Intellectual merit. Lipids and proteins in the plasma membrane are often distributed in widely differing patches of close spatial correlation, as exemplified by the well known association of particular membrane proteins to lipid patches enriched in sphingolipids and cholesterol, also known as lipid rafts. Unfortunately, many of the underlying molecular processes related to functionally important protein-lipid interactions remain unclear due to the challenges involved in characterizing functional lipid heterogeneities in plasma membranes. To overcome these difficulties, the current project is investigating specific aspects of protein-lipid interactions by looking at raft-mimicking liquid-ordered/liquid-disordered phase separations which span bilayers and which only exist in one leaflet of a bilayer, and determining how inter-monolayer coupling affects the sequestration and functionality of select membrane proteins. To achieve this objective, the project uses polymer-tethered lipid bilayers of well-defined lipid compositions. The research includes three specific aims, which are: (1) to explore the packing density of lipids in both monolayer and bilayer-spanning domains; (2) to study the sequestering of select glycosylphosphatidylinositol-anchored proteins and membrane-spanning proteins in the presence of both monolayer and bilayer-spanning lipid domains; and (3) to investigate the recruitment of these proteins in the presence of their native ligands. The packing density of lipids will be determined by fluorescence intensity and anisotropy analysis using a highly sensitive confocal fluorescence detection system. The behavior of membrane proteins will be monitored with single molecule sensitivity using confocal fluorescence intensity analysis and wide-field single molecule fluorescence microscopy. By enabling the analysis of molecular processes at the single protein level, the approach is well suited to explore several of the poorly understood processes of protein-lipid interplay in plasma membranes. For example, the impact of transbilayer coupling of lipids on the sequestering of membrane proteins in lipid rafts will be elucidated. Furthermore, new perspectives about the roles of asymmetric bilayer composition and inter-leaflet coupling of raft domains on protein-protein interactions and protein recruitment processes should be anticipated as an outcome of these investigations.
Broader impacts. The project provides a powerful experimental and training tool to study molecular processes of a wide variety of membrane proteins in well-defined environments. The interdisciplinary character of the research project will provide excellent training for graduate and undergraduate students, thus preparing them for professional careers in emerging areas at the interface between traditional sciences, materials science, engineering, and medicine. Being located at an urban campus with a significant minority student population, the principal investigator will remain committed to minority student education and will expand outreach activities at the high-school level (internships, high-school science/math summer camp) and through the institutional Nanoscale Imaging Center. Research results will also be integrated into several specific undergraduate- and graduate-level courses (physical chemistry, biomimetic chemistry, and biomaterials science).
