Bicelle and vesicle preparation

All lipids and detergents were obtained from Avanti Polar Lipids (Alabaster, AL) and used without any additional purification. Bicelles with a q value of 0.25 (q = [DMPC]/[DHPC]) and a total lipid concentration of 150 mM were generated using 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC). An appropriate amount of DMPC was mixed with 25 mM sodium phosphate buffer, pH 6.8, followed by continuously vortexing and centrifuging the mixture until a homogeneous slurry formed, and adding a suitable amount of a 400 mM DHPC solution. This mixture was then subjected to several cycles of centrifugation and vortexing until a clear non-viscous solution was attained. DMPC vesicles were prepared by dissolving an appropriate amount of lipid in 3:1 chloroform:methanol to reach a concentration of 10 mg/mL, and subsequently dried via a rotary evaporator to acquire a thin lipid film. The film was then reconstituted in 25 mM sodium phosphate buffer, pH 6.8, to a final concentration of 3.3 mg/mL and treated to four freeze–thaw cycles, followed by sonication for 2 min. Prior to use, the DMPC vesicles were extruded through polycarbonate membranes (Avanti Polar Lipids) with pore diameters of 100 nm. Vesicles of diameter < 30 nm containing DMPC, DHPC and 1,2-dimyristoyl-sn-glycero-3-phospho-(1-rac-glycerol) (DMPG) were prepared by adapting a protocol previously described by Yue et al.⁴⁷ Briefly, two solutions containing 4:1 DMPC:DHPC and 60:2:15 DMPC:DMPG:DHPC and a total lipid concentration of 50 mg/mL each, were repeatedly vortexed and temperature cycled from 4 to 50 °C in order to dissolve the lipids and then combined to achieve a final composition of 60:1:15 DMPC:DMPG:DHPC. This mixture was then allowed to equilibrate at 4 °C for 24 h before diluting to a final total lipid concentration of 10 mg/mL and subjecting to one freeze–thaw cycle. Vesicles with the same composition but larger sizes were allowed to equilibrate for up to one week instead of 24 h and extruded through either a 50 or a 100 nm polycarbonate membrane before use. All reported diameters were verified via dynamic light scattering.