured) species, I 1, I2 and I3, with the rest of the unstructured species being converted to fully reduced protein. Mass spectrometry indicates that I1 has two disulfide bonds, whereas I2 and I 3 have three disulfide bonds each. A disulfide mapping method, based on cyanylation, was used to identify I2 and I3 as des-[30-75] and des-[19-68], respectively. On enzymatic digestion using trypsin, I1 was identified as des-[19-68, 30-75]. Differences in the intermediates that are generated during the oxidative folding of the two structural homologues, RNase A and ONC, demonstrate that regenerative pathways are not necessarily influenced by tertiary structure. This indicates that the lack of a disulfide bond in ONC, analogous to the (65-72) disulfide bond in RNase A which plays an important role in its oxidative regeneration, does not adversely affect the oxidative folding of ONC. © The Author 2008. Published by Oxford University Press. All rights reserved.