Each lane contains 25 μg of membrane protein (CadC derivatives are

in the same order as in the graph). CadC was detected by a monoclonal mouse antibody against the His-Tag and an alkaline phosphatase coupled anti-mouse antibody. In order to detect intermolecular disulfide bonds, membrane vesicles containing wild-type CadC or CadC derivatives with cysteine replacements were treated with copper phenanthroline, Bafilomycin A1 manufacturer a Cys null crosslinker. Subsequent Western blot analysis revealed that in case of wild-type CadC and CadC with a single Cys at position 172, a fraction of the protein was transformed into an oligomeric form which might be related to the formation of an intermolecular disulfide bond at position 172 (data not shown). Since replacement of Cys172 was without effect on the CadC-mediated cadBA expression (Figure 1), it is concluded

that an intermolecular disulfide bond is without functional importance for CadC. An intramolecular disulfide bond between C208 and C272 is found at pH 7.6 in vivo To analyze whether a disulfide bond is formed in CadC, an in vivo differential thiol trapping approach with iodoacetamide and PEG-maleimide was used [16]. For simplification, these studies were performed with CadC_C172A which contains only the two periplasmic cysteines. The method is based on the fact that both iodoacetamide and PEG-maleimide react only with free thiol groups. First, E. coli cells Selleckchem GSK872 producing CadC_C172A were labeled with iodoacetamide during growth Thymidylate synthase at pH 7.6 or pH 5.8. Subsequently, free iodoacetamide was removed, and all disulfide bonds were reduced by treatment with dithiothreitol see more (DTT). Free thiol groups were labeled with PEG-maleimide in a second step. In consequence, only cysteines that are present in an oxidized form and thus protected from iodoacetamide labeling in the first step, are labeled with PEG-maleimide resulting in a detectable increase of the molecular weight. At pH 7.6 differential labeling of CadC_C172A clearly resulted in a labeling with PEG-maleimide (Figure 2). The band for unlabeled CadC decreased, and an additional higher molecular band appeared demonstrating labeling of C208 and C272 with PEG-maleimide

(Figure 2a, lane 2). This additional band was only detectable when cells were treated with DTT (Figure 2a, lane 3 in comparison to lane 2). The PEG-ylated CadC_C172A runs as a smeared and broadened band which is probably due to the interaction between PEG and SDS [17]. Addition of PEG-maleimide (regardless of the treatment with DTT) resulted in an additional labeling product that also appeared in cells producing the cysteine-free CadC. Therefore, this signal can be regarded as unspecific labeling product which might be related to a reactivity of maleimide with other residues (e.g., lysine or tyrosine) in CadC (Figure 2a, lanes 2, 3, and 7, 8). Labeling of CadC_C172A with PEG-maleimide implies that iodoacetamide was unable to react with the periplasmic cysteines.