Characterization of kinetic and thermodynamic phases in the prefolding process of bovine pancreatic ribonuclease A coupled with fast SS formation and SS reshuffling.
In the redox-coupled oxidative folding of a protein having several SS bonds, two folding phases are usually observed, corresponding to SS formation (oxidation) with generation of weakly stabilized heterogeneous structures (a chain-entropy losing phase) and the subsequent intramolecular SS rearrangement to search for the native SS linkages (a conformational folding phase). By taking advantage of DHS(ox) as a highly strong and selective oxidant, the former SS formation phase was investigated in detail in the oxidative folding of RNase A. The folding intermediates obtained at 25 °C and pH 4.0 within 1 min (1S°-4S°) showed different profiles in the HPLC chromatograms from those of the intermediates obtained at pH 7.0 and 10.0 (1S-4S). However, upon prolonged incubation at pH 4.0 the profiles of 1S°-3S° transformed slowly to those similar to 1S-3S intermediate ensembles via intramolecular SS reshuffling, accompanying significant changes in the UV and fluorescence spectra but not in the CD spectrum. Similar conversion of the intermediates was observed by pH jump from 4.0 to 8.0, while the opposite conversion from 1S-4S was observed by addition of guanidine hydrochloride to the folding solution at pH 8.0. The results demonstrated that the preconformational folding phase coupled with SS formation can be divided into two distinct subphases, a kinetic (or stochastic) SS formation phase and a thermodynamic SS reshuffling phase. The transition from kinetically formed to thermodynamically stabilized SS intermediates would be induced by hydrophobic nucleation as well as generation of the native interactions.
Department of Chemistry, School of Science, Tokai University, Kitakaname, Hiratsuka-shi, Kanagawa 259-1292, Japan.
Kenta
K
Arai
Fumio
F
Kumakura
Michio
M
Iwaoka
D000818:Animals; D002417:Cattle; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics; D019277:Entropy; D007700:Kinetics; D010084:Oxidation-Reduction; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics*; D013816:Thermodynamics
D004220:Disulfides; D012259:Ribonuclease, Pancreatic