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 s ecreted proteins. Its active site contains one accessible and one buri ed cysteine residue, separated in the primary structure by only two ot her residues. These cysteine residues can form a very unstable disulfi de bond that is 10(3)-fold more reactive toward thiols than normal. Mo reover, the mixed disulfide between the accessible cysteine residue an d glutathione is 10(4)-fold more reactive than normal. Site-directed m utagenesis was carried out to replace either one or both cysteine resi dues 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 s table in the native conformation than when DsbA was unfolded. If the n ative conformation destabilizes the mixed disulfide, the mixed disulfi de must destabilize the folded conformation to the same extent. This w as confirmed by demonstrating that the folded conformation of the mixe d 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 originat ed almost exclusively in the folded conformation. Replacing the cystei ne residues by serine destabilized the folded conformation of the redu ced protein to varying extents. This suggests that the thiol groups ar e involved ia interactions that stabilize the folded conformation, whi ch would cause any disulfide bonds, either inter- or intramolecular, t hat involve these groups to be unstable.