{"id":{"repo_id":"utmb","oai_identifier":"oai:utmb-ir.tdl.org:2152.3/98"},"canonical_url":"https://search.dev.ndltd.org/etd/utmb/oai:utmb-ir.tdl.org:2152.3/98","repository":{"repo_id":"utmb","name":"University of Texas Medical Branch","base_url":"https://utmb-ir.tdl.org/server/oai/request"},"display":{"title":"Structural characterization of synaptotagmin I","abstract":"Synaptotagmin I is the most abundant Ca+2 binding protein present on synaptic vesicles accounting for 7% of total vesicle protein and is widely accepted as the Ca2+ sensor in fast synchronous neurotransmitter release. The protein is composed of one trans-membrane domain, an unstructured linker followed by two C2A domains identified as C2A and C2B. Each C2 domain is composed of an 8 stranded β-sandwich joined by a 9 amino acid linker. The Ca+2 binding pocket is composed of three loops located at the apex of the protein. In the Syt I C2AB structure, we see evidence of a domain structural change in the absence of Ca2+. Analysis of interacting residues between C2A and C2B show a network of highly conserved residues within the C2 domain that regulates Ca+2/phohspholipid binding in C2A. Analysis of the Syt I C2A structure, as well as, previous C2A structures shows a strong H-bond between Tyr 180 and His 237 in C2A. By removing this H-bond, disorder of Loop 3 is increased and the thermodynamic stability of the C2 domain decreases. Our hypothesis is that the absolute position of the Ca2+ binding loops of C2 domains affects Ca+2 affinity and, and ultimately domain stability. We used several different biochemical approaches to test the hypothesis. We assessed the importance of Loop 3 mutations using X-ray crystallography methods, bulk thermodynamic measurements using lifetime fluorescence, and analyzed the mechanical properties of the C2-domains using single molecule force spectroscopy.\\r\\nWe studied the mechanical stability of the C2A and C2B domains of human Syt1 using single-molecule atomic force microscopy. We found that stretching the C2AB domains of Syt1 resulted in two distinct unfolding force peaks. The larger force peak of ~100pN was identified as C2B and the second peak of ~50pN as C2A. Further, a significant fraction of C2A domains unfolded through a low force intermediate that was not observed in C2B. We conclude that these domains have different mechanical properties. We hypothesize that a relatively small stretching force may be sufficient to deform the effector-binding regions of C2A domain and modulate the affinity for SNAREs, phospholipids and Ca+2.\\r\\n","abstract_html":"Synaptotagmin I is the most abundant Ca+2 binding protein present on synaptic vesicles accounting for 7% of total vesicle protein and is widely accepted as the Ca2+ sensor in fast synchronous neurotransmitter release. The protein is composed of one trans-membrane domain, an unstructured linker followed by two C2A domains identified as C2A and C2B. Each C2 domain is composed of an 8 stranded β-sandwich joined by a 9 amino acid linker. The Ca+2 binding pocket is composed of three loops located at the apex of the protein. In the Syt I C2AB structure, we see evidence of a domain structural change in the absence of Ca2+. Analysis of interacting residues between C2A and C2B show a network of highly conserved residues within the C2 domain that regulates Ca+2/phohspholipid binding in C2A. Analysis of the Syt I C2A structure, as well as, previous C2A structures shows a strong H-bond between Tyr 180 and His 237 in C2A. By removing this H-bond, disorder of Loop 3 is increased and the thermodynamic stability of the C2 domain decreases. Our hypothesis is that the absolute position of the Ca2+ binding loops of C2 domains affects Ca+2 affinity and, and ultimately domain stability. We used several different biochemical approaches to test the hypothesis. We assessed the importance of Loop 3 mutations using X-ray crystallography methods, bulk thermodynamic measurements using lifetime fluorescence, and analyzed the mechanical properties of the C2-domains using single molecule force spectroscopy.\\r\\nWe studied the mechanical stability of the C2A and C2B domains of human Syt1 using single-molecule atomic force microscopy. We found that stretching the C2AB domains of Syt1 resulted in two distinct unfolding force peaks. The larger force peak of ~100pN was identified as C2B and the second peak of ~50pN as C2A. Further, a significant fraction of C2A domains unfolded through a low force intermediate that was not observed in C2B. We conclude that these domains have different mechanical properties. We hypothesize that a relatively small stretching force may be sufficient to deform the effector-binding regions of C2A domain and modulate the affinity for SNAREs, phospholipids and Ca+2.\\r\\n","abstract_has_math":false,"creators":["Kerry Fuson"],"institution":"The University of Texas Medical Branch","degree_name":"PhD","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Andres Oberhauser"],"committee_chairs":[],"committee_members":["Xiaodong Cheng","R. Bryan Sutton","Mark Hellmich","Darren Boehning"],"year":2009,"date_issued":"2009-02-13","date_published":"2009-02-13","updated_at":"2026-07-24T05:51:09Z","subjects":["X-ray crystallography","synaptotagmin","protein structure","exocytosis"],"languages":["eng"],"rights":["Copyright © is held by the author. Presentation of this material on the TDL web site by The University of Texas Medical Branch at Galveston was made possible under a limited license grant from the author who has retained all copyrights in the works."],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-04152009-130217"],"render_values":[{"text":"etd-04152009-130217","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2152.3/98","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Andres Oberhauser"]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Xiaodong Cheng","R. 