Opening and closing of the periplasmic gate in lactose permease

Zhou et al. 10.1073/pnas.0800825105.

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Fig. 5. Cross-linking and lactose transport with double-Cys mutant I32C/N245C. All experiments were performed with RSO vesicles as described in Materials and Methods. C, control sample with no treatment (filled squares); 1, MTS-3-MTS (filled circles); 2, MTS-6-MTS (filled triangles); 3, MTS-8-O2-MTS (inverted filled triangles); 4, MTS-11-O3-MTS (filled diamonds); 5, MTS-14-O4-MTS (left-pointing, filled triangles); 6, MTS-17-O5-MTS (right-pointing, filled triangles); 7, NDM (open triangles); pT7-5, RSO membrane vesicles containing empty vector pT7-5 (stars). (A)SDS/PAGE and Western blots with anti-C-terminal antibody. (B) Time courses of transport after cross-linking with various homobifunctional cross-linking agents. Lactose accumulation with RSO membrane vesicles containing given mutant or empty vector pT7-5 was assayed as described in Materials and Methods. (C) Correlation between maximum levels of accumulation and cross-linker length. Percentage of lactose accumulation at 5 min from at least three independent measurements of the untreated control with error bars are plotted against the length of the cross-linkers. (D) Reversal of transport and cross-linking by DTT. RSO vesicles containing given mutant were assayed for transport and cross-linking (Inset) without or with treatment of DTT. Effects of DTT on lactose transport were carried out as described in Materials and Methods. Open circles, DTT only; open squares, MTS-3-MTS then DTT.

SI Figure 6

Fig. 6. Cross-linking and lactose transport with double-Cys mutant T45C/N245C. All experiments were performed with RSO vesicles as described in Materials and Methods and in SI Fig. 5.