Influence of Cholesterol on Phase Behavior and Nonrandom Mixing of Membrane Components

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This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Cholesterol is the single most abundant lipid species in mammalian plasma membranes. Nonetheless, the underlying nature of its interactions with neighboring membrane molecules has remained obscure, as have its effects on membrane biochemistry. Cholesterol is known to mix nonrandomly with other membrane lipids, and yet in most cases a clear understanding is missing for the lateral distribution of cholesterol, and the way cholesterol influences the lateral distribution of other membrane components. This project has four main conceptual elements: (i) 3-component bilayer lipid mixtures serve as models for the outer and the inner leaflets of mammalian plasma membranes. These 3-component mixtures are sufficiently complex to model real biomembranes, yet are chemically well-defined; (ii) Several independent, complementary methods are used to thoroughly map and characterize the compositional phase behavior of these model membranes; (iii) Some, perhaps all, of the most realistic model membranes have compositional regions with phase separated "nanodomains". These tiny domains, not the same as the clusters found in ordinary nonideal mixing, must be characterized as to size, and their chemical properties correlated with the well-studied macroscopic phases; (iv) Binding of membrane proteins influences the size of nanodomains, and conversely the presence of these nanodomains influences protein binding. Experimentally, this work involves large data sets of fluorescence resonance energy transfer measurements to find the lipid mixture phase boundaries and the partition behavior of fluorescent probes, including membrane-bound peptides. Confocal fluorescence microscopy is used to visualize and identify coexisting phases. X-ray diffraction experiments at the Cornell High Energy Synchrotron Source can detect nanodomains without the use of fluorescent probes. A long-range objective of this project is to discover the fundamental nature of the information that is contained in the structure of lipid polar and hydrocarbon moieties, thereby serving the broader scientific community. In this way, membrane lipids should become as well understood as are nucleic acids and amino acids. Previous work shows that lipid structural information, in part, is manifested in the phase behavior of lipid mixtures, and this project is designed to elucidate this mixture behavior. This research also will yield systematic information about properties of membrane probes that will be of wide use to the community of cell biologists who use fluorescence microscopy. As an integral feature of this project, in order to promote scientific education and training, a group of undergraduate students will receive lectures in lipid physical chemistry and fluorescence spectroscopy, followed by systematic training in lipid analytical chemistry, lipid organic synthesis, and spectroscopic and microscope techniques, which they will then use in their assigned independent research.