Chemical approaches for the investigation of protein-lipid binding interactions synthesis, modification, and evaluation of novel azido-based lipid probes

Lipids such as diacylglycerol (DAG) and phosphatidic acid (PA) have been shown to play key roles in cell signaling events. The anchoring of proteins to the membrane via DAG or PA binding helps regulate protein function and localization and is also involved in normal and disease-related cellular processes. In order to fully understand these pathways, it is necessary to develop synthetic analogues that mimic the natural species, but that also have a means which allows investigation of the process at the molecular level. This dissertation will detail the design and synthesis of novel lipid-based probes possessing a reactive azide tag, which can then be functionalized via the 1,3-dipolar cycloaddition reaction ("click" chemistry) to produce a series of analogues with fluorescence and photo-cross-linking capabilities. Initial receptor-ligand binding results will be presented, along with the description of future studies that will provide insight into the mechanism of such interactions. In addition, the immobilization of whole liposomes onto a surface will be discussed. Techniques such as measuring the fluorescence-after-immobilization and dye leakage assays were developed and optimized to prove that immobilization onto a surface had been achieved. This approach is then geared towards the development of a high-throughput, micro-plate assay to detect the binding of a protein to a membrane. Here, liposomes which express a specific ligand at the surface will be immobilized and introduced to various receptors. Appropriate techniques will be employed to determine whether a given receptor binds to the expressed ligand. Lastly, an initial attempt at the development of an artificial vesicle fusion system using "click" chemistry will be discussed.