Biophysical Studies of Cholesterol in Unsaturated Phospholipid Model Membranes

Williams, Justin A. Ph.D., Purdue University, December 2013. Biophysical Studies of Cholesterol in Unsaturated Phospholipid Model Membranes. Major Professor: Stephen R. Wassall. Cellular membranes contain a staggering diversity of lipids. The lipids are heterogeneously distributed to create regions, or domains, whose physical properties differ from the bulk membrane and play an essential role in modulating the function of resident proteins. Many basic questions pertaining to the formation of these lateral assemblies remain. This research employs model membranes of well-defined composition to focus on the potential role of polyunsaturated fatty acids (PUFAs) and their interaction with cholesterol (chol) in restructuring the membrane environment. Omega-3 (n-3) PUFAs are the main bioactive components of fish oil, whose consumption alleviates a variety of health problems by a molecular mechanism that is unclear. We hypothesize that the incorporation of PUFAs into membrane lipids and the effect they have on molecular organization may be, in part, responsible. Chol is a major constituent in the plasma membrane of mammals. It determines the arrangement and collective properties of neighboring lipids, driving the formation of domains via differential affinity for different lipids. The molecular organization of 1-[H31]palmitoyl-2-eicosapentaenoylphosphatidylcholine (PEPCd31) and 1-[H31]palmitoyl-2-docosahexaenoylphosphatidylcholine (PDPC-d31) in membranes with sphingomyelin (SM) and chol (1:1:1 mol) was compared by solid-state H NMR spectroscopy. Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are the two major n-3 PUFAs found in fish oil, while PEPC-d31 and PDPC-d31 are phospholipids containing the respective PUFAs at the sn-2 position and a perdeuterated palmitic acid at the sn-1 position. Analysis of spectra recorded as a function of temperature indicates that in both cases, formation of PUFA-rich (less ordered) and SM-rich (more ordered) domains occurred. A surprisingly substantial proportion of PUFA was found to infiltrate the more ordered domain. There was almost twice as much DHA (65%) as EPA (30%). The implication is that n-3 PUFAs can incorporate into lipid rafts, which are domains enriched in SM and chol in the plasma membrane, and potentially disrupt the activity