Fig. 4: Dynamics of the membrane potential and PMF.

a The fluorescent probe 3,3’-dipropylthiadicarbocyanine iodide (DiSC₃(5)) distributes in response to a membrane potential (∆Ψ). b DiSC₃(5) fluorescence curves generated for MleP LPR 250 proteoliposomes containing MleS (n = 3) or no MleS (n = 2). After equilibration of DiSC₃(5), L-malate was added at t = 0 and the fluorescence quenching effect was recorded. At the end valinomycin was added to dissipate the ∆Ψ. c Effect of pH on the DiSC₃(5) fluorescence curve for the L-malate decarboxylation pathway (n = 1). Conditions of the measurements at pH 6 are the same as those at pH 7, but the internal and external buffer was K phosphate pH 6. Measurements were performed at 30 °C. Solid lines correspond to the fluorescence data normalized to the point immediately before L-malate addition and are presented in arbitrary units (arb. u.). Data in green curves represent the mean of fluorescence from independent experiments with n different preparations of proteoliposomes. Shaded areas in (b, c). indicate ± SD. Membrane potential was calculated from calibration data (Supplementary Fig. 16) and is presented in the right axis. d Gradient forces calculated from the pH gradient and membrane potential data at pH 7. e Gradient forces calculated from the ΔpH and membrane potential data at pH 6. pH curves after addition of 10 mM L-malate (violet) were taken from Fig. 3d and the driving forces were calculated from \({{{\rm{Z}}}}\Delta {{{\rm{pH}}}}=2.303({{{\rm{RT}}}}/{{{\rm{F}}}})\Delta {{{\rm{pH}}}}\), where R is the gas constant (8.31 J mol⁻¹ K⁻¹), T is temperature in Kelvin (303 K) and F is the Faraday constant (96485 C mol⁻¹). ΔpH was determined assuming that at t = 0 pH_i = pH_o, and that the external pH does not change substantially. Since ΔpH is a positive value (pH_i – pH_o), the plotted curve corresponds to −ZΔpH. Membrane potential (ΔΨ) data (green) were calculated by interpolation of the fluorescence quenching from panel c and Supplementary Fig. 18b, using the calibration curve from Supplementary Fig. 16b. Proton motive force (PMF) curves were calculated from −ZΔpH plus ΔΨ. a was created with Biorender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.