Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
In the cell, there are about 60% of the total proteins are membrane and membrane-associated proteins. Exocytosis, or its reverse phase endocytosis, contains a variety of membrane proteins such as synaptobrevin2, syntaxin-1A etc and membrane-associated proteins, for example SNAP25 and epsin1 etc. Synaptobrein2 is also called vesicle-associated protein or v-SANRE which interacts with t-SNAREs syntaxin-1A and SNAP25 binary complex to form a four helical-bundle complex, which was the minimal fusion machinery for synaptic vesicle fused with plasma membrane in neuron. In addition, many other proteins such as Munc18, synaptotagmin, complexin etc are essential regulators to control neuronal transmitter release spatially and temporally. Epsin1, a membrane-associated protein binding with the plasma membrane by interaction with lipid phosphatidylinositol 4, 5-biphosphate (PIP2), on the contrary, plays a role in the clathrin-mediated endocytosis process.
The purpose of this work is to investigate the structures of TMD (transmembrane domain) of synaptobrevin2 and N-termini (residues 1-14) of ENTH domain of epsin1 by EPR (electron paramagnetic resonance) technique. In addition, lipid mixing assay with fluorescence dyes was also applied to mimic the kinetics of membrane fusion with recombinant SNARE proteins on the liposome in vitro. EPR studies with TMD of synaptobrevin2 revealed that TMD was characterized as a loose dimmer with interaction mostly at the N-half of TMD, while C-half interacted with each other to form a dimmer only on the vesicle with 40% cholesterol. Thus, a scissors mechanism exists for TMD of synaptobrevin2 with the stimulation of cholesterol molecule. Moreover, disulfide cross-linked lipid mixing assay confirmed different functions for these two confirmations of TMD. Meanwhile, the EPR work of N-termini of ENTH domain provided evidence that the N-termini of ENTH domain forms an amphiphilic helix when it inserts into the vesicle contained 3% of lipid PIP2. Our power saturation study unraveled the topology of this helix with hydrophobic residues L6 and M10 deep insertion into the membrane. Most interestingly, the work helps us to identify the anti-parallel dimerization of N-termini of ENTH domain on the surface of membrane. Functional studies both in vitro and in vivo confirmed that such a self-association of ENTH domains on the membrane surface plays an important role to tubulate the membrane during endocytosis process.
Tong, Jiansong, "Structural and functional studies with membrane-associated proteins in synaptic exocytosis and endocytosis" (2009). Graduate Theses and Dissertations. 10975.