Shifting the competition between the intramolecular Reshuffling reaction and the direct oxidation reaction during the oxidative folding of kinetically trapped disulfide-insecure intermediates.
The oxidative folding pathway(s) of single-domain proteins can be characterized by the existence, stability, and structural nature of the intermediates that populate the regeneration pathway. Structured intermediates can be disulfide-secure in that they are able to protect their existing (native) disulfide bonds from SH/SS reshuffling and reduction reactions, and thereby form the native protein directly, i.e., by oxidation of their exposed (or locally exposable) thiols. Alternatively, they can be disulfide-insecure, usually requiring global unfolding to expose their free thiols. However, such an unfolding event also exposes the existing native disulfide bonds. Thus, the subsequent oxidation reaction to form the native protein in a disulfide-insecure intermediate competes with the intramolecular attack by the thiols of the macromolecule on its own native disulfide bonds, resulting in a large population of intermediates that are reshuffled instead of being oxidized. Under stabilizing conditions, disulfide-insecure species become long-lived kinetically trapped intermediates that slowly and only indirectly convert to the native protein through reshuffling reactions. In this study, trans-[Pt(en)(2)Cl(2)](2+), a strong oxidizing agent which has not traditionally been used in oxidative folding, was applied to shift the competition between reshuffling and oxidation reactions in des [58-110] and des [26-84], two long-lived disulfide-insecure intermediates of RNase A, and oxidize them directly under stable conditions to form the native protein. Such a successful direct conversion of kinetically trapped intermediates to the native molecule by trans-[Pt(en)(2)Cl(2)](2+) may be helpful in facilitating the oxidative folding processes of multi-disulfide-containing proteins in general.
Narayan
Mahesh
M
Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
Welker
Ervin
E
Wanjalla
Celestine
C
Xu
Guoqiang
G
Scheraga
Harold A
HA
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry*; D004226:Dithioerythritol / Q000737:chemistry; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives* / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D009944:Organoplatinum Compounds / Q000737:chemistry; D010084:Oxidation-Reduction; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D004220:Disulfides; D009944:Organoplatinum Compounds; D013438:Sulfhydryl Compounds; C093171:methanethiosulfonate ethylammonium; D004226:Dithioerythritol; D005020:Ethyl Methanesulfonate; D012259:Ribonuclease, Pancreatic; D019791:Guanidine