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Binding of calcium ions to synaptotagmin measured with fluorescence label and label-free

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1 Protein-Ion Interaction Analysis Application Note NT-MO-006 Binding of Calcium Ions to Synaptotagmin measured with fluorescence label and label-free Karsten Meyenberg 1 and Geert van den Boogaart 2 1 University Göttingen, Institut für Organische und Biomolekulare Chemie, Tammannstrasse 2, D-37077 Göttingen, Germany 2 Max Planck Institute for biophysical Chemistry, Department of Neurobiology, Am Faßberg 11, D-37077 Göttingen, Germany Abstract The synaptic vesicle protein synaptotagmin 1 is the main calcium sensor of neuronal exocytosis. Calcium binds to its cytosolic portion that consists of tandem C2-type domains. In this work we show that MicroScale Thermophoresis is a valuable tool to measure binding of ions to proteins, with and without the use of a fluorescent label. Introduction Neuronal excocytosis is the process were small synaptic vesicle fuse with the plasma membrane, thereby releasing neurotransmitter in the synaptic cleft. The process is triggered by a rapid increase of the cytoplasmic calcium concentration. Fig. 1 Calcium binding site of synaptotagmin (Radhakrishnan et al 2009) Calcium binds to synaptotagmin 1, the calcium sensor of neuronal exocytosis, which than actively promotes exocytotic fusion (for review, see Chapman et al. 2008). Synaptotagmin 1 consists of a single transmembrane region followed by a large unstructured linker and two C2-type domains, called C2A and C2B. We employed MicroScale Thermophoresis to measure the intrinsic calcium binding affinities of the calcium binding synaptotagmin 1 C2AB domain and found a very good agreement with Isothermal Titration Calorimetry (ITC) experiments. Results As shown in Fig. 2 and 3 the binding of calcium ions to synaptotagmin was observed as a clear and strong response in MST signal, while no signal was observed at increasing magnesium ion concentrations. Fig. 2 Binding of Calcium ions to NT-647 labeled synaptotagmin 1 C2AB. The difference in thermophoretic mobility is measured by attaching a fluorescence label to the synaptotagmin 1 (n = 2 measurements).

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