Phase studies of model biomembranes: complex behavior of DSPC/DOPC/cholesterol

We have undertaken a series of experiments to examine the behavior of individual components of cell membranes. Here we report an initial stage of these experiments, in which the properties of a chemically simple lipid mixture are carefully mapped onto a phase diagram. Four different experimental methods were used to establish the phase behavior of the 3-component mixture DSPC/DOPC/chol: (1) confocal fluorescence microscopy observation of giant unilamellar vesicles, GUVs; (2) FRET from perylene to C20:0-DiI; (3) fluorescence of dilute dyes C18:2-DiO and C20:0-DiI; and (4) wide angle X-ray diffraction. This particular 3-component mixture was chosen, in part, for a high level of immiscibility of the components in order to facilitate solving the phase behavior at all compositions. At 23 degrees C, a large fraction of the possible compositions for this mixture give rise to a solid phase. A region of 3-phase coexistence of {Lalpha+Lbeta+Lo} was detected and defined based on a combination of fluorescence microscopy of GUVs, FRET, and dilute C20:0-DiI fluorescence. At very low cholesterol concentrations, the solid phase is the tilted-chain phase Lbeta'. Most of the phase boundaries have been determined to be within a few percent of the composition. Measurements of the perturbations of the boundaries of this accurate phase diagram could serve as a means to understand the behaviors of a range of added lipids and proteins.