A new method for rapid characterization of the folding pathways of multidisulfide-containing proteins.
Oxidative folding is a composite process that consists of both the conformational folding to the native three-dimensional structure and the regeneration of the native disulfide bonds of a protein, frequently involving over 100 disulfide intermediate species. Understanding the oxidative folding pathways of a multiple-disulfide-containing protein is a very difficult task that often requires years of devoted research due to the high complexity of the process and the very similar features of the large number of intermediates. Here we developed a method for rapidly delineating the major features of the oxidative folding pathways of a protein. The method examines the temperature dependence of the oxidative folding rate of the protein in combination with reduction pulses. Reduction pulses expose the presence of structured intermediates along the pathways. The correlation between the regeneration rate at different temperatures and the stability of the structured intermediates reveals the role that the intermediates play in determining the pathway. The method was first tested with bovine pancreatic ribonuclease A whose folding pathways were defined earlier. Then, it was explored to discern some of the major features of the folding pathways of its homologue, frog Onconase. The results suggest that the stability of the three-dimensional structure of the native protein is a major determinant of the folding rate in oxidative folding.
Welker
Ervin
E
Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
Hathaway
Laura
L
Scheraga
Harold A
HA
D000818:Animals; D001001:Anura; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D010179:Pancreas / Q000201:enzymology; D011487:Protein Conformation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D012260:Ribonucleases / Q000737:chemistry*; D013696:Temperature
D004220:Disulfides; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; C058703:ranpirnase