Effects of DsbA on the disulfide folding of bovine pancreatic trypsin inhibitor and alpha-lactalbumin.
DsbA is a protein found in the periplasm of Escherichia coli that is required for the formation of disulfide bonds in secreted proteins. It contains only two cysteine residues, which can form reversibly a very unstable disulfide bond that has been proposed to be the oxidant that introduces disulfide bonds into secreted proteins. The present study investigates the effect of DsbA on the well-characterized disulfide-coupled refolding processes of BPTI and of alpha-lactalbumin. Disulfide-bonded DsbA in stoichiometric amounts proved to be a very potent donor of disulfide bonds to reduced BPTI but showed little catalytic activity at neutral pH in the presence of a glutathione redox buffer. In contrast to the related eukaryotic enzyme protein disulfide isomerase, DsbA did not substantially catalyze the usual intramolecular disulfide bond rearrangements of quasi-native folding intermediates of BPTI. Neither did DsbA catalyze the intramolecular rearrangements observed in the three disulfide-bonded "molten globule" form of alpha-lactalbumin at neutral pH. Thiol-disulfide exchange is normally very slow at acidic pH but occurs rapidly with DsbA; consequently, DsbA catalyzed the disulfide folding of BPTI under acidic conditions. It was then possible to detect some increase in the rates of disulfide rearrangements, but only with stoichiometric amounts of DsbA and on the hour time scale. These results suggest that the primary role of DsbA in the bacterial periplasm is to introduce disulfide bonds into newly secreted proteins.
Zapun
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European Molecular Biology Laboratory, Heidelberg, Germany.
Creighton
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D000595:Amino Acid Sequence; D000818:Animals; D007611:Aprotinin / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D004220:Disulfides; D007535:Isomerases / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding
D004220:Disulfides; D007768:Lactalbumin; D007611:Aprotinin; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D003545:Cysteine