Fig. 3: Biophysical characterisation of proton conduction in dissected antiporter-like modules induced by addition of weak acid.

a Proteoliposome assay for probing the proton conduction kinetics in the dissected antiporter-like subunits with pyranine (HPTS) by addition of K⁺CH₃COO^-. Addition of 10 mM acetate to (b) Nqo12^ΔTH- and (c) Nqo13- proteoliposomes induced conduction of protons across the membrane. The ionophore nigericin was added to dissipate the generated ∆pH. The final steady-state ∆pH level before addition of nigericin is shown for the (d) Nqo12^ΔTH and (e) Nqo13 constructs. The protonation conduction rate is sensitive to substitution of the conserved ion-pair and residues along the proton pathway. The data is compared to proton conduction assayed with empty liposomes (EL, n = 3) and aquaporin (AqpZ) reconstituted proteoliposome. Note that the initial pH gradient pre-equilibrates upon mixing the proteoliposomes in the buffer, leading to a higher initial baseline for the fast-conducting constructs (E123Q¹³, n = 3/ E132Q¹²), while the final steady-state pH (d, e), obtained prior to addition of nigericin, is independent of the baseline. Data shown are derived from independent experiments where n = 6 (mean ± SD) unless specified differently. See Supplementary Fig. 11 for addition of weak base, and Supplementary Fig. 13b, c for ∆pH and Δψ-mediated proton transport. Data are provided in the Source Data file.