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The protein DsbA facilitates disulfide bond formation in the periplasm of Escherichia coli. It has only two cysteine residues that are separated in the sequence by two other residues and are shown to form a disulfide bond reversibly. Chemical modification studies demonstrate that only one of the cysteine residues has an accessible thiol group in the reduced protein. Equilibrium and kinetic characterization of thiol-disulfide exchange between DsbA and glutathione showed that the DsbA disulfide bond was 10(3)-fold more reactive than a normal protein disulfide. Similarly, the mixed disulfide between the accessible cysteine residue and glutathione was 10(4)-fold more reactive than normal. The overall equilibrium constant for DsbA disulfide bond formation from GSSG was only 8 x 10(-5) M. These properties indicate that disulfide-bonded DsbA is a potent oxidant and ideally suited for generating protein disulfide 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 unstable 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 more stable than that with the disulfide bond.

DsbA is a periplasmic protein of Escherichia coli that appears to be the immediate donor of disulfide bonds to proteins that are secreted. Its active site contains one accessible and one buried cysteine residue, Cys30 and Cys33, respectively, which can form a very unstable disulfide bond between them that is 10(3)-fold more reactive toward thiol groups than normal. The two cysteine residues have normal properties when in a short peptide. In DsbA, the Cys30 thiol group is shown to be reactive toward alkylating reagents down to pH 4 and to be fully ionized, on the basis of the UV absorbance of the thiolate anion at 240 nm. Its reactivity is altered by another, unknown group on the reduced protein titrating with a pKa of about 6.7. The other cysteine residue is buried and unreactive and has a high pKa value. The ionization properties of the DsbA thiol groups can explain, at least partly, the high reactivity of its disulfide bonds and thiol groups at both neutral and acidic pH values.

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DsbC is a soluble protein of the bacterial periplasm that was identified genetically as being involved in protein disulfide formation. The gene sequence was corrected to include an additional proline residue and was then consistent with the molecular weight of the purified protein. Gel filtration and subunit hybridization indicate that DsbC is a stable dimer of identical subunits. Each subunit has a -Cys-Gly-Tyr-Cys- segment that forms an unstable and reactive disulfide bond; only the first cysteine residue is accessible, similar to thioredoxin and DsbA. The other two cysteine residues of DsbC form a buried, structural disulfide bond. The reactivities and stabilities of the active site disulfide bond of DsbC have been characterized and compared to that of DsbA. Both are very unstable and can be transferred rapidly to reduced proteins and peptides, although they differ somewhat in their kinetic reactivities. The two active sites of the DsbC dimer appear to function independently. DsbC is much more active than DsbA in catalyzing protein disulfide rearrangements, and this may be its main function in vivo.

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Our determination of the global fold of the b domain of PDI by NMR reveals that, like the a domain, the b domain contains the thioredoxin motif, even though the b domain has no significant amino-acid sequence similarities to any members of the thioredoxin family. This observation, together with indications that the b' domain adopts a similar fold, suggests that PDI consists of active and inactive thioredoxin modules. These modules may have been adapted during evolution to provide PDI with its complete spectrum of enzymatic activities.

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