Figure A1.

Characterization of AHA2 proteoliposomes by thin-layer chromatography and SDS-PAGE. (A) Schematic diagram illustrating the reconstitution of AHA2. Preformed liposomes (LUV) composed of PC in mixture with the indicated amounts of anionic phospholipids are detergent-destabilized and mixed with the detergent-solubilized H⁺-ATPase (BG). Subsequent removal of the detergent by Sephadex G-50 gel filtration (AG) and Bio-Bead treatment result in the formation of sealed proteoliposomes (PL). (B) Liposome solubilization is shown schematically (upper panel): liposomes (I) are destabilized upon detergent addition until completely saturated with detergent (II). Further additions lead to partly solubilization and occurrence of lipid–detergent micelles (III) until liposomes are complete solubilized (IV). The procedure can be followed by light scattering at 600 nm (lower panel) as solubilization of LUVs is accompanied by less light scattering. The obtained signal was fitted to a Boltzmann equation (Equation (1) in Material and Methods) to derive characteristic parameters such as the turning point, which was used for reconstitution and was usually slightly above the critical micelle concentration of used detergent OG. The amount of OG needed to solubilize the vesicles did not differ between lipid compositions (C) Exemplary Coomassie-Brilliant-Blue-Stained SDS-PAGE on AHA2 proteoliposomes, demonstrating equal amounts of protein reconstituted. Molecular weight markers are indicated on the left. Samples were loaded in duplicates (PS) or triplicates (PG, PA). Gels were run for a minimum of two independent reconstitutions. (D) Liposome composition analysis before and after reconstitution using thin layer chromatography. Chromatograms shown were dried completely before photographing after staining with primuline. Difference in Rf values derive from varying temperature and running chamber sizes. OG and DDM was compared to a standard spot of 0.5 mM allowing to conclude that less than 0.5 mM of each detergent remained in the sample. TLC were performed on two independent reconstitutions.; Abbreviations: AG: after gel filtration, BG: before gel filtration, DDM, N-dodecyl-β-maltoside; F, solvent front, of the chromatograms; O, origin; OG, octyl glucoside; PA, phosphatidic acid; PC, phosphatidylcholine; PL: proteoliposome, PS, phosphatidylserine; Sat: onset of solubilisation, Sol: total solubilisation.