Electrostatic Interaction of Synaptotagmin I with PIP2 Containing Membranes

Kuo, Weiwei, Department of Chemistry, University of Virginia
Cafiso, David, Department of Chemistry, University of Virginia
Bryant, Robert, Department of Chemistry, University of Virginia
Macdonald, Timothy, Department of Chemistry, University of Virginia
Tamm, Lukas, Department of Chemistry, University of Virginia
Castle, David, Department of Cell Biology, University of Virginia

Site-directed spin labeling Electron Paramagnetic Resonance (EPR) and a vesicle sedimentation assay were used to investigate the docking orientations and membranebinding properties of synaptotagmin 1 (Syt 1). Syt 1 is a synaptic vesicle membrane protein that functions as the Ca 2+ sensor for synchronous neuronal exocytosis. Multivalent phosphatidylinositol 4,5-bisphosphate (PIP 2 ) on the presynaptic membrane is essential for exocytosis but its role in membrane fusion remains to be determined. The shared nature of Syt 1 binding with Ca 2+ and anionic phospholipids, including phosphatidylserine (PS) and PIP 2 , is electrostatics. The Ca 2+ -independent and -dependent membrane binding properties of Syt 1 were characterized by combining cw-EPR power saturation and pulse-EPR spectroscopy with simulated annealing to generate a modeling of Syt 1 C2AB domain on PS and PIP 2 containing bilayers and to examine the effect of Ca 2+ and PIP 2 on changes in membrane association using a vesicle sedimentation assay. Data on membrane affinity and depth indicate that the isolated C2A domain does not associate with PS containing membranes without Ca 2+ , but that C2B can electrostatically interact under Ca 2+ -free conditions with membrane interface through its polybasic region (PBR). Models generated from EPR depth and inter-domain restraints suggest that the C2B re-orientates itself to an upright position upon influx of Ca 2+ with Ca 2+ -binding loops (CBL) penetrating the POPC:POPS bilayers. With additional 1 mol % PIP 2 , Syt 1 bridges the two membranes and decreases the separation between planes from 43 Å to 35 Å with CBL inserting deeper and PBR sitting parallel to the membrane surface. The alterations on membrane binding properties iv and docking orientations of Syt 1 C2AB due to Ca 2+ and PIP 2 may be the essential part of fusion mechanism. Models proposed in this dissertation provide a possible step-wise sequence of events at the fusion site before and after participations of Ca 2+ and PIP 2 .

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PHD (Doctor of Philosophy)
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