Replacement of the active-site cysteine residues of DsbA, a protein required for disulfide bond formation in vivo.
DsbA is a periplasmic protein of Escherichia coli that was identified genetically as being involved in the formation of disulfide bonds in secreted proteins. Its active site contains one accessible and one buried cysteine residue, separated in the primary structure by only two other residues. These cysteine residues can form a very unstable disulfide bond that is 10(3)-fold more reactive toward thiols than normal. Moreover, the mixed disulfide between the accessible cysteine residue and glutathione is 10(4)-fold more reactive than normal. Site-directed mutagenesis was carried out to replace either one or both cysteine residues by serine. Cys30 is shown to be the accessible thiol, while Cys33 is shielded from the solvent. Even though the thiol group of Cys30 is exposed and reactive, it formed a very unstable mixed disulfide with glutathione. This disulfide bond was 2.17 +/- 0.02 kcal mol-1 less stable in the native conformation than when DsbA was unfolded. If the native conformation destabilizes the mixed disulfide, the mixed disulfide must destabilize the folded conformation to the same extent. This was confirmed by demonstrating that the folded conformation of the mixed disulfide form of the mutant DsbA was 2.7 +/- 0.9 kcal mol-1 less stable than that of the reduced form; these stability effects originated almost exclusively in the folded conformation. Replacing the cysteine residues by serine destabilized the folded conformation of the reduced protein to varying extents. This suggests that the thiol groups are involved in interactions that stabilize the folded conformation, which would cause any disulfide bonds, either inter- or intramolecular, that involve these groups to be unstable.
Zapun
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European Molecular Biology Laboratory, Heidelberg, Germany.
Cooper
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Creighton
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D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D005978:Glutathione / Q000378:metabolism; D007460:Iodoacetamide / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007535:Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases; D012694:Serine / Q000737:chemistry; D013816:Thermodynamics
D004220:Disulfides; D007461:Iodoacetates; D012694:Serine; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D003545:Cysteine; D004229:Dithiothreitol; D019807:Iodoacetic Acid; D007460:Iodoacetamide