PREFERENTIAL BINDING OF AN UNFOLDED PROTEIN TO DSBA

The oxidoreductase DsbA from the periplasm of Escherichia coli introdu ces disulfide bonds into proteins at an extremely high rate, During ox idation, a mixed disulfide is formed between DsbA and the folding prot ein chain, and this covalent intermediate reacts very rapidly either t o form the oxidized protein or to revert back to oxidized DsbA, To inv estigate its properties, a stable form of the intermediate was produce d 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 conform ational stability of DsbA is increased by 5 kJ/mol, This reciprocal ef fect suggests strongly that DsbA interacts with the unfolded substrate protein not only by the covalent disulfide bond, but also by preferen tial 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 impo rtant 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.