Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Intracellular membrane fusion is essential for eukaryotic cell life. Synaptic vesicle exocytosis is an exquisitely regulated form of intracellular membrane fusion. In vivo, neurotransmitter release is triggered by the Ca2+ influx within submillisecond timescale. SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are believed to be the minimal fusion machinery which serves as the energy source to overcome the energy barrier between fusion intermediates. In addition to SNAREs, many other regulatory proteins and lipids also exert their effects in this calcium-dependent regulated exocytosis.
Site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy are well established techniques to study the structure and topology of membrane proteins in lipid bilayers. Fluorescent labeled lipid mixing assay is employed to test the functional activity of SNARE proteins and regulators in vitro. To characterize the specific steps in the fusion pathway, the single-vesicle lipid and content mixing assay are developed based on the total internal reflection fluorescence (TIRF) microscopy.
With the advantage of these techniques, we can investigate the interplay between SNAREs and regulatory proteins and lipids in synaptic vesicle exocytosis and the mechanisms of how the interplay between them regulates the rapid membrane fusion. In this dissertation, the conformational change of SNAP-25 along the SNARE assembly pathway has been studied and the mechanism of neurotoxins blocking neurotransmitter release has been examined. The function of the N terminus and accessory alpha-helix of complexin has been disclosed based on the structural analysis of complexin/SNARE quaternary complex. One of key lipid components in the membrane, phosphatidylethanolamine (PE), is also involved in the study to figure out the mechanism of regulatory lipids in the arrangement of SNAREs beyond curvature effect. The information gathered from these detailed investigations will contribute to elaborate the molecular mechanism of SNARE-mediated membrane fusion.
Lu, Bin, "Investigation of the molecular mechanism of SNARE-mediated membrane fusion" (2010). Graduate Theses and Dissertations. 11654.