Figure 4.

Cholesterol thresholds for ALO and PFO are determined by membrane phospholipids. (A and B) Recombinant FL and D4 of PFO and ALO were overexpressed and purified as described in Materials and Methods. Each reaction mixture, in a total volume of 200 μL of buffer B, contained 4.4 μM of the indicated protein and 600 μM liposomes composed of DOPC or DPhyPC and varying mole fractions of cholesterol. After incubation for 1 h at room temperature, the intrinsic tryptophan fluorescence of the samples was measured (excitation wavelength, 290 nm; emission wavelength, 340 nm). For each combination of protein and phospholipid, fluorescence values were normalized to range from 0 to 1.