Interaction of Cholesterol in Ternary Lipid Mixtures Investigated Using Single-Molecule Fluorescence

Fluorescence measurements of the sterol analog 23-(dipyrrometheneboron difluoride)-24-norcholesterol (BODIPY-cholesterol) are used to compare the effects of cholesterol (Chol) in monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Chol and chicken egg sphingomyelin (SM)/DOPC/Chol. Monolayers are formed using the Langmuir-Blodgett technique and compared at surface pressures of 8 and 30 mN/m. In particular, these ternary lipid mixtures are compared using both ensemble and single-molecule fluorescence measurements of BODIPY-cholesterol. In mixed monolayers incorporating 0.10 mol % BODIPY-cholesterol, fluorescence microscopy measurements as a function of cholesterol added reveal similar trends in monolayer phase structure for both DPPC/DOPC/Chol and SM/DOPC/Chol films. With a probe concentration reduced to ∼10(-8) mol % BODIPY-cholesterol, single-molecule fluorescence measurements using defocused polarized total internal reflection microscopy are used to characterize the orientations of BODIPY-cholesterol in the monolayers. Population histograms of the BODIPY emission dipole tilt angle away from the membrane normal reveal distinct insertion geometries with a preferred angle observed near 78°. The measured angles and populations are relatively insensitive to added cholesterol and changes in surface pressure for monolayers of SM/DOPC/Chol. For monolayers of DPPC/DOPC/Chol, however, the single-molecule measurements reveal significant changes in the BODIPY-cholesterol insertion geometry when the surface pressure is increased to 30 mN/m. These changes are discussed in terms of a squeeze-out mechanism for BODIPY-cholesterol in these monolayers and provide insight into the partitioning and arrangement of BODIPY-cholesterol in ternary lipid mixtures.