Characterizing the unstructured intermediates in oxidative folding.
A recently developed method is used here to characterize some of the folding intermediates, and the oxidative folding processes, of RNase A. This method is based on the ability of trans-[Pt(en)(2)Cl(2)](2+) to oxidize cysteine residues to form disulfide bonds faster than the disulfide bonds can be rearranged by reshuffling or reduction. Variations of this method have enabled us to address three issues. (i) How the nature of the residual structure and/or conformational order that is present, or develops, during the initial stages of folding can be elucidated. It is shown here that there is a 10-fold increase in the propensity of the unfolded reduced forms of RNase A to form the native set of disulfides directly, compared to the propensity under strongly denaturing conditions (4-6 M GdnHCl). Thus, the unfolded reduced forms of RNase A are not statistical coils with a more condensed form than in the GdnHCl-denatured state; rather, it is suggested that reduced RNase A has a little bias toward a native topology. (ii) The structural characterization of oxidative folding intermediates in terms of disulfide pairing is demonstrated; specifically, a lower-limit estimate is made of the percentage of native disulfide-containing molecules in the two-disulfide ensemble of RNase A. (iii) The critical role of structured intermediate species in determining the oxidative folding pathways of proteins was shown previously. Here, we demonstrate that the presence of a structured intermediate in the oxidative folding of proteins can be revealed by this method.
Narayan
Mahesh
M
Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
Welker
Ervin
E
Scheraga
Harold A
HA
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D008954:Models, Biological; D009944:Organoplatinum Compounds / Q000737:chemistry; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*
D004220:Disulfides; D009944:Organoplatinum Compounds; D012259:Ribonuclease, Pancreatic; D019791:Guanidine; D003545:Cysteine