Reconstitution of a disulfide isomerization system.
Isomerization of disulfide bonds is vital for the proper folding of proteins that possess multiple disulfides. In prokaryotes, the catalytic pathway responsible for disulfide isomerization involves thioredoxin, thioredoxin reductase, and the DsbC, DsbG, and DsbD proteins. To be active as isomerases, DsbC and DsbG must be kept reduced. This task is performed by the cytoplasmic membrane protein DsbD. DsbD in turn is reduced by the cytoplasmic thioredoxin and is composed of three domains. The beta domain is membrane-embedded, whereas the alpha and gamma domains are localized to the periplasm. It had been proposed that electrons are transferred within DsbD by a succession of disulfide exchange reactions between the three domains. To test this model using biochemical methods, we purified to homogeneity different polypeptides corresponding to the alpha, beta, gamma, and betagamma domains. Using these domains, we could reconstitute a DsbD activity and, for the first time, reconstitute in vitro the electron transport pathway from NADPH and thioredoxin to DsbC and DsbG. We showed that electrons are transferred from thioredoxin to the beta domain then successively to the gamma domain, the alpha domain, and finally on to DsbC or DsbG. We also determined the redox potential of the gamma domain to be -241 mV, and that of the alpha domain was found to be -229 mV. This shows that the direction of electron flow within DsbD is thermodynamically driven.
Collet
Jean-François
JF
Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 N. University, Ann Arbor, MI 48109-1048, USA.
Riemer
Jan
J
Bader
Martin W
MW
Bardwell
James C A
JC
D002384:Catalysis; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003593:Cytoplasm / Q000378:metabolism; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D007700:Kinetics; D008954:Models, Biological; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D033903:Periplasmic Proteins; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013816:Thermodynamics; D013879:Thioredoxins / Q000378:metabolism; D013997:Time Factors
D004220:Disulfides; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins; D010088:Oxidoreductases; C109831:DsbG protein, E coli; D019704:Protein Disulfide-Isomerases