Figure 3

In vivo monitoring of the thiol/disulfide state of the periplasmic cysteines of CadC at pH 5.8 (a) and illustration of the results (b). (a) CadC_C172A or CadC_C172A,C208A,C272A were overproduced in E. coli BL21(DE3)pLysS grown in phosphate buffered minimal medium pH 5.8. The labeling procedure was essentially the same as described in Figure 2, with the difference that the alkylation time was prolonged. Control experiments were done without DTT (lanes 3, 8), or PEG-mal (lanes 1, 5, 6) or iam (lane 4, 5). As a negative control the cysteine-free CadC derivative CadC_C172A,C208A,C272A was used. iam = iodoacetamide, DTT = dithiothreitol, PEG = PEG-maleimide. (b) The results are schematically illustrated.