Phase-Separated Giant Liposomes for Stable Elevation of & alpha;-Hemolysin Concentration in Lipid Membranes

Staphylococcus aureus & alpha;-hemolysin(& alpha;HL) is one of the most popular proteins in nanopore experimentswithin lipid membranes. Higher concentrations of & alpha;HL withinthe lipid membrane are desirable to enhance the mass transport capacitythrough nanopores. However, the reconstitution of & alpha;HL at highconcentrations is associated with the problem of membrane lytic disruption.In this study, we present a method that effectively increases & alpha;HLconcentration while maintaining membrane stability. This method isachieved by using phase-separated giant liposomes, where coexistingliquid-disordered (Ld) and liquid-ordered phases (Lo) are enrichedin unsaturated lipids and saturated lipids with cholesterol (Chol),respectively. Fluorescence observation of & alpha;HL in liposomes revealedthat the presence of Chol facilitates & alpha;HL insertion into themembrane. Despite the preferential localization of & alpha;HL in theLd phase rather than the Lo phase, the coexistence of both Lo andLd phases prevents membrane disruption in the presence of concentrated & alpha;HL. We have explained this stabilization mechanism consideringthe lower membrane tension exhibited by phase-separated liposomescompared to homogeneous liposomes. Under hypertonic conditions, wehave successfully increased the local concentration of & alpha;HL byinvagination of the lipid-only region in the Ld phase, leaving & alpha;HLbehind. This method exhibits potential for the reconstitution of variousnanochannels and membrane proteins that prefer the Ld phase over theLo phase, thus enabling the production of giant liposomes at highconcentrations and the replication of the membrane-crowding conditionobserved in cells.