Figure 2

In vivo monitoring of the thiol/disulfide state of the periplasmic cysteines of CadC at pH 7.6 (a) and illustration of the results (b). (a) CadC_C172A, CadC_C172A,C208A or CadC_C172A,C208A,C272A (cysteine-free CadC) were overproduced in E. coli BL21(DE3)pLysS grown in phosphate buffered minimal medium at pH 7.6. To label free thiol groups irreversibly, 5 mM iodoacetamide was added directly to the living cells. After TCA precipitation and extensive washing, oxidized thiol groups were reduced by addition of 10 mM DTT in denaturing buffer. These reduced cysteines were then alkylated by addition of 10 mM PEG-maleimide. Samples were mixed with non-reducing SDS-sample buffer and 30 μg total cell protein were loaded onto 12.5% SDS-polyacrylamide gels. CadC was detected by Western blot analysis of the His-tagged proteins. Control experiments were done without DTT (lanes 3, 8) or PEG-mal (lanes 1, 6) or iam (lane 4). As a negative control the cysteine-free CadC derivative CadC_C172A,C208A,C272A was used. The iam control was performed with a CadC derivative that contains only one cysteine (CadC_C172A,C208A). iam = iodoacetamide, DTT = dithiothreitol, PEG = PEG-maleimide. (b) The results are schematically illustrated.