Abstract
Disulfide bond formation protein B (DsbBS-S,S-S) is an inner membrane protein in Escherichia coli that has two disulfide bonds (S-S, S-S) that play a role in oxidization of a pair of cysteine residues (SH, SH) in disulfide bond formation protein A (DsbASH,SH). The oxidized DsbAS-S, with one disulfide bond (S-S), can oxidize proteins with SH groups for maturation of a folding preprotein. Here, we have described the transient kinetics of the oxidation reaction between DsbASH,SH and DsbBS-S,S-S. We immobilized DsbBS-S,S-S embedded in lipid bilayers on the surface of a 27-MHz quartz crystal microbalance (QCM) device to detect both formation and degradation of the reaction intermediate (DsbA-DsbB), formed via intermolecular disulfide bonds, as a mass change in real time. The obtained kinetic parameters (intermediate formation, reverse, and oxidation rate constants (kf, kr, and kcat, respectively) indicated that the two pairs of cysteine residues in DsbBS-S,S-S were more important for the stability of the DsbA-DsbB intermediate than ubiquinone, an electron acceptor for DsbBS-S,S-S. Our data suggested that the reaction pathway of almost all DsbASH,SH oxidation processes would proceed through this stable intermediate, avoiding the requirement for ubiquinone.
Highlights
A device of Quartz crystal microbalance (QCM) can be used in the transient kinetics of oxidation of a pair of cysteine residues in DsbA by DsbB
In this paper we have described the immobilization of a membrane protein, DsbB, embedded in supported lipid bilayers on a QCM plate and the detection of an intermediate complex between DsbASH,SH and DsbBS-S,S-S according to mass changes on the QCM device (Fig. 2)
To observe and quantify thiol-disulfide exchange reactions between soluble DsbASH,SH and membrane-embedded DsbBS-S,S-S, we examined the construction of DsbBS-S,S-S-embedded supported lipid bilayers on a QCM plate (Fig. 2)
Summary
DsbA belongs to the thioredoxin superfamily and contains a disulfide bond in the thioredoxin domain (Cys-30 –X-X–Cys-33) that is known to act as an acceptor of two electrons (and two protons) from a preprotein containing cysteine residues during the transfer of the disulfide bond from the oxidized DsbAS-S to the preprotein (Fig. 1) [2,3,4,5]. To repeat this disulfide bond-introducing reaction in the catalytic cycle, re-oxidation of DsbASH,SH is required. Nonreducing SDS-PAGE analysis has been used to estimate the amounts of
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