Abstract:
In Escherichia coli, a family of disulfide bond-forming proteins catalyse disulfide bond (Dsb) formation and rearrangement during protein folding. The two disulfide bond isomerases DsbC and DsbG, and the membrane protein DsbD (which catalyses the rereduction of DsbC and DsbG) are involved in rearranging non-native disulfide bonds of proteins in the periplasm. Both DsbC and DsbG are V-shaped homodimers, sharing similarities both at functional and structural levels. Dimerisation is essential for the function of both enzymes. Dimerisation of DsbC (and probably also DsbG) plays a role in distinguishing the disulfide bond formation and isomerisation pathways. Although the structure and the function of DsbC and DsbG have been characterised, little analysis has been made of the dimerisation properties of these enzymes. To characterise DsbC and DsbG dimerisation, the atomic structures of the isolated DsbC and DsbG dimerisation domains have been determined at a resolution of 2.0 and 1.9 A respectively, using X-ray crystallographic techniques. Both structures reveal compact homodimeric molecules that are similar to the corresponding domains in the full-length molecules, indicating that these dimerisation domains can fold and dimerise independently. Biophysical characterisation of both the DsbC and DsbG dimerisation domains show that they are able to undergo monomer exchange under physiological conditions. The dimerisation domain mutant DsbGI17N undergoes monomer exchange over a broader temperature range than wild-type DsbG. Curiously, the effect of the I17N mutation appears to only influence monomer exchange for the full length DsbG molecule. The formation of BFP-GFP heterodimers, linked by the N-terminal DsbC (or DsbG) dimerisation domain, has been demonstrated by monitoring the fluorescent resonance energy transfer (FRET) between recombinant fusion proteins. These molecules were used in FRET-based experiments to investigate dimerisation kinetics under a range of conditions, and to determine the activation energy for monomer exchange.
