Phase diagram and tie-line determination for the ternary mixture DOPC/eSM/cholesterol.

We propose a novel, to our knowledge, method for the determination of tie lines in a phase diagram of ternary lipid mixtures. The method was applied to a system consisting of dioleoylphosphatidylcholine (DOPC), egg sphingomyelin (eSM), and cholesterol (Chol). The approach is based on electrofusion of single- or two-component homogeneous giant vesicles in the fluid phase and analyses of the domain areas of the fused vesicle. The electrofusion approach enables us to create three-component vesicles with precisely controlled composition, in contrast to conventional methods for giant vesicle formation. The tie lines determined in the two-liquid-phase coexistence region are found to be not parallel, suggesting that the dominant mechanism of lipid phase separation in this region changes with the membrane composition. We provide a phase diagram of the DOPC/eSM/Chol mixture and predict the location of the critical point. Finally, we evaluate the Gibbs free energy of transfer of individual lipid components from one phase to the other.

Effect of the HIV-1 fusion peptide on the mechanical properties and leaflet coupling of lipid bilayers.

The fusion peptide (FP) of the human immunodeficiency virus (HIV) is part of the N-terminus of the viral envelope glycoprotein gp41 and is believed to play an important role in the viral entry process. To understand the immediate effect of this peptide on the cell membrane, we have studied the influence of the synthetic FP sequence FP23 on the mechanical properties of model lipid bilayers. For this purpose, giant unilamellar vesicles were prepared from the unsaturated lipid dioleoylphosphatidylcholine mixed in various molar ratios with FP23. The bending stiffness of the vesicles was measured with two different methods: fluctuation analysis and aspiration with micropipettes. The data obtained from both of these approaches show that the bending stiffness of the membrane decreases gradually with increasing concentration of the FP23 in the bilayer. Low concentrations of only a few mol% FP23 are sufficient to decrease the bending stiffness of the lipid bilayer by about a factor of 2. Finally, data obtained for the stretching elasticity modulus of the membrane suggest that the peptide insertion decreases the coupling between the two leaflets of the bilayer.