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Grazing incidence X-ray diffraction measurements were performed on single hydrated bilayers and monolayers of DPPC:Cholesterol:POPC at varying concentrations. There are substantial differences in the phase and structure behavior of the crystalline domains formed within the bilayers relative to the corresponding monolayers, due to interactions between the opposing leaflets. Depending on the lipid composition, these interactions led to phase separation, changes in molecular tilt angle, or formation of cholesterol crystals. In monolayers, DPPC and cholesterol form a single crystalline phase at all compositions studied. In bilayers, a second crystalline phase appears when cholesterol levels are increased: domains of cholesterol and DPPC form monolayer thick crystals where each of the lipid leaflets diffracts independently, whereas excess cholesterol forms cholesterol bilayer thick crystals at a DPPC:Chol ratio &lt; 46:54 +/- 2 mol %. The nucleation of the cholesterol crystals occurs at concentrations relevant to the actual cell plasma membrane composition.

Crystalline Domain Structure and Cholesterol Crystal Nucleation in Single Hydrated DPPC:Cholesterol:POPC Bilayers