Reconstitution of KCNE1 in POPC:POPG Liposomes

A POPC:POPG lipid bilayer was used to mimic phospholipids typically found in mammalian membranes^{[9 (link), 12 (link), 15 (link)–17 (link), 25 (link), 26 (link)]}. POPC and POPG powdered lipids (Avanti, Alabaster, AL) were dissolved to a total final concentration of 5 mM (9/1 POPC/POPG molar ratio) in 5 mM HEPES and 0.5% DM (n-Dodecyl-β-D-maltoside) (pH 7.0) by repeating at least 10 freeze/thaw cycles until the solutions were clear^{[12 (link)]}. SM2 Bio Beads (Bio-Rad) were washed with 8× 20 mL volumes of methanol followed by 8× 20 mL volumes of water prior to use. Samples were reconstituted by mixing 1 mL of the lipid slurry with 157 μg of KCNE1 protein in 0.2% SDS (500/1 lipid/protein molar ratio) and repeating 2 freeze/thaw cycles. The lipid/protein mixture was then added to 300 mg of wet Bio Beads and nutated at room temperature for 2 hours. Previous studies showed that this amount of Bio Beads effectively removes all detergent and forms MLVs^{[27 (link)]}. The resulting sample was centrifuged at 2,000 g for 5 minutes to remove excess Bio Beads. The supernatant was concentrated by ultracentrifugation at 300,000 g for 30 minutes. The proteoliposome pellet was thoroughly resuspended in 50 μL of 100 mM NaH₂PO₄ (pH 7.2) and immediately used in spectroscopic studies.