FIG. 4.

Labeling of the gating loop cysteines with F-M (A). Cells of E. coli UL3 fhuA were transformed with one of the following plasmids: pT7-6 (vector without fhuA), pfhuA9 [FhuA(C318S C329S C692S C698S); No Cys], pfhuA8 [FhuA(wild type); w.t.], pfhuA5 [FhuA(C329S) Cys-318], pfhuA4 [FhuA(C318S) Cys-329], pfhuA6 [FhuA(D336C)], and pfhuA7 [FhuA(V347C)], as indicated. Cells were incubated for 15 min with 0.5 mM F-M at 30°C in darkness, and the proteins of whole cells were separated by SDS-PAGE. In addition, purified protein of FhuA(V347C) was heated for 3 min in 4% SDS and subsequently labeled with F-M (purified prot.). Proteins were separated by SDS-PAGE, illuminated at 302 nm, and photographed with a Cybertech video camera (A). The proteins were then stained with Serva R to show that equal amounts were applied to the lanes (B).