Proton flux into vesicles containing Hv channels at various protein to lipid ratios.

**(A)** Fluorescence-based H

^{+} flux assay for vesicles containing a decreasing number of Hv channels. Protein to lipid ratios of 1:100 (dark blue, n = 5), 1:500 (pink, n = 2), 1:1000 (orange, n = 3), 1:5000 (yellow, n = 3), 1:10,000 (cyan, n = 4), 1:20,000 (light green, n = 3), 1:40,000 (green, n =4), 1:60,000 (violet, n = 3) and empty vesicles (red squares, n = 4) are plotted (error bars represent the standard error of the mean, range of mean for 1:500). Protein and vesicles were prepared as described in the legend.

**(B)** Sucrose cushion of vesicles containing Hv channels. Numbers denote the fractions collected from top to bottom. Lipid vesicles containing Hv1, with protein to lipid ratio 1: 100 (wt:wt), were layered on a sucrose gradient (From top to bottom, 140 µl sample plus 60 µl dialysis buffer, 600 µl 7 % sucrose, and 1 ml 27 % sucrose in dialysis buffer). The gradients were then centrifuged at 135,000 × g in a Sorvall RP55-S swinging bucket rotor for 2 hours and then fractionated into 8 × 225 µl fractions. A 15 µl sample of each fraction was then mixed with 15 µl 2x running buffer and run on a 12% gel (SDS-PAGE) and stained with Coomassie blue.

**(C)** Determination of the fraction of functional Hv channels. Plot of μ versus the ratio of fluorescence decay contributed by Hv containing vesicles over the total fluorescence decay by addition of CCCP where μ is the ratio of the number of channels over number of vesicles calculated with

where g

_{Hv} and g

_{L} are the grams of Hv channel and lipid added, r is the estimated average radius of a vesicle (100 nm), M

_{L} is the molecular weight of the average lipid molecule (754 Da), σ is the estimated area per lipid molecule (63 Å

^{2} ) and M

_{Hv} is the molecular mass of the Hv channel dimer (70,000 Da). Protein to lipid ratios are as in , 1:100 (μ = 43.0, n = 5), 1:500 (μ = 8.59, n = 2), 1:1000 (μ = 4.30, n = 3), 1:5000 (μ = 0.86, n = 3), 1:10,000 (μ = 0.43, n = 4), 1:20,000 (μ = 0.21, n = 3), 1:40,000 (μ = 0.11, n =4), 1:60,000 (μ = 0.07, n = 3) error bars represent the standard error of the mean (range of mean for 1:500). The two curves are derived from with φ (fraction of functional Hv) = 1.0, θ (fraction of reconstitution deficient vesicles) = 0.15 (red) and ϕ = 0.5, θ = 0.15 (green). The inset is a close-up view along the x-axis indicating that the fit is superior with a curve corresponding to ϕ = 1.0, θ = 0.15.

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