Non-redox-active small-molecules can accelerate oxidative protein folding by novel mechanisms.
Multi-disulfide-bond-containing proteins acquire their native structures through an oxidative folding reaction which involves formation of native disulfide bonds through thiol-disulfide exchange reactions between cysteines and disulfides coupled to a conformational folding event. Oxidative folding rates of the four-disulfide-bond-containing protein bovine pancreatic ribonuclease A (RNase A) in the presence of the synthetic redox-active molecule, (+/-)-trans-1,2-bis(2-mercaptoacetamido)cyclohexane (BMC), and in combination with non-redox-active trimethylamine-N-oxide (TMAO), and trifluorethanol were determined by HPLC analysis. The data indicate that regeneration of RNase A is enhanced 2-fold by BMC (50 microM) and 3-fold upon addition of TMAO (0.2 M) and TFE (3% v/v) relative to control experiments performed in the absence of small-molecules. Examination of the native tendency of the fully-reduced polypeptide and the stability of key folding intermediates suggests that the increased oxidative folding rate can be attributed to native-like elements induced within the fully-reduced polypeptide and the stabilization of native-like species by added non-redox-active molecules.
Department of Chemistry, University of Texas at El Paso, Texas 79968, United States.
Matthew
M
Fink
Paul
P
Nieves
Saemin
S
Chang
Mahesh
M
Narayan
D000818:Animals; D002417:Cattle; D004220:Disulfides; D004795:Enzyme Stability; D007700:Kinetics; D009930:Organic Chemicals; D010084:Oxidation-Reduction; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry
D004220:Disulfides; D009930:Organic Chemicals; D012259:Ribonuclease, Pancreatic