Preferential binding of an unfolded protein to DsbA.
The oxidoreductase DsbA from the periplasm of escherichia coli introduces disulfide bonds into proteins at an extremely high rate. During oxidation, a mixed disulfide is formed between DsbA and the folding protein chain, and this covalent intermediate reacts very rapidly either to form the oxidized protein or to revert back to oxidized DsbA. To investigate its properties, a stable form of the intermediate was produced by reacting the C33A variant of DsbA with a variant of RNase T1. We find that in this stable mixed disulfide the conformational stability of the substrate protein is decreased by 5 kJ/mol, whereas the conformational stability of DsbA is increased by 5 kJ/mol. This reciprocal effect suggests strongly that DsbA interacts with the unfolded substrate protein not only by the covalent disulfide bond, but also by preferential non-covalent interactions. The existence of a polypeptide binding site explains why DsbA oxidizes protein substrates much more rapidly than small thiol compounds. Such a very fast reaction is probably important for protein folding in the periplasm, because the accessibility of the thiol groups for DsbA can decrease rapidly when newly exported polypeptide chains begin to fold.
Frech
C
C
Laboratorium fur Biochemie, Universitat Bayreuth, Germany.
Wunderlich
M
M
Glockshuber
R
R
Schmid
F X
FX
D004220:Disulfides; D004926:Escherichia coli / Q000201:enzymology; D005978:Glutathione; D019791:Guanidine; D006146:Guanidines; D007535:Isomerases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D017354:Point Mutation; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D006163:Ribonuclease T1 / Q000737:chemistry* / Q000378:metabolism*; D012965:Sodium Chloride; D013997:Time Factors
D004220:Disulfides; D006146:Guanidines; D011994:Recombinant Proteins; D012965:Sodium Chloride; D006163:Ribonuclease T1; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D019791:Guanidine