Measurement of Cys245 Labeling Kinetics

RSO membrane vesicles containing pseudo-WT or a given LacY mutant were prepared from a 0.5 L culture of E. coli T184 cells as described previously.^{34 (link),35} For each mutant with a single Cys245, 50 μL of RSO membrane vesicles (0.1 mg of total protein) was incubated with 40 μM TMRM in the absence or presence of 2 mM α-NPG at 25 °C for 0, 5 10, 15, and 20 s. Labeling was terminated by addition of 10 mM (final concentration) dithiothreitol (DTT). The membranes were solubilized with 2% n-dodecyl β-D-maltopyranoside (DDM), and LacY was purified by avidin affinity chromatography as described previously.^{14 (link)} A purified LacY sample from each labeling reaction mixture was subjected to SDS–PAGE. The intensity of labeling and the amount of purified protein on the gel were determined by using an Amersham Typhoon 9410 Workstation and silver staining, respectively. The volumes of the labeled protein bands on the SDS–PAGE gel were quantified with ImageQuant 5.0 (GE Healthcare).^{14 (link),15 (link),17 (link)} Relative labeling was calculated as described previously.^{14 (link)} The rate of labeling was obtained by plotting relative labeling as a function of labeling time. Data were fitted by linear fit or a firstorder rate equation [A_t = A_∞(1 – e^−kt)] with GraFit 6 (Erithacus Software).