13522 DOI: 10.1021/la9020299 Langmuir 2009, 25(23), 13522–13527 Published on Web 08/13/2009 pubs.acs.org/Langmuir Published 2009 by the American Chemical Society Asymmetric Distribution of Cholesterol in Unilamellar Vesicles of Monounsaturated Phospholipids Norbert Ku cerka,* ,†,‡ Mu-Ping Nieh, † and John Katsaras* ,†,§,^ † Canadian Neutron Beam Centre, National Research Council, Chalk River, Ontario K0J 1J0, Canada, ‡ Department of Physical Chemistry of Drugs, Faculty of Pharmacy, Comenius University, 832 32 Bratislava, Slovakia, § Department of Physics, Brock University, St. Catharines, Ontario L2S 3A1, Canada, and ^ Guelph-Waterloo Physics Institute and Biophysics Interdepartmental Group, University of Guelph, Guelph, Ontario N1G 2W1, Canada Received June 5, 2009. Revised Manuscript Received July 22, 2009 We have studied the effect of cholesterol on curved bilayers using 600 A ˚ unilamellar vesicles made of mono- unsaturated lipids. From small-angle X-ray scattering experiments we were able to detect an asymmetric distribution of lipid densities across certain bilayers. We discovered that, with the exception of diC24:1PC bilayers, monounsaturated diacylphosphatidylcholine lipids (diCn:1PC, n=14, 16, 18, 20, and 22) form symmetric bilayers. However, the addition of 44 mol % cholesterol resulted in some of these bilayers (i.e., n = 14, 16, and 18) to become asymmetric, where cholesterol was found to distribute unequally between the bilayer’s two leaflets. This finding is potentially of relevance to biological membranes made up of different types of lipids and whose local curvature may be dictated by lipid composition. Introduction Membrane curvature is an essential feature for inter- and intracellular communication as various cell membrane functions (i.e., budding and fusion) are performed when specific geometric conditions are present. 1,2 Changes to a cell’s membrane local curvature have been observed to drive the lateral organization in model systems 3 and may also lead to lateral heterogeneities in cell membranes. 4 A membrane’s local curvature and its lateral orga- nization are often modulated by membrane-associated proteins; on the other hand, curvature alone enables mechanisms for organizing mobile membrane molecules. 2 Of note is that mem- brane lateral heterogeneities in model systems have been observed to produce local variations in membrane curvature. 5,6 There have been numerous studies on single-component model membranes assessing the influence of curvature on a membrane’s thermodynamic properties and its asymmetry. 7-13 Meanwhile, similar studies of multicomponent membranes have addressed questions related to lipid miscibility. 14-16 Data from these studies show that an increase in membrane curvature results in a shift and broadening of the phase transition temperature of single-compo- nent vesicles and influences the miscibility and bilayer asymmetry in mixed lipid systems. Though it is not known how a cell regulates the lateral orga- nization of its plasma membrane, cholesterol-dependent phase separation is believed to play a key role. 2 The connection between cholesterol content and membrane thickness has been suggested in the sorting and trafficking of membrane proteins along the exocytic pathway through the Golgi apparatus. For example, transmem- brane domains of plasma membrane proteins are, on average, five amino acids longer than those of the Golgi, and membranes along the exocytic pathway increasingly thicken from the endoplasmic reticulum to the plasma membrane. 17 This progressive membrane thickening has been correlated with a concomitant increase in cholesterol content along the secretory pathway, suggesting that cholesterol determines the membrane’s thickness and controls the destination of proteins based on hydrophobic matching. 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