Proteoliposomes were prepared by co-micellisation [31 (link)]. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) and cholesterol (Avanti Polar Lipids) were mixed in chloroform/methanol (2:1 (v/v)) at a molar ratio of 5:2:2:1 and dried using a rotary evaporator [21 (link)]. The dried lipid film was hydrated in 20 mM HEPES, pH 7.4, 150 mM KCl, 0.1 mM TCEP, and 5% (w/v) sodium cholate yielding multilamellar vesicles with a total lipid concentration of 15 mM. Syb(1-116) in 1% chaps was added at a lipid to protein ratio of 300:1 (n/n) and incubated for 1 h at 25 °C in a rotary evaporator without evaporation. Detergent removal and spontaneous proteoliposome formation was achieved by size exclusion chromatography using a self-packed Sephadex G25 Superfine column (5/100 mm, bed volume 2 mL) on an Äkta Pure system (GE Healthcare) equilibrated in 20 mM HEPES, pH 7.4, 150 mM KCl, and 0.1 mM TCEP. Proteoliposome-containing fractions were pooled and subjected to a liposome recruitment assay (‘liposome flotation assay’) based on sucrose density gradient centrifugation (sucrose gradient 0 M sucrose (top) – 1.2 M sucrose (bottom)) [32 (link)]. The size distribution of the reconstituted liposomes was determined by dynamic light scattering using a Zetasizer Nano (Malvern Instruments).
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