Figure 5.

Chemical crosslinking of helices VII and II. Membranes were prepared from E. coli T184 expressing N₆ and C₆ with paired Cys residue at position 245 in helix VII and 52 (left) or 53 (right) in helix II. Iodine-catalyzed crosslinking was carried out as described in Fig. 2. For BMH crosslinking, membranes (2 mg protein per ml) were incubated with 0.5 mM BMH at room temperature for 10 min, and the reaction was terminated by adding dithiothreitol to a final concentration of 10 mM. Samples were probed with anti-C-terminal antibody. Although data are not shown, results similar to those observed with iodine were also obtained with BMH, as well as N,N′-p-phenylenedimaleimide, and each of the other paired Cys mutants described.