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Reconstitution of a disulfide isomerization system

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Collet, Jean-François [UCL] Riemer, Jan Bader, Martin Bardwell, James

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 a 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 a domain was found to be -229 mV. This shows that the direction of electron flow within DsbD is thermodynamically driven.

Document type Article de périodique (Journal article) – Article de recherche
Access type Accès restreint
Publication date 2002
Language Anglais
Journal information "Journal of Biological Chemistry" - Vol. 277, no. 30, p. 26886-26892 (2002)
Peer reviewed yes
Publisher Amer Soc Biochemistry Molecular Biology Inc (Bethesda)
issn 0021-9258
e-issn 1083-351X
Publication status Publié
Affiliations UCL - MD/BICL - Département de biochimie et de biologie cellulaire
University of Michigan - Department of Molecular, Cellular and Developmental Biology
Links
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Bibliographic reference Collet, Jean-François ; Riemer, Jan ; Bader, Martin ; Bardwell, James. Reconstitution of a disulfide isomerization system. In: Journal of Biological Chemistry, Vol. 277, no. 30, p. 26886-26892 (2002)
Permanent URL http://hdl.handle.net/2078.1/41804