The reactive and destabilizing disulfide bond of DsbA, a protein required for protein disulfide bond formation in vivo

Abstract

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.

Intrachain disulfide bond in the core hinge region of human IgG4

“…For example, the disulfide bond in the thioredoxin active site was found to be quite stable with an equilibrium constant for forming the disulfide bond (effective concentration) of 10 M using glutathione as a reference species (Lin & Kim, 1989). In contrast, the active disulfides of protein disulfide isomerase are extremely unstable and oxidizing, with a value of 42-60 p M for the equilibrium constant with glutathione (Hawkins et al, 1991;Lyles & Gilbert, 1991), similar to the value measured for DsbA (Zapun et al, 1993).…”

Roles of cysteine residues of DsbB in its activity to reoxidize DsbA, the protein disulphide bond catalyst of Escherichia coli

“…The Cys 30 residue of DsbA is unusually reactive (Zapun et al 1993;Nelson & Creighton 1994: Darby & Creighton 1995. Therefore, the DsbA33S mutant protein which retains Cys 30 as the sole cysteine residue forms disulphide bridges with a number of cellular proteins when cells are grown on broth or in the presence of GSSG (Kishigami et al 1995b).…”

Eukaryotic translation elongation factor 1γ contains a glutathione transferase domain—Study of a diverse, ancient protein super family using motif search and structural modeling

“…The equilibrium between GSH and GSSG is an important regulator of the state of thiol groups in proteins, according to the reaction Cys-S-S-Cys + 2GSH = 2Cys-SH + GSSG (Gilbert, 1990;Hwang et al, 1992;Zapun et al, 1993, and references therein). In turn, the balance between free thiol groups of cysteines and disulfide bonds is important for protein folding.…”