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arXiv · 1801.09318

Towards monitoring conformational changes of the GPCR neurotensin receptor 1 by single-molecule FRET

Thomas Heitkamp·Reinhard Grisshammer·Michael Börsch
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Abstract

Neurotensin receptor 1 (NTSR1) is a G protein-coupled receptor that is important for signaling in the brain and the gut. Its agonist ligand neurotensin (NTS), a 13-amino-acid peptide, binds with nanomolar affinity from the extracellular side to NTSR1 and induces conformational changes that trigger intracellular signaling processes. Our goal is to monitor the conformational dynamics of single fluorescently labeled NTSR1. For this, we fused the fluorescent protein mNeonGreen to the C terminus of NTSR1, purified the receptor fusion protein from E. coli membranes, and reconstituted NTSR1 into liposomes with E. coli polar lipids. Using single-molecule anisotropy measurements, NTSR1 was found to be monomeric in liposomes, with a small fraction being dimeric and oligomeric, showing homoFRET. Similar results were obtained for NTSR1 in detergent solution. Furthermore, we demonstrated agonist binding to NTSR1 by time-resolved single-molecule F\"orster resonance energy transfer (smFRET), using neurotensin labeled with the fluorophore ATTO594.

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Thomas Heitkamp, Reinhard Grisshammer, Michael Börsch. 2018-01-28. Towards monitoring conformational changes of the GPCR neurotensin receptor 1 by single-molecule FRET. https://arxiv.org/abs/1801.09318

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