Biochemistry 1990, 29, 1025-1038 1025 Organization and Interaction of Cholesterol and Phosphatidylcholine in Model Bilayer Membranes Paul A. Hyslop,*i* Benoit Morel,§ and Richard D. Sauerheberll Department of Central Nervous System Pharmacology, Lilly Research Laboratories, Indianapolis, Indiana 46285, Department of Engineering and Public Policy, Carnegie- Mellon University, Pittsburgh, Pennsylvania 1521 3, and Rees-Stealy Research Received April 18, 1989; Revised Manuscript Received August 31, 1989 Foundation, 2001 4th Avenue, San Diego, California 92101 ABSTRACT: The molecular organization of sterols in liposomes of 1 -palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) at 37 OC is examined by utilizing the fluorescent analogue of cholesterol choles- ta-5,7,9-trien-3/3-01 (cholestatrienol). (1) Cholestatrienol is shown to be indistinguishable from native cholesterol in terms of its ability to condense POPC, as determined by (i) pressure/area studies of mixed-lipid monolayers and (ii) its ability to increase the order of POPC bilayers (determined by electron spin resonance studies) whether on its own or admixed with cholesterol at various ratios. (2) By analysis of the perturbation of the absorption spectra, cholestatrienol was found to be freely miscible in aggregates of cholesterol in buffer. In contrast, a lack of any detectable direct interaction of the sterol molecules in POPC bilayers was detected. (3) Fluorescence intensity and lifetime measurements of POPC/sterol (1:l mol/mol) at various chole- sterol/cholestatrienol molar ratios (0.5: 1 up to 1: 1 cholestatrienol/POPC) confirmed that sterol molecules in the membrane matrix were not associated to any great degree. (4) A quantitative estimate of how close sterol molecules approach each other in the membrane matrix was evaluated from the concentration de- pendence of the steady-state depolarization of fluorescence and was found to be 10.6 A. From geometrical considerations, the sterol/phospholipid phase at 1: 1 mol/mol is depicted as each sterol having four POPC molecules as nearest neighbors. We term this arrangement of the lipid matrix an "ordered bimolecular mesomorphic lattice". (5) The concentration dependence of depolarization of fluorescence of cholestatrienol in POPC liposomes in the absence of cholesterol yielded results that were consistent with the cholestatrienol molecules being homogeneously dispersed throughout the phospholipid phase at sterol/POPC ratios of less than 1:l. (6) From qualitative calculations of the van der Waals' hydrophobic interactions of the lipid species, the phospholipid condensing effect of cholesterol is postulated to arise from increased interpenetration of the flexible methylene segments of the acyl chains, as a direct result of their greater mutual attraction compared to their attraction for neighboring sterol molecules. (7) The interdependence of the ordered bimolecular mesomorphic lattice and the acyl chain condensation is discussed in an effort to understand the ability of cholesterol to modulate the physical and mechanical properties of biological membranes. xe major functions of cholesterol in the plasma membrane appear to involve minimizing the permeability to small mol- ecules and ions (Demel et al., 1971) and modulating the physical and mechanical properties of the membrane (Stockton & Smith, 1976; Jacobs & Oldfield, 1979; El-Sayed et al., 1986). These effects of cholesterol arise from the reduction in the degrees of motional freedom of the acyl chains of the phospholipids (Stockton & Smith, 1976), resulting in an in- creased in-plane elastic stiffness and viscosity of the membrane (El-Sayed et al., 1986). Cholesterol also can modulate the activity (Whetton et al., 1983; Connolly et al., 1986) and distribution of membrane proteins (Rottem et al., 1973). Also, heterogeneous phos- pholipid classes and species within the membrane matrix give rise to laterally inhomogeneous cholesterol distribution (de Kruyff et al., 1974) as well as to an asymmetric transbilayer distribution of cholesterol (Hale & Schroeder, 1982). These effects of cholesterol on plasma membrane structure and function appear to have wide pathological implications. For example, modulation of the cholesterol content of the plasma membrane influences platelet aggregation (McLeod et al., 1982). Also, manipulation of the fluidity and cholesterol * To whom correspondence should be addressed. * Lilly Research Laboratories. 1 Carnegie-Mellon University. 11 Rees-Stealy Research Foundation. content of the lipid envelopes of both vesicular stomatitis (Pal et al., 1981) and HIV (Aloia et al., 1988; Crews et al., 1988) markedly influences the infectivity of these retroviruses. Insights to the physical principles by which cholesterol modulates the molecular motion of the acyl chains have come from an understanding of both the organization and motional characteristics of the lipid components, utilizing model lipid membranes [Yeagle (1985) and references cited therein]. The cholesterol molecule is located with its hydroxyl in the ester carbonyl region of the hydrophilic/hydrophobic interface (Worcester & Franks, 1976) and its long axis perpendicular to the membrane surface. On the time scale of interest (i.e., with respect to acyl chain anisotropic segmental motion; Davis, 1983), the cholesterol molecule rotates unhindered about its long axis with a correlation time of about 100 ps (Taylor et al., 1981; Yeagle, 1981). The phospholipid molecules have much longer rotational correlational times (- 10 ns), which preclude any long-lived complex with the sterol (Yeagle, 1981). The rigid cholesterol molecule exhibits rapid, highly hin- dered angular fluctuations about its short axis with a rotational correlation time of around 500 ps (Taylor et al., 1982). The relatively rigid fused ring system and the nearly axially sym- metric reorientation of cholesterol make it possible to describe its angular fluctuations in terms of a single molecular order parameter of 0.78 in DMPC liposomes at 37 OC (Dufourc et al., 1984). The segmental order parameter measurements of the acyl chain methylene deuterons from C2 to CII are about 0006-2960/90/0429-1025$02.50/0 0 1990 American Chemical Society1026 0.45 and 0.7 in the absence and presence of cholesterol, re- spectively (Stockton & Smith, 1976; Dufourc et al., 1984). Although the mechanism by which