Electrostatic Interaction of Synaptotagmin I with PIP2 Containing Membranes

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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