Item Details

Investigation of the Solution and Membrane-Bound Structure of Synaptotagmin1 via Electron Paramagnetic Resonance Spectroscopy

Herrick, Dawn Zbell
Thesis/Dissertation; Online
Herrick, Dawn Zbell
Bryant, Robert
Bushweller, John
Columbus, Linda
Tamm, Lukas
Cafiso, David
Synaptotagmin 1 (syt1) is a synaptic vesicle membrane protein that functions as the Ca 2+ -sensor in neuronal exocytosis. Site-directed spin labeling (SDSL) was used to investigate the conformation and membrane-binding properties of syt1. First, the Ca 2+ -dependent membrane interactions of a water soluble fragment of syt1C2AB that contains its two C2 domains (C2A and C2B) were determined using SDSL. Depth parameters were obtained for spin labeled mutants of C2AB when bound to negatively-charged membranes, and distance constraints were used to generate a model for the orientation and position of syt1 on the bilayer interface. Both C2A and C2B penetrate the membrane interface with their first and third Ca 2+ -binding loops. The two domains are positioned deeper into the bilayer interior when present in the tandem construct. These data indicate that C2A and C2B do not act independently, but influence their mutual membrane penetration. Second, the Ca 2+ -independent membrane interactions of syt1C2AB were characterized. It is shown that C2B binds to negatively-charged membranes in a Ca 2+ - independent manner. SDSL was used to obtain bilayer depth restraints and a simulated annealing routine was used to generate a model for the membrane docking of the syt1C2B(C2A). In this model, the polybasic strand of C2B forms the membrane binding surface for the domain through an electrostatic interaction without penetrating the bilayer. In the presence of Ca 2+ , the domain rotates from roughly parallel to perpendicular to the bilayer, thus syt1C2AB may act as a switch. iii Third, SDSL was used to generate models for the solution and membrane-bound structures of syt1C2AB. In solution, distances between the two C2 domains were measured using double electron-electron resonance (DEER) and were used in a simulated annealing routine. The data indicate that the two C2 domains are flexibly linked, do not interact with each other (with or without Ca 2+ ) and favor an antiparallel orientation. A similar approach was taken for membrane-associated C2AB, combining both distances and bilayer depth restraints. The restraints only are satisfied when C2A and C2B are antiparallel and docked to opposing bilayers. The result suggests that syt1 functions to bridge across the vesicle and plasma membrane surfaces in a Ca 2+ -dependent manner. Note: Abstract extracted from PDF text
University of Virginia, Department of Chemistry, PHD (Doctor of Philosophy), 2009
Published Date
PHD (Doctor of Philosophy)
Libra ETD Repository
Logo for In CopyrightIn Copyright


Read Online