Characterization of the fast-forming intermediate, des [30-75], in the reductive unfolding of onconase.
A fast-forming intermediate in the reductive unfolding of frog onconase (ONC), des [30-75], analogous to the des [40-95] intermediate found in the reductive unfolding of its structural homologue, bovine pancreatic ribonuclease A (RNase A), has been isolated and characterized. The midpoints of the thermal transition and chemical denaturing curves (representing global unfolding) indicate that the conformation of des [30-75] is considerably less stable than that of the parent molecule, suggesting that the (30-75) disulfide bond plays a significant role in the conformational stability of ONC. While des [30-75] is formed very quickly by a partial reduction of the parent molecule in a local unfolding step, it is not as easily susceptible to further reduction, indicating that its three disulfides are much more buried compared to the (30-75) disulfide bond in the parent protein. The nature of des [30-75] is similar to that of des [40-95] RNase A, in that des [30-75] ONC is also a disulfide-secure species. In addition, based on the resistance to mild reducing conditions, structured des species appear to form in ONC from unstructured three-disulfide-containing ensembles. This step is key in the oxidative folding of RNaseA, and is much faster in ONC than the formation of the structured des [40-95] species in RNase A.
Xu
Guoqiang
G
Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
Narayan
Mahesh
M
Welker
Ervin
E
Scheraga
Harold A
HA
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry; D019277:Entropy; D004795:Enzyme Stability; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D011894:Rana pipiens; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D012260:Ribonucleases / Q000737:chemistry*; D040681:Structural Homology, Protein
D004220:Disulfides; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; D019791:Guanidine; C058703:ranpirnase