In vivo control of redox potential during protein folding catalyzed by bacterial protein disulfide-isomerase (DsbA).
The formation of disulfide bonds in Escherichia coli is catalyzed by periplasmic protein disulfide-isomerase (DsbA). When the alpha-amylase/trypsin inhibitor from Ragi, a protein containing five intramolecular disulfide bridges, is secreted into the periplasm of E. coli, large amounts of misfolded inhibitor with incomplete or incorrect disulfides are accumulated. Folding of the inhibitor in the periplasm is not improved when DsbA is coexpressed and cosecreted. However, an up to 14-fold increase in correctly folded inhibitor is observed by co-expression of DsbA in conjugation with the addition of reduced glutathione to the growth medium. This peptide acts as a disulfide-shuffling reagent and can pass the outer membrane of E. coli. Since the influence of DsbA on the folding yield of the inhibitor is reduced in the presence of oxidized glutathione, the in vivo function of DsbA appears to be dependent on the ratio between oxidizing and reducing thiol equivalents in the periplasm. The high stability of thiol reagents against air oxidation during growth of E. coli allows the investigation of oxidative protein folding in vivo under controlled, thiol-dependent redox conditions.
Wunderlich
M
M
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Glockshuber
R
R
D000595:Amino Acid Sequence; D000681:Amylases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D002523:Edible Grain / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005978:Glutathione / Q000378:metabolism; D007535:Isomerases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010940:Plant Proteins; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000378:metabolism*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
D010940:Plant Proteins; D014361:Trypsin Inhibitors; D000681:Amylases; D000516:alpha-Amylases; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione