Site-directed alkylation of LacY: effect of the proton electrochemical gradient

Abstract

Previous N-ethylmaleimide-labeling studies show that ligand binding increases the reactivity of single-Cys mutants located predominantly on the periplasmic side of LacY and decreases reactivity of mutants located for the most part of the cytoplasmic side. Thus, sugar binding appears to induce opening of a periplasmic pathway with closing of the cytoplasmic cavity resulting in alternative access of the sugar-binding site to either side of the membrane. Here we describe the use of a fluorescent alkylating reagent that reproduces the previous observations with respect to sugar binding. We then show that generation of an H(+) electrochemical gradient (Delta(mu (H)+), interior negative) increases the reactivity of single-Cys mutants on the periplasmic side of the sugar-binding site and in the putative hydrophilic pathway. The results suggest that Delta(mu (H)+), like sugar, acts to increase the probability of opening on the periplasmic side of LacY.

Figure 1

Structures of NEM (A) and TMRM (B).

Figure 2

Effect of TDG and/or temperature on TMRM reactivity of single-Cys mutants. RSO membrane vesicles (0.1 mg of protein in 50 μl) prepared from E. coli with given single-Cys replacements [(A) mutants T45C and A88C; (B) mutant N245C] were incubated for 15 min at 25 °C or 0 °C in the absence or presence of TDG with 40 μM TMRM. DTT was added to terminate the reactions, and DDM was used to solubilize the membrane. Biotinylated proteins were purified, subjected to SDS-PAGE. TMRM-labeled (upper panels) and silver-stained (lower panels) bands corresponding to LacY were imaged and measured qualitatively as described in Materials and Methods.

Figure 3

Distribution of single-Cys replacements exhibiting TDG-induced changes in TMRM reactivity. Seventy-one single-Cys LacY mutants were labeled with TMRM in the absence and presence of TDG as described in Fig. 2. Positions of Cys replacements that exhibit changes in TMRM reactivity are superimposed on the backbone of LacY (Protein Data Bank ID code 1PV7; www.pdb.org). LacY is viewed perpendicular to the membrane with the N-terminal helix bundle on the left and the C-terminal bundle on the right. (A) green spheres, increased TMRM reactivity at positions 2, 3, 8, 12, 14, 17, 24, 25, 28, 29, 30, 31, 32, 42, 44, 45, 49, 53, 70, 71, 96, 100, 136, 157, 158, 159, 160, 161, 241, 242, 244, 245, 246, 248, 265, 291, 295, 298, 308, 315, 359, 361, 362, 363 and 364; (B) blue spheres, decreased TMRM reactivity at positions 4, 5, 11, 15, 21, 22, 27, 34, 60, 81, 84, 86, 87, 88, 122, 141, 145, 148, 264, 268, 272, 327, 329, 331, 356 and 357. TDG is shown as a stick model at the apex of the inward-facing cavity (yellow sticks).

Figure 4

Effect of ^{Δμ̄_H +} on time-course of TMRM labeling. RSO membrane vesicles (0.1 mg of protein in 50 μl) prepared from E. coli with given single-Cys replacements, L4C (A), N8C (B), M11C (C), F30C (D), P31C (E), K42C (F), F49C (G) S53C (H), Q60C (I), I160C (J), M161C (K), N245C (L), T248C (M) and T265C (N), were incubated for 5, 15, 30, and 60 sec at 25 °C with 40 μM TMRM alone (control), in the presence of 20 mM sodium ascorbate and 0.2 mM PMS under oxygen (+^{Δμ̄_H +}) or in the presence of 250 μM valinomycin and 0.5 μM nigericin to abolish ^{Δμ̄_H +} (^{Δμ̄_H +} / V+N). After terminating the reactions with DTT at the indicated time, the samples were treated as described in Fig. 2 and in Materials and Methods. The data calculated according to eq. 1 are presented as labeling relative to the amount measured at 60 sec in the absence of ^{Δμ̄_H +} for each mutant. Each data set was fit into eq 2. RSO membrane vesicles with C-less LacY containing the biotin acceptor domain were incubated with 40 μM TMRM for 15 min at 25 °C and biotinylated C-less LacY was purified and loaded into the first lane of each gel, as a negative control. ■, no additions; ●, plus ascorbate/PMS; ▲, plus ascorbate/PMS, valinomycin and nigericin.

Figure 4

Effect of ^{Δμ̄_H +} on time-course of TMRM labeling. RSO membrane vesicles (0.1 mg of protein in 50 μl) prepared from E. coli with given single-Cys replacements, L4C (A), N8C (B), M11C (C), F30C (D), P31C (E), K42C (F), F49C (G) S53C (H), Q60C (I), I160C (J), M161C (K), N245C (L), T248C (M) and T265C (N), were incubated for 5, 15, 30, and 60 sec at 25 °C with 40 μM TMRM alone (control), in the presence of 20 mM sodium ascorbate and 0.2 mM PMS under oxygen (+^{Δμ̄_H +}) or in the presence of 250 μM valinomycin and 0.5 μM nigericin to abolish ^{Δμ̄_H +} (^{Δμ̄_H +} / V+N). After terminating the reactions with DTT at the indicated time, the samples were treated as described in Fig. 2 and in Materials and Methods. The data calculated according to eq. 1 are presented as labeling relative to the amount measured at 60 sec in the absence of ^{Δμ̄_H +} for each mutant. Each data set was fit into eq 2. RSO membrane vesicles with C-less LacY containing the biotin acceptor domain were incubated with 40 μM TMRM for 15 min at 25 °C and biotinylated C-less LacY was purified and loaded into the first lane of each gel, as a negative control. ■, no additions; ●, plus ascorbate/PMS; ▲, plus ascorbate/PMS, valinomycin and nigericin.

Figure 5

Effect of ^{Δμ̄_H +} on TMRM reactivity of single-Cys mutants. Positions of single- Cys mutants that exhibit increased TMRM reactivity are superimposed on the backbone of LacY viewed perpendicular to the membrane. TDG is shown as a stick model at the apex of the inward-facing cavity (yellow sticks). The Cys replacements that exhibit ^{Δμ̄_H +} -induced increases in TMRM reactivity are L4C, N8C, M11C, F30C, P31C, K42C, F49C, S53C, Q60C, I160C, M161C, N245C, T248C and T265C (magenta spheres).