Fig. 4.
Site-directed alkylation of a Cys replacement at position 245 in the periplasmic pathway. The target Cys (mutant N245C) is shown at the top for protein with a closed (Left) or open (Right) periplasmic cavity. Time courses of labeling were recorded at excitation and emission wavelengths of 380 and 465 nm, respectively, in 50 mM NaPi/0.02% DDM (pH 7.5) at 1 μM BM, added to 0.5 μM protein at 30 s, as indicated by an inverted arrow. (Left and Right) BM labeling of WT or mutant G46W/G262W, respectively. The highly reactive/accessible native Cys148 in each mutant was replaced with Met. In each panel, trace 1 was recorded with no protein added and traces 2 and 3 correspond to labeling of Cys245 in the absence of sugar or after addition of 6 mM TDG, respectively. Labeling of control proteins with no Cys replacement at position 245 is shown by trace 4 (no sugar) and trace 5 (6 mM TDG). Labeling of Cys245 with BM was tested with proteins in DDM (A and B), with mutants reconstituted into proteoliposomes (C and D) or with the same proteoliposomes dissolved in DDM (E and F).