Figure 2.

Disulfide crosslinking of N₆/C₆ permease fragments containing paired Cys residues in helices VII and I. Membranes were prepared from E. coli T184 expressing N₆ and C₆, each with a single Cys residue at a given position as indicated. Disulfide crosslinking was carried out at room temperature by incubating membranes (2 mg protein per ml) with 0.5 mM iodine for 10 min. Reactions were terminated by adding NEM to a final concentration of 10 mM. Samples containing approximately 20 μg of protein were subjected to NaDodSO₄/PAGE and electroblotted. C₆ was detected with anti-C-terminal antibody (A) and N₆ with HRP-avidin (B).