Formation of the liquid-ordered phase in fully hydrated mixtures of cholesterol and lysopalmitoylphosphatidylcholine Deborah L. Gater, John M. Seddon* and Robert V. Law* Received 13th July 2007, Accepted 18th October 2007 First published as an Advance Article on the web 9th November 2007 DOI: 10.1039/b710726a The role of cholesterol (Chol) in promoting lamellar phase formation in mixtures with 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso-PPC) in excess water was investigated using multinuclear solid-state NMR and X-ray scattering. It was found that mixtures containing Chol and Lyso-PPC form a liquid-ordered (L o ) lamellar phase over a range of temperatures and concentrations, as previously observed in mixtures of Chol with various diacylphospholipids. The maximum quadrupolar splitting of the 2 H-NMR powder patterns for samples containing per- deuterated Lyso-PPC were 40–50 kHz which is strongly indicative of an L o phase. This evidence was supported by wide angle X-ray scattering data which showed a characteristic diffuse peak centred at 4.2 A ˚ . The L o phase coexists with an isotropic Lyso-PPC phase at Chol concentrations up to 70 mol% Chol, and with Chol crystals at Chol concentrations above this value. Below 70 mol% Chol, an increase in the concentration of Chol in the system caused a corresponding increase in the proportion of the L o phase present compared with the amount of isotropic Lyso- PPC. The chemical-shift anisotropy (CSA) of the static 31 P-NMR spectra of the L o phase showed the symmetry of a lamellar phase, but the linewidth, Ds, was much narrower than CSA powder patterns obtained for diacylphospholipids in similar conditions, being y20 ppm as opposed to y40 ppm, respectively. Introduction Cholesterol (Chol) is the ubiquitous sterol in mammalian cell membranes. 1 Its effects on the lipid bilayer are complex, and range from reducing the permeability of the bilayer, 1 to moderating phase behaviour, 2 and altering the bilayer micro- mechanical stability, bending moduli and area expansivity. 3,4 Chol is also implicated in the formation of the biologically relevant liquid-ordered (L o ) phase. 5 Along with all of these properties, Chol can also drive the formation of lamellar bilayer structures when mixed with non-lamellar amphiphiles such as fatty acids. 6,7 It is this property in which we are most interested. In the case of both fatty acids and the 1-palmitoyl- 2-hydroxy-sn-glycero-3-phosphocholine (Lyso-PPC) studied here, the lamellar phase formed by the addition of Chol is the L o phase. The L o phase 8–11 is a lamellar liquid-crystalline phase with properties different to those of both the liquid-disordered (L a ) and the gel (L b ) phases. The L o phase has long-axis rotational correlation times, and lateral diffusion rates, comparable to the L a phase, but the acyl chains of the amphiphiles present in the system adopt an ordered, largely all-trans conformation similar to that found in the L b phase, rather than the highly disordered conformations found in the L a phase. The L o phase has been linked to the phenomenon of ‘lipid rafts’ in biological membranes. 5,12,13 In particular, it has been proposed that Chol interacts preferentially with certain long- chain saturated phospholipids such as sphingomyelins to form regions of the L o phase in a membrane which is predominantly in the L a phase. 14 This hypothesis arose as a result of the presence of high proportions of Chol and saturated long-chain phospholipids as insoluble fractions in biological extracts isolated with detergent from whole membranes. 14 Some of these conclusions are now in doubt as a result of recent evidence suggesting that membrane extracts made using this technique contain artefacts. 15–18 However, since this time, other studies have used fluorescence techniques to look directly at both intact and reconstituted biological membranes. In these systems it has been observed that the addition of cyclodextrin, which acts to remove Chol from the membrane, to a membrane exhibiting ‘raft-like’ behaviour often leads to the disruption of the rafts and the appearance of a more homogeneous phase. 19–21 The important role of Chol in these biologically occurring regions of ordered fluid lamellar phase has been supported and expanded upon by studies using model membranes. 10,22–25 It has been found that Chol packs against the chains of saturated phospholipids and causes an increase in the order of the chains, as measured by 2 H-NMR order parameters, S CD , where S CD reports on the orientational ordering of the C– 2 H bonds in the methylene groups down the acyl chain, on the NMR timescale. The phase behaviour of various mixtures of lysopho- spholipids, alkanes, alkanols and diacylphosphatidylcholine (PC) and diacylphosphatidylethanolamine (PE) has been studied previously. 26–28 The ability of Chol to induce L o phase formation in fatty acid systems has also been reported. 6,7 A previously recorded 31 P-NMR spectrum of the single composi- Department of Chemistry, Imperial College, South Kensington Campus, London, UK SW7 2AZ. E-mail: j.seddon@imperial.ac.uk; r.law@imperial.ac.uk; Fax: +44 (0)20 7594 5804; Tel: +44 (0)20 7594 5860 for R. V. Law PAPER www.rsc.org/softmatter | Soft Matter This journal is ß The Royal Society of Chemistry 2008 Soft Matter, 2008, 4, 263–267 | 263