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PEDS Advance Access published online on January 31, 2008
Protein Engineering Design and Selection, doi:10.1093/protein/gzm093
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

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

Robert F. Gahl, Mahesh Narayan1, Guoqiang Xu2 and Harold A. Scheraga3

Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853-1301

3 To whom correspondence should be addressed. E-mail: has5{at}cornell.edu

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, I1, 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 I3 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.

Keywords: onconase/oxidative folding/RNase A/structured intermediate

Received November 14, 2007; revised November 14, 2007; accepted November 20, 2007.

1 Present address: Department of Chemistry, University of Texas at El Paso, El Paso, TX 79968-0513

2 Present address: Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10065, USA


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