Dissimilarity in the oxidative folding of onconase and ribonuclease A, two structural homologues.
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), I(2) and I(3), with the rest of the unstructured species being converted to fully reduced protein. Mass spectrometry indicates that I(1) has two disulfide bonds, whereas I(2) and I(3) have three disulfide bonds each. A disulfide mapping method, based on cyanylation, was used to identify I(2) and I(3) as des-[30-75] and des-[19-68], respectively. On enzymatic digestion using trypsin, I(1) 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.
Gahl
Robert F
RF
Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853-1301, USA.
Narayan
Mahesh
M
Xu
Guoqiang
G
Scheraga
Harold A
HA
D000818:Animals; D001001:Anura / Q000378:metabolism; D002417:Cattle; D004220:Disulfides; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism*; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000378:metabolism
D004220:Disulfides; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; D014357:Trypsin; C058703:ranpirnase