Protein reconstitution into liposomes

Glt_Ph protein samples were reconstituted into liposomes as previously described (Boudker et al., 2007; Ryan et al., 2009). In brief, 0.1% L-α-Phosphatidylethanolamine-N-lissamine rhodamine B sulfonyl (Avanti Polar Lipids) was added to with 3:1 (w/w) mixture of E.coli polar lipid extract and egg phosphatidylcholine and dried on rotary evaporator and under vacuum overnight. The mixture was hydrated using 20 mM Hepes/Tris, pH 7.4 and 200 mM KCl buffer at final lipid concentration of 5 mg/ml by 10 freeze/thaw cycles. The suspensions were extruded through 400 nm filter membranes (Whatman) 10 times to form unilamellar liposomes. The rhodamine fluorescence of the liposomes was measured to generate a concentration calibration curve; excitation and emission wavelengths were 560 and 583 nm, respectively. The liposomes were destabilized by addition of Triton X-100 0.5:1 (w:w) detergent to lipid ratio. Proteins were added to the destabilized liposome at a ratio of 1:1000 (w/w) of protein to lipid, and incubated for 30 min at room temperature. Detergent was removed by three rounds of incubation with 80 mg/ml of pre-washed SM-2 beads (Bio-Rad): 2 hr at room temperature, 2 hr at 4°C and overnight at 4°C with gentle agitation. The proteoliposomes were extruded 10 times through 400 nm membranes and concentrated by ultracentrifugation at 86,000 g for 45 min at 4°C. To replace the internal buffer, proteoliposomes were re-suspended in 1 mL of appropriate buffer and subjected to three freeze/thaw cycles followed by extrusion. Final concentrations of proteoliposomes were determined by rhodamine fluorescence.