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).
