Figure 2.

Characterization of mutants with Ala → Gly mutations in helices IV and V. (A–E) Time courses of lactose transport by E. coli T184 expressing wild-type (WT) permease or mutants with Ala in place of Gly at positions 115 (A), 121 (B), 141 (C), 147 (D), or 150 (E) in the wild-type or E126D background, as indicated. Experiments were carried out as described in Materials and Methods. (A*–E*) Flow dialysis profiles comparing binding of NPG to RSO vesicles containing mutants G115A (A*), G121A (B*), G141A (C*), G147A (D*), or G150A (E*) in the wild-type or E126D background, as indicated. An aliquot of RSO vesicles (250 μl) in 0.1 M KP_i (pH 7.5) at a concentration of 25 mg/ml protein was placed in the upper chamber, and 5 μl of [³H]NPG (0.95 mCi/mmol) was added to a final concentration of 20 μM at fraction 1. Where indicated by the arrow, 2.5 μl of d-galactopyranosyl-1-thio-β,d-galactopyranoside (TDG) was added to the upper chamber to yield a final concentration of 10 mM.