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Quantitating the Energetics and Binding of Syt1 C2 Domain to Lipid Bilayers

Thapa, Anusa
Thesis/Dissertation; Online
Thapa, Anusa
Cafiso, David
Signaling in the central nervous system is mediated by the fusion of synaptic vesicles to the presynaptic membrane of the neuron. This fusion event is driven by membrane-anchored SNAREs (Soluble NSF Attachment Protein Receptors), but requires a number of additional proteins including munc18, complexin and Synaptotagmin 1 (Syt 1). Synaptotagmin 1 functions as the Ca2+ sensor in neuronal fusion, and it may act by bridging bilayers and assisting SNARE-mediated fusion. We have determined the role of electrostatics and electrostatic screening in determining the membrane association of a tandem fragment of Syt1 containing its two C2 domains. Charge neutralization of two highly conserved arginine residues opposite the Ca2+ binding sites (R398Q/R399Q) did not alter the binding of C2AB to membranes composed of mixtures of PS and PIP2; however, neutralization of charge in the conserved polybasic face (K326A/K327A) dramatically reduced binding. The polybasic face also demonstrated specificity for PIP2 both with and without Ca2+ through a mechanism that is likely driven by electrostatics. ATP is known to modulate the interactions of Syt1 (Park et al. (2012), NSMB 19, 991) and ATP dramatically alters the membrane binding of C2AB. 2D HSQC NMR experiments were used to determine the sites of interaction of ATP and of the PIP2 headgroup, IP3. At levels that are physiologically relevant, both ATP and IP3 modulate chemical shifts in the polybasic face of C2B, suggesting that these electrolytes reversibly associate at these sites in Syt1. Polyelectrolytes are also seen to interact with the membrane binding loops of both C2A and C2B. EPR power saturation depth measurements indicate that the binding mode of C2AB is different in PCPS vesicles versus PCPIP2 vesicles. It was found that the Ca2+-binding loops do not insert into bilayers where PIP2 is the only anionic lipid present. This was in contrast to PCPS suggesting that the mechanism of Syt1 binding is significantly different in PIP2 bilayers. The results suggested that certain regions in C2B domain are specific to either PS or PIP2. These interactions are likely to play an important role in controlling the membrane interactions of Syt1 in vivo.
University of Virginia, Department of Chemistry, PHD, 2014
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