Structural characterization of synaptotagmin I

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dc.contributor.advisorAndres Oberhauseren_US
dc.contributor.committeeMemberXiaodong Chengen_US
dc.contributor.committeeMemberR. Bryan Suttonen_US
dc.contributor.committeeMemberMark Hellmichen_US
dc.contributor.committeeMemberDarren Boehningen_US
dc.creatorKerry Fusonen_US
dc.date.accessioned2011-12-20T16:04:38Z
dc.date.accessioned2014-02-19T22:05:06Z
dc.date.available2010-09-28en_US
dc.date.available2011-12-20T16:04:38Z
dc.date.available2014-02-19T22:05:06Z
dc.date.created2009-04-15en_US
dc.date.issued2009-02-13en_US
dc.description.abstractSynaptotagmin 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\nen_US
dc.format.mediumelectronicen_US
dc.identifier.otheretd-04152009-130217en_US
dc.identifier.urihttp://hdl.handle.net/2152.3/98
dc.language.isoengen_US
dc.rightsCopyright © 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.en_US
dc.subjectX-ray crystallographyen_US
dc.subjectsynaptotagminen_US
dc.subjectprotein structureen_US
dc.subjectexocytosisen_US
dc.titleStructural characterization of synaptotagmin Ien_US
dc.type.genredissertationen_US
dc.type.materialtexten_US
thesis.degree.departmentHuman Biological Chemistry and Geneticsen_US
thesis.degree.grantorThe University of Texas Medical Branchen_US
thesis.degree.levelDoctoralen_US
thesis.degree.namePhDen_US

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