Molecular Organization of Cholesterol in Polyunsaturated Membranes: Microdomain Formation

Abstract

The molecular organization of cholesterol in phospholipid bilayers composed of 1,2-diarachidonylphosphatidylcholine (20:4–20:4PC), 1-stearoyl-2-arachidonylphosphatidylcholine (18:0–20:4PC), and 20:4–20:4PC/18:0–20:4PC (1/1 mol) was investigated by solid-state 2H NMR and by low- and wide-angle x-ray diffraction (XRD). On the basis of distinct quadrupolar powder patterns arising from [3 α- 2H 1]cholesterol intercalated into the membrane and phase separated as solid, solubility χ chol NMR = 17 ± 2 mol% and tilt angle α 0 = 25 ± 1° in 20:4–20:4PC were determined. The corresponding values in 18:0–20:4PC were χ chol NMR ≥ 50 mol% and α 0 = 16 ± 1°. Cholesterol solubility determined by XRD was χ chol XRD = 15 ± 2 mol% and χ chol XRD = 49 ± 1 mol% for 20:4–20:4PC and 18:0–20:4PC, respectively. XRD experiments show that the solid sterol is monohydrate crystals presumably residing outside the bilayer. The 2H NMR spectrum for equimolar [3 α- 2H 1]cholesterol added to mixed 20:4–20:4PC/18:0–20:4PC (1/1 mol) membranes is consistent with segregation of cholesterol into 20:4–20:4PC and 18:0–20:4PC microdomains of <160 Å in size that preserve the molecular organization of sterol in the individual phospholipid constituents. Our results demonstrate unambiguously that cholesterol has low affinity to polyunsaturated fatty acids and support hypotheses of lateral phase separation of membrane constituents into sterol-poor/polyunsaturated fatty acid-rich and sterol-rich/saturated fatty acid-rich microdomains.