Application Notes

Interactions of liposome embedded SNARE proteins

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membrane as well as synaptobrevin which is integrated into the vesicle membrane. By forming the SNARE complex the vesicles are closely attached to the pre-synaptic plasma membrane and form a pool of neurotransmitter containing releasable vesicles (Fig.2). Results For the experiment, two different liposome populations were combined. One liposome population contains the neuronal SNARE protein synaptobrevin-2 (syb-2), while the other contains a receptor complex consisting of SNAP-25 and syntaxin-1A (N-complex) associated with a syb49-96 peptide labeled with Alexa Fluor 488 (Fig.3). Full-length syb-2 binds to the N-complex (acceptor SNARE complex) and a cis-SNARE complex is formed. This results in the replacement of the fluorescently labeled syb49-96 fragment and is directly followed by membrane fusion. For this particular experiment a competitive approach (labeled peptide competition instead of incorporating a label in a liposome) has been used to focus on the interaction between the membrane receptors and not the following process of liposome fusion (Fig.3). Fig. 3 Liposome embedded membrane receptor interactions. The interaction of two liposome embedded membrane receptors – synaptobrevin-2 (blue) and the N- complex consisting of syntaxin (red) and SNAP-25 (green) (acceptor SNARE complex) associated with a labeled syb49- 96 peptide leads to the release of the syb49-96 peptide due to higher affinity of the full-length syb-2 receptor. The process of liposome docking is followed by liposome fusion. The result of a thermophoresis experiment as a function of the concentration of unlabeled syb-2 liposomes is shown in Fig.4. The concentration of N-complex/labeled syb49-96 liposomes has been kept constant. The 50 % point of the binding curve is found at about 450 nM. The change in thermophoretic amplitude shows the dissociation of the syb49-96 fragment due to competition with the liposome embedded full-length synaptobrevin, which has a higher affinity to the N-complex. Since this dissociation is irreversible, the result reflects the point at which 50 % of active acceptor SNAREs are bound. The binding curve that is obtained (in equilibrium) shows a relatively strong change from the region of very high concentrations of (unlabeled) syb-2 liposomes towards low concentrations where the MST signal change is only small because little of the syb49-96 is dissociated. As a control, plain liposomes containing no synaptobrevin have been titrated to the N- complex/syb49-96 liposomes. As expected no change of the thermophoretic signal is observed (Fig.4). Fig. 4 Liposome embedded membrane receptor interactions measured with MicroScale Thermophoresis. The interaction of two liposome embedded membrane receptors – synaptobrevin-2 (blue) and the ∆N-complex associated with labeled syb49-96 peptide is measured with MicroScale Thermophoresis. Upon receptor-receptor interaction of the ∆N-complex and full-length synaptobrevin, the fluorescently labeled syb49-96 peptide is released (black diamonds). As a control plain liposomes containing no syb-2 receptor have been used (black triangles). Error bars of synaptobrevin liposomes represent standard error of n = 3 measurements. Conclusion This experiment demonstrates that even complexes with a size of several 100nm can be analyzed with MST. The use of liposomes allows to measure membrane associated proteins and trans-membrane proteins at conditions that are, in comparison to other approaches, close to the native conditions.

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