Investigating Lipid-Protein Interactions Using Supported Lipid Bilayers
Membrane proteins play vital roles in cell function and as such represent the targets of over 60% of pharmaceuticals on the market1. Lipid interactions with membrane proteins are a crucial but often overlooked effector of cell physiology. Lipids impart conformational stability and modulation on membrane proteins, which is closely tied to their function. In addition, there is increasing evidence that the cell membrane is heterogeneous with dynamic "raft" domains with different compositions and the dynamics of these domains could regulate membrane protein function via lipid-protein interactions. When studying membrane proteins in cells, these types of lipid-protein interactions are preserved, but decoupling to elucidate specific mechanisms is difficult because of the complexity of the cell system. Other techniques use detergent solubilized membrane proteins to reduce complexity, but lipid interactions are also lost. The proposed bridging technique is the supported lipid bilayer (SLB). It combines a large library of techniques with a simplified, yet sufficient mimic of the properties of the cell membrane; however key functionalities have remained unaddressed. In this work I discuss development of additional function for the SLB platform in two key areas: 1) generation of patterned supported lipid bilayers that mimic membrane heterogeneities such as the "lipid raft" to evaluate associated dynamics and 2) the inclusion of membrane proteins in a robust and simplified manner. In Chapter 1, I conduct a thorough literature review of the field. In Chapter 2, I describe a laminar flow patterned two-phase bilayer used to perform an affinity based separation of membrane species. In Chapter 3, I characterize a novel technique for delivery of membrane proteins from cell blebs into a cushioned supported lipid bilayer. In Chapter 4, I detail patterning of two-phase supported lipid bilayers as a mimic for lipid rafts. In Chapter 5, I provide an outlook of how our work has increased the biological relevance of the supported lipid bilayer and has increased its utility for studying a wide range of biological processes.
Membrane proteins; Lipid-Protein Interactions; Lipid Bilayers
Delisa,Matthew; Travis,Alexander J.
Ph. D., Chemical Engineering
Doctor of Philosophy
dissertation or thesis