THE REACTIVE AND DESTABILIZING DISULFIDE BOND OF DSBA, A PROTEIN REQUIRED FOR PROTEIN DISULFIDE BOND FORMATION INVIVO

The protein DsbA facilitates disulfide bond formation in the periplasm of Escherichia coli. It has only two cysteine residues that are separ ated in the sequence by two other residues and are shown to form a dis ulfide bond reversibly. Chemical modification studies demonstrate that only one of the cysteine residues has an accessible thiol group in th e reduced protein. Equilibrium and kinetic characterization of thiol-d isulfide exchange between DsbA and glutathione showed that the DsbA di sulfide bond was 10(3)-fold more reactive than a normal protein disulf ide. Similarly, the mixed disulfide between the accessible cysteine re sidue and glutathione was 10(4)-fold more reactive than normal. The ov erall equilibrium constant for DsbA disulfide bond formation from GSSG was only 8 X 10(-5) M. These properties indicate that disulfide-bonde d DsbA is a potent oxidant and ideally suited for generating protein d isulfide bonds. Disulfide bonds generally increase the stabilities of folded proteins, when the folded conformation reciprocally stabilizes the disulfide bonds. In contrast, the disulfide bond of DsbA was so un stable in the folded state that its stability increased by 4.5 +/- 0.1 kcal.mol-1 when the protein unfolded. This implies that the disulfide bond destabilizes the folded conformation of DsbA. This was confirmed by demonstrating that the reduced protein was 3.6 +/-1.4 kcal.mol-1 m ore stable than that with the disulfide bond.