Structural and Functional Studies of C2-Domain Proteins Involved In Neurotransmitter Release
dc.contributor.advisor | Rizo-Rey, Jose | en |
dc.creator | Dai, Han | en |
dc.date.accessioned | 2010-07-12T19:00:25Z | en |
dc.date.accessioned | 2014-02-19T22:00:02Z | |
dc.date.available | 2010-07-12T19:00:25Z | en |
dc.date.available | 2014-02-19T22:00:02Z | |
dc.date.issued | 2007-05-22 | en |
dc.description.abstract | Neurotransmitter release is mediated by synaptic vesicle exocytosis at presynaptic nerve terminals. This process is extremely fast and strictly regulated by the intracellular Ca2+ concentration. To achieve this exquisite regulation, many proteins are involved. The central fusion machinery includes the SNARE proteins synaptobrevin, syntaxin and SNAP-25, as well as Munc18. Besides these universal components, many other neuronal specific proteins are also involved in regulating Ca2+-triggered release. Interestingly, most of these regulatory proteins contain C2 domains, versatile protein modules with Ca2+-dependent and Ca2+- independent activities. However, the mechanism of regulation by these C2-domain proteins remains unclear. My research has focused on understanding the structural and functional properties of two types of C2-domain proteins, synaptotagmins and RIMs. With NMR spectroscopy and biochemical assays, we demonstrated that synaptotagmin 4 is a Ca2+ sensor in Drosophila but not in rat, in contrast to the prediction based on sequence alignments. X-ray crystallography revealed that changes in the orientations of critical Ca2+-ligands render the rat synaptotagmin 4 C2B domain unable to form full Ca2+-binding sites. We also analyzed the structural and biochemical properties of the RIM2 C2A domain. NMR spectroscopy and FRET experiments demonstrated no interaction between the RIM2 C2A domain and Ca2+, phospholipid, synaptotagmin 1, and SNAP-25. However, the crystal structure of RIM C2A domain exhibits a strikingly dipolar distribution of the surface electrostatic potential. Several lines of evidence from the crystal structure suggested a potential target binding site around the bottom 310-helix. With fluorescence microscopy and microfluidic channel technology, we demonstrated that synaptotagmin 1 binds simultaneously to phospholipids and the SNARE complex reconstituted in membranes in the presence of Ca2+, forming a quaternary SSCAP complex, and that the membrane penetration of synaptotagmin 1 into phospholipids is independent of the reconstituted SNARE complex. We also showed that synaptotagmin 1 displaces complexin from the reconstituted SNARE complex in the presence of Ca2+, and that the synaptotagmin 1 C2B domain is primarily responsible for SNARE binding. Moreover, NMR spectroscopy and site-directed mutagenesis studies yielded structural information of the potential binding interface, allowing us to use computational modeling and docking to generate a preliminary model of the SSCAP complex. | en |
dc.format.digitalOrigin | born digital | en |
dc.format.medium | Electronic | en |
dc.format.mimetype | application/pdf | en |
dc.identifier.other | 755008665 | en |
dc.identifier.uri | http://hdl.handle.net/2152.5/763 | en |
dc.language.iso | en | en |
dc.subject | SNARE Proteins | en |
dc.subject | Nerve Tissue Proteins | en |
dc.subject | Nuclear Magnetic Resonance, Biomolecular | en |
dc.title | Structural and Functional Studies of C2-Domain Proteins Involved In Neurotransmitter Release | en |
dc.type.genre | dissertation | en |
dc.type.material | Text | en |
thesis.date.available | 2008-05-22 | en |
thesis.degree.department | en | |
thesis.degree.discipline | Molecular Biophysics | en |
thesis.degree.grantor | Graduate School of Biomedical Sciences | en |
thesis.degree.level | Ph.D. | en |
thesis.degree.name | Doctor of Philosophy | en |