The reactive and destabilizing disulfide bond of DsbA, a protein required for protein disulfide bond formation in vivo.
The protein DsbA facilitates disulfide bond formation in the periplasm of Escherichia coli. It has only two cysteine residues that are separated in the sequence by two other residues and are shown to form a disulfide bond reversibly. Chemical modification studies demonstrate that only one of the cysteine residues has an accessible thiol group in the reduced protein. Equilibrium and kinetic characterization of thiol-disulfide exchange between DsbA and glutathione showed that the DsbA disulfide bond was 10(3)-fold more reactive than a normal protein disulfide. Similarly, the mixed disulfide between the accessible cysteine residue and glutathione was 10(4)-fold more reactive than normal. The overall equilibrium constant for DsbA disulfide bond formation from GSSG was only 8 x 10(-5) M. These properties indicate that disulfide-bonded DsbA is a potent oxidant and ideally suited for generating protein disulfide bonds. Disulfide bonds generally increase the stabilities of folded proteins, when the folded conformation reciprocally stabilizes the disulfide bonds. In contrast, the disulfide bond of DsbA was so unstable in the folded state that its stability increased by 4.5 +/- 0.1 kcal.mol-1 when the protein unfolded. This implies that the disulfide bond destabilizes the folded conformation of DsbA. This was confirmed by demonstrating that the reduced protein was 3.6 +/- 1.4 kcal.mol-1 more stable than that with the disulfide bond.
European Molecular Biology Labortory, Heidelberg, Germany.
A
A
Zapun
J C
JC
Bardwell
T E
TE
Creighton
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004355:Drug Stability; D004926:Escherichia coli / Q000737:chemistry*; D005978:Glutathione / Q000378:metabolism; D007460:Iodoacetamide / Q000494:pharmacology; D007535:Isomerases / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013816:Thermodynamics
D001426:Bacterial Proteins; D004220:Disulfides; D013438:Sulfhydryl Compounds; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D003545:Cysteine; D007460:Iodoacetamide