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. 2006 Jul;188(14):5066–5076. doi: 10.1128/JB.00368-06

FIG. 2.

FIG. 2.

Membrane topology of the membrane-embedded domain (β) of DsbD and in vivo mutational effects. (A) The topology of DsbDβ was predicted using HMMTOP. The two reactive cysteines are represented by the large boldface letters, and the conserved amino acid residues, which are substituted with alanines, are represented as letters inside black rectangles. The residue numbers represent the start or end of the TM helixes. (B and C) The in vivo redox states of DsbC (B) and DsbD (C) in the strains expressing 19 mutant DsbDs. Expression of the genes was induced by 20 μM IPTG, harvested at mid-log phase, precipitated with TCA, and subjected to AMS alkylation. Proteins were separated by SDS-PAGE and visualized by Western blotting, using antibodies against DsbC (B) and DsbDα (C). DsbC and DsbD that are reduced become alkylated and display a higher molecular weight than the oxidized forms. (D) Reduced redox states of DsbD in the strains expressing 19 mutant DsbDs. Expression of the genes was induced by 20 μM IPTG, harvested at mid-log phase, reduced by 50 mM DTT, precipitated with TCA, and subjected to AMS alkylation. Proteins were separated by SDS-PAGE and visualized by Western blotting, using an antibody against DsbDα. For lane 23 [wild-type (100)], expression of the genes was induced with 100 μM IPTG and processed in the same way except that there was no reduction with DTT. Wild-type′ is the strain which retains the chromosomal dsbD gene. The strain background used was SEN45 (the dsbD mutant) in all lanes except lane 22 (SEN44). The following plasmids were used: pSC01-1 (G155A, lane 1), pSC01-2 (P162A, lane 2), pSC01-3 (P166A, lane 3), pSC01-4 (P169A, lane 4), pSC01-5 (Y194A, lane 5), pSC01-6 (M198A, lane 6), pSC01-7 (Y202A, lane 7), pSC01-8 (G206A, lane 8), pSC01-12 (L290A, lane 9), pSC01-9 (G275A, lane 10), pSC01-10 (P284A, lane 11), pSC01-11 (P289A, lane 13), pSC01-13 (L294A, lane 14), pSC01-14 (G316A, lane 15), pSC01-15 (P320A, lane 16), pSC01-16 (G336A, lane 17), pSC01-17 (W338A, lane 18), pSC01-18 (M339A, lane 19), pSC01-19 (G347A, lane 20), pAM238 [(−), lane 21, and wild-type′, lane 22], and pSC01 (wild-type, lane 12, and wild-type (100), lane 23). P162A, P284A, P289A, and G316A are marked by superscript stars because they were analyzed further for Fig. 5.