NMR investigations of solution and membrane-binding properties of synaptotagmin1 C2A domain

NMR approach to measure electrostatic potentials based upon paramagnetic enhancements of nuclear relaxation on the backbone amide protons of C2A domain from synaptotagmin1 (Syt1 C2A) was explored. Syt1 C2A domain has been implicated as the Ca2+ sensor for neuronal exocytosis and Ca2+ dependent membrane binding. Spin-lattice relaxation rates at the backbone amide protons were measured from a 2D heteronuclear correlation spectrum of 15N labeled C2A domain in the presence and absence of oppositely charged water-soluble nitroxide spin labels. The difference in relaxation rates between positively and negatively charged nitroxides provided an estimate of local electrostatic potentials given that these nitroxides do not bind to the protein and that differences in paramagnetic relaxation enhancements are due entirely to differences in the spin label charge. Given these assumptions, electrostatic potentials were estimated indicating that the C2A domain has an overall negative potential in the absence of Ca2+. In the presence of Ca2+, regions near the membrane interacting surface of the domain become positively charged. These findings are in approximate agreement with the results of computational work and indicate that long-range Coulombic interactions play a role in the Ca2+-dependent association of these domains.;Membrane binding properties of Syt1 C2A domain were examined by measuring nuclear spin-lattice relaxation rate changes induced by a freely diffusing paramagnet such as oxygen when the target protein is in solution or bound to a membrane. In the solution case, the intramolecular cross-relaxation rates were modest and large differences are observed in the oxygen-induced protein proton relaxation rates. In the case where a dynamic equilibrium between solution and membrane bound environments was established, the intramolecular 1H cross-relaxation rates increased substantially due to the slow re-orientational motion of the protein in the membrane-bound environment. As a consequence, all protein protons relaxed with nearly similar spin-lattice relaxation rate constants under the membrane bound conditions, thereby negating any site-specific or local effects of the diffusing paramagnet.