{"id":{"repo_id":"utswmed","oai_identifier":"oai:utswmed-ir.tdl.org:2152.5/763"},"canonical_url":"https://search.dev.ndltd.org/etd/utswmed/oai:utswmed-ir.tdl.org:2152.5/763","repository":{"repo_id":"utswmed","name":"University of Texas Southwestern Medical Center","base_url":"https://utswmed-ir.tdl.org/server/oai/request"},"display":{"title":"Structural and Functional Studies of C2-Domain Proteins Involved In Neurotransmitter Release","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.","abstract_html":"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.","abstract_has_math":false,"creators":["Dai, Han"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Rizo-Rey, José"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2010,"date_issued":"2010-07-12T19:00:25Z","date_published":"2010-07-12T19:00:25Z","updated_at":"2026-07-24T05:52:38Z","subjects":["SNARE Proteins","Nerve Tissue Proteins","Nuclear Magnetic Resonance, Biomolecular"],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["755008665"],"render_values":[{"text":"755008665","href":null,"code":true}]}]},"links":{"outbound_url":"https://hdl.handle.net/2152.5/763","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Rizo-Rey, José"]},{"key":"dc:creator","label":"Author","values":["Dai, Han"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2010-07-12T19:00:25Z","2007-05-22"]},{"key":"dc:type","label":"Dc Type","values":["Thesis","Text","dissertation"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["SNARE Proteins","Nerve Tissue Proteins","Nuclear Magnetic Resonance, Biomolecular"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://hdl.handle.net/2152.5/763","755008665"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["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."]},{"key":"dc:format","label":"Dc Format","values":["Electronic","application/pdf","born digital"]},{"key":"dc:title","label":"Title","values":["Structural and Functional Studies of C2-Domain Proteins Involved In Neurotransmitter Release"]}]}],"canonical_facts":{"dc:contributor":["Rizo-Rey, José"],"dc:creator":["Dai, Han"],"dc:date":["2010-07-12T19:00:25Z","2007-05-22"],"dc:description":["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."],"dc:format":["Electronic","application/pdf","born digital"],"dc:identifier":["https://hdl.handle.net/2152.5/763","755008665"],"dc:language":["en"],"dc:subject":["SNARE Proteins","Nerve Tissue Proteins","Nuclear Magnetic Resonance, Biomolecular"],"dc:title":["Structural and Functional Studies of C2-Domain Proteins Involved In Neurotransmitter Release"],"dc:type":["Thesis","Text","dissertation"]},"updated_at":"2026-07-24T05:52:38Z"}