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Presentation of this material on the TDL web site by The University of Texas Medical Branch at Galveston was made possible under a limited license grant from the author who has retained all copyrights in the works."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-04152009-130217"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/2152.3/98"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Synaptotagmin I is the most abundant Ca+2 binding protein present on synaptic vesicles accounting for 7% of total vesicle protein and is widely accepted as the Ca2+ sensor in fast synchronous neurotransmitter release. The protein is composed of one trans-membrane domain, an unstructured linker followed by two C2A domains identified as C2A and C2B. Each C2 domain is composed of an 8 stranded β-sandwich joined by a 9 amino acid linker. The Ca+2 binding pocket is composed of three loops located at the apex of the protein. In the Syt I C2AB structure, we see evidence of a domain structural change in the absence of Ca2+. Analysis of interacting residues between C2A and C2B show a network of highly conserved residues within the C2 domain that regulates Ca+2/phohspholipid binding in C2A. Analysis of the Syt I C2A structure, as well as, previous C2A structures shows a strong H-bond between Tyr 180 and His 237 in C2A. By removing this H-bond, disorder of Loop 3 is increased and the thermodynamic stability of the C2 domain decreases. Our hypothesis is that the absolute position of the Ca2+ binding loops of C2 domains affects Ca+2 affinity and, and ultimately domain stability. We used several different biochemical approaches to test the hypothesis. We assessed the importance of Loop 3 mutations using X-ray crystallography methods, bulk thermodynamic measurements using lifetime fluorescence, and analyzed the mechanical properties of the C2-domains using single molecule force spectroscopy.\\r\\nWe studied the mechanical stability of the C2A and C2B domains of human Syt1 using single-molecule atomic force microscopy. We found that stretching the C2AB domains of Syt1 resulted in two distinct unfolding force peaks. The larger force peak of ~100pN was identified as C2B and the second peak of ~50pN as C2A. Further, a significant fraction of C2A domains unfolded through a low force intermediate that was not observed in C2B. We conclude that these domains have different mechanical properties. We hypothesize that a relatively small stretching force may be sufficient to deform the effector-binding regions of C2A domain and modulate the affinity for SNAREs, phospholipids and Ca+2.\\r\\n"]},{"key":"dc:format.medium","label":"Dc Format Medium","values":["electronic"]},{"key":"dc:title","label":"Title","values":["Structural characterization of synaptotagmin I"]}]}],"canonical_facts":{"dc:contributor.advisor":["Andres Oberhauser"],"dc:contributor.committeemember":["Xiaodong Cheng","R. Bryan Sutton","Mark Hellmich","Darren Boehning"],"dc:creator":["Kerry Fuson"],"dc:date.accessioned":["2011-12-20T16:04:38Z"],"dc:date.available":["2010-09-28","2011-12-20T16:04:38Z"],"dc:date.issued":["2009-02-13"],"dc:description.abstract":["Synaptotagmin I is the most abundant Ca+2 binding protein present on synaptic vesicles accounting for 7% of total vesicle protein and is widely accepted as the Ca2+ sensor in fast synchronous neurotransmitter release. The protein is composed of one trans-membrane domain, an unstructured linker followed by two C2A domains identified as C2A and C2B. Each C2 domain is composed of an 8 stranded β-sandwich joined by a 9 amino acid linker. The Ca+2 binding pocket is composed of three loops located at the apex of the protein. In the Syt I C2AB structure, we see evidence of a domain structural change in the absence of Ca2+. Analysis of interacting residues between C2A and C2B show a network of highly conserved residues within the C2 domain that regulates Ca+2/phohspholipid binding in C2A. Analysis of the Syt I C2A structure, as well as, previous C2A structures shows a strong H-bond between Tyr 180 and His 237 in C2A. By removing this H-bond, disorder of Loop 3 is increased and the thermodynamic stability of the C2 domain decreases. Our hypothesis is that the absolute position of the Ca2+ binding loops of C2 domains affects Ca+2 affinity and, and ultimately domain stability. We used several different biochemical approaches to test the hypothesis. We assessed the importance of Loop 3 mutations using X-ray crystallography methods, bulk thermodynamic measurements using lifetime fluorescence, and analyzed the mechanical properties of the C2-domains using single molecule force spectroscopy.\\r\\nWe studied the mechanical stability of the C2A and C2B domains of human Syt1 using single-molecule atomic force microscopy. We found that stretching the C2AB domains of Syt1 resulted in two distinct unfolding force peaks. The larger force peak of ~100pN was identified as C2B and the second peak of ~50pN as C2A. Further, a significant fraction of C2A domains unfolded through a low force intermediate that was not observed in C2B. We conclude that these domains have different mechanical properties. We hypothesize that a relatively small stretching force may be sufficient to deform the effector-binding regions of C2A domain and modulate the affinity for SNAREs, phospholipids and Ca+2.\\r\\n"],"dc:format.medium":["electronic"],"dc:identifier.other":["etd-04152009-130217"],"dc:identifier.uri":["http://hdl.handle.net/2152.3/98"],"dc:language.iso":["eng"],"dc:rights":["Copyright © is held by the author. Presentation of this material on the TDL web site by The University of Texas Medical Branch at Galveston was made possible under a limited license grant from the author who has retained all copyrights in the works."],"dc:subject":["X-ray crystallography","synaptotagmin","protein structure","exocytosis"],"dc:title":["Structural characterization of synaptotagmin I"],"thesis:degree_level":["Doctoral"],"thesis:degree_name":["PhD"],"thesis:institution_name":["The University of Texas Medical Branch"]},"updated_at":"2026-07-24T05:51:09Z"}