I. Causes of Multiple Diffusing Populations of Fluorescently Labeled Probes in Lipid Membranes II. Evaluation of Phospholipid Membranes Incorporating the Polymerizable Lipid Bis-Denpc (16, 16) and Suitability as Ultra-Stable Platforms for Ion Channel Based Sensors

Abstract

This dissertation is composed of two major projects, though some capabilities and findings from the first project were applied to the second. Project I focuses on advancements made in the understanding of the chemical interactions of a number of commonly used fluorescently labeled phospholipid probes. These probes are used for a variety of studies, including labeling of cellular or artificial membranes, examining transport and communication between different membranes, and determining membrane fluidity. Understanding the chemical behavior and interactions of these probes in membranes can be key for the proper interpretation of experimental data. Utilizing fluorescent recovery after photobleaching (FRAP), in combination with other spectroscopic techniques, multiple diffusing populations of commonly used probes in various artificial lipid membrane formats were identified, as were the causes for these populations. This allows for a fuller description of the fluidity of lipid membranes. These findings are the focus of Chapters 3 and 4 while the hardware developed that enabled critical measurements is the focus of Chapter 2. Project II focuses on addressing key limitations in developing ion channel (IC) based biosensors utilizing artificial lipid membranes. Among these limitations are the weak mechanical, chemical, and electrical stabilities of artificial lipid bilayers due to the weak noncovalent interactions involved in the membrane. To address these limitations, the polymerizable lipid bis-dienoyl phosphatidylcholine (bis-DenPC(16, 16)) was characterized for its ability to form ultra-stable membranes suitable for IC based sensors using the model IC gramicidin A (gA). Special attention was given to determining the membrane fluidity given the requirement of gA that two subunits must laterally diffuse to converge and dimerize to form a conductive pore. These studies are the focus of Chapters 5 and 6.