Preparation of liposomes, bicelles, and nanodiscs

All lipids were purchased from Avanti Polar Lipids, Inc., including POPC (1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine), DOPG (1,2-dioleoyl-sn-glycero-3-phospho1’-rac-glycerol), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanol-amine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DMPG (1,2-dimyristoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (sodium salt)), DHPC (1,2-dihexanoyl-sn-glycero-3-phosphocholine), LPC (1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine), LPG (1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1’-rac-glycerol) (sodium salt)), and LPE (1-myristoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine).

For liposome preparation, lipids in chloroform were added to a glass tube and dried to a thin film by spinning with heat for an hour using a condenser rotor SpeedVac, followed by lyophilization overnight once the volatile organics were removed. Lipids were rehydrated with H₂O for 1 h at 42 °C and vortexed every 15 min. Rehydrated lipids were transferred to a 1.5 mL plastic tube and put through 5 cycles of freeze-thaw using a dry ice bath and a room temperature water bath. The mixture was then transferred to a clean glass tube for bath sonication (BRANSON 3510R-MT Bransonic Ultrasonic Cleaner). The lipids were sonicated for 15-min intervals until clear, then stored at room temp. If liposomes were not consumed within 2 days, they were sonicated for an additional 5–10 min before use. Extruded liposomes were prepared as described previously⁴². Briefly, lipids were prepared as above, but instead of sonication, the rehydrated lipids were passed through a 0.05 μm filter 11 times (SPARTAN HPLC Syringe Filter) in a liposome extruder (Avanti Polar Lipids) after the dry ice bath. The extruded liposomes were collected and stored at room temperature.

Bicelles and nanodiscs (cNW9, circulated nanodisc width of 9 nm) were prepared as described previously^43–45. Briefly, for the preparation of bicelles, DMPC, DMPG, and LPE were mixed together with double distilled H₂O (ddH₂O), frozen on ethanol/dry ice, and thawed at 42 °C for 3 freeze-thaw cycles. DHPC in ddH₂O was then added into the mixture and quickly vortexed to form a clear solution. The solution was frozen on ethanol/dry ice and slowly thawed at room temperature for use. For the preparation of nanodiscs, purified NW9 was circularized to form cNW9 (circularized NW9) using freshly made sortase in buffer (300 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl₂). cNW9 was purified through Ni column and then S200 16/60 column. The purified cNW9 was exchanged into a buffer of 20 mM Tris, 100 mM NaCl, pH 8.0, and 20 mM sodium cholate, and incubated with lipids (POPC/POPE/POPG in a molar ratio of 2/2/1, cNW9/lipid in a molar ratio of 1/60) on ice for 1 h. Sodium cholate was then removed by incubation with Bio-beads SM-2 (Bio-Rad) for 1 h on ice followed by incubation overnight at 4 °C. Bio-beads were removed through 0.22 μm nitrocellulose filter. The nanodisc preparations were further purified by size-exclusion chromatography and assessed using SDS–PAGE.