Oxidative protein folding in bacteria

A critical step in the folding of newly synthesized proteins is the formation of native disulfide bonds between the thiol groups of two cysteine residues. In Escherichia coli, this disulfide bond formation occurs in the periplasm and is catalyzed by a protein called DsbA (Dsb stands for Disulfide Bond). DsbA is reoxidized by DsbB which generates disulfides de novo from oxidized quinones. DsbA is a powerful and rather nonspecific oxidant that has the potential of introducing non-native disulfides into proteins with multiple cysteines. In order to correct these non-native disulfides, the cell needs a disulfide isomerization system. The isomerization system in E. coli involves three other Dsb proteins: DsbC, DsbG and DsbD. DsbC and DsbG are two soluble protein disulfide isomerases that have a conserved pair of cysteines. The cysteines of DsbC and DsbG are found reduced in the periplasm and the protein responsible for the reduction of DsbC and DsbG is the inner-membrane protein DsbD. DsbD is a 59 kDa protein which has 3 different domains. Two domains, cc and gamma, are located in the periplasm and are connected by the membranous P domain. Each domain of DsbD possesses a conserved pair of cysteine residues, which are required for activity. Genetic studies have shown that DsbD receives its electrons from cytoplasmic thioredoxin, which is kept reduced by thioredoxin reductase and NADPH. We successfully reconstituted the isomerization pathway in vitro. We produced and purified separately the 3 domains of DsbD. Using the purified domains, we could reconstitute a DsbD activity in vitro. We showed that electrons are transferred from thioredoxin to the P domain then successively to the gamma domain, the alpha domain and finally on to DsbC or DsbG. We determined the redox potential of both the gamma and the alpha domains and showed that the electron now is both kinetically and thermodynamically driven. Moreover, our results indicate that the isomerization and oxidation pathways are kept separated from each other in the periplasm.