Disulfide-dependent folding and export of Escherichia coli DsbC.
DsbC, a member of the Dsb family in the periplasm of Gram-negative bacteria, is not only a disulfide isomerase but also a chaperone. Five DsbC mutants with Cys in the active site sequence of Cys(98)-Gly-Tyr-Cys(101) and the nonactive site disulfide Cys(141)-Cys(163) replaced by Ser have been studied. The results show that the active site Cys residues are necessary for enzyme activities but not required for chaperone activity, while the lack of the nonactive site disulfide results in a decreased chaperone activity in assisting the reactivation of denatured d-glyceraldehyde-3-phosphate dehydrogenase but has no effect on enzyme activities. Wild-type DsbC was overexpressed and correctly processed as a soluble periplasmic protein. Mutation in one of these Cys residues results in aggregation or extracellular/membrane locations, but does not affect the proper processing. DsbC mutated in either Cys residue of nonactive site disulfide shows higher sensitivity to unfolding by guanidine hydrochloride and slower refolding compared with wild-type DsbC and the active site Cys mutants. The above results provide experimental evidence for structural role of the nonactive site disulfide in folding and biological activities of DsbC.
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National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, 15 Datun Road, Beijing 100101, China.
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D019943:Amino Acid Substitution; D004220:Disulfides / Q000494:pharmacology*; D019286:Endopeptidase K; D004926:Escherichia coli / Q000201:enzymology*; D019791:Guanidine / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D004220:Disulfides; D007328:Insulin; D011994:Recombinant Proteins; D019286:Endopeptidase K; D019704:Protein Disulfide-Isomerases; D019791:Guanidine