Phase-Separated Giant Liposomes for Stable Elevation of α-Hemolysin Concentration in Lipid Membranes

Abstract

Staphylococcus aureus α-hemolysin (αHL) is one of the most popular proteins in nanopore experiments within lipid membranes. Higher concentrations of αHL within the lipid membrane are desirable to enhance the mass transport capacity through nanopores. However, the reconstitution of αHL at high concentrations is associated with the problem of membrane lytic disruption. In this study, we present a method that effectively increases αHL concentration while maintaining membrane stability. This method is achieved by using phase-separated giant liposomes, where coexisting liquid-disordered (Ld) and liquid-ordered phases (Lo) are enriched in unsaturated lipids and saturated lipids with cholesterol (Chol), respectively. Fluorescence observation of αHL in liposomes revealed that the presence of Chol facilitates αHL insertion into the membrane. Despite the preferential localization of αHL in the Ld phase rather than the Lo phase, the coexistence of both Lo and Ld phases prevents membrane disruption in the presence of concentrated αHL. We have explained this stabilization mechanism considering the lower membrane tension exhibited by phase-separated liposomes compared to homogeneous liposomes. Under hypertonic conditions, we have successfully increased the local concentration of αHL by invagination of the lipid-only region in the Ld phase, leaving αHL behind. This method exhibits potential for the reconstitution of various nanochannels and membrane proteins that prefer the Ld phase over the Lo phase, thus enabling the production of giant liposomes at high concentrations and the replication of the membrane-crowding condition observed in cells.

Effects of the Molecular Structure of Malodor Substances and Their Masking on 1,2-Dioleoyl-sn-glycero-3-phosphocholine Molecular Layers

“…On the basis of the experimental results mentioned above and related papers, ,− we discuss the characteristic responses of TS molecules to the DOPC molecular layer. Π for all of the samples utilized, that is, NE, NA, DE, DA, BA, and CA, was maintained at zero for A > 15 Å 2 molecule –1 (Figure S4 of the Supporting Information).…”

“…The phospholipid artificial membrane is used as a model cell membrane to investigate the mechanism of cell response because it is difficult to factor out intrinsic elements and their nature on the mechanism of chemical sensation in living organisms as a result of their complexity. − Indeed, interactions between chemical species and functional groups in phospholipid artificial membranes have been reported to be associated with barrier homeostasis of cellular membranes in skin . It has also been reported that membrane fluidity of liposomes is affected by ODs. − We recently evaluated the interaction between OD molecules and phospholipid molecular layers based on the spectroscopies and surface pressure ( Π )–area ( A ) isotherms .…”