Extended Data Fig. 9: Characterization of activating mutations in CLC-ec1 and CLC-7.
From: Structural basis of pH-dependent activation in a CLC transporter
(a) Theoretical (top panels - dimer & monomer) and experimental (middle and bottom panels – WT & ΔN29 pH 7.5 & 4.5) photobleaching steps probability distributions (P1, P2, P3+) for CLC-ec1 in lipid bilayers as a function of the experimental subunit/lipid mole fraction, χobs. Data for WT and ΔN29 pH 7.5 & 4.5 are means ± SEM for n = 4 for WT pH 4.5 and n = 3 for all other systems, where n is the number of independently prepared samples. (b, d) Representative normalized time courses of chloride efflux from liposomes reconstituted at pH 4.5 (B) or 7.5 (D) with CLC-ec1 WT (black), Q24C (orange), I201W (purple), E202Q (red) and E202Y (grey) at 0.2 μg protein/mg lipid. Traces were normalized to the total chloride content of the liposomes determined following the addition of detergent2. (c-e) Initial velocity of Cl− efflux at pH 4.5 (C) and 7.5 (E) of proteoliposomes reconstituted with CLC-ec1 WT (white), Q24C (orange), I201W (purple), E202Q (red) and E202Y (grey). All values are shown as means ± S.D. The number of repeats of independent experiments and preparations of proteo-liposome samples for all constructs are reported in Supplementary Table 1. (f, g) smFRET population contour plots of CLC-ec1 E202Q (F) and E202Y (G) labelled at E385C at pH 7.5 (left) and 4.5 (right) in 100 mM Cl−. Data are average of 3 independent repeats, and errors are SEM. (h) Forward and reverse potential of mean force (PMF) profile as a function of the minimum distance between αA of monomer A and αR of monomer B with E202 protonated, E202(0), or deprotonated, E202(-), at pH 4.5 or pH 7.5 with no Cl− in the central binding site. Molecular systems are listed in Supplementary Table 4. Error bars (in SD) are estimated by calculating the PMF values in four consecutive blocks of the trajectories. (i) Free energy difference between local energy minima for αA ‘bound’ to and ‘loose’ from αR with E202 protonated (E2020) or deprotonated (E202−) at pH 4.5 or pH 7.5 with no Cl− in the central binding site. ΔG is taken from the free energy profiles in panel H. Error bars are estimated in the same fashion as in panel H. (j) Current density-voltage curves for CLC-7PM WT (black), E311Q (red), L310W (blue), G578W (pink), and L310W/G578W (WW, orange). Data are averages of 10 independent repeats, and error bars are SEM.
