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Proceedings Paper

Towards monitoring conformational changes of the GPCR neurotensin receptor 1 by single-molecule FRET
Author(s): Thomas Heitkamp; Reinhard Grisshammer; Michael Börsch
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Paper 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 Förster resonance energy transfer (smFRET), using neurotensin labeled with the fluorophore ATTO594.

Paper Details

Date Published: 23 February 2018
PDF: 15 pages
Proc. SPIE 10498, Multiphoton Microscopy in the Biomedical Sciences XVIII, 104980T (23 February 2018); doi: 10.1117/12.2286787
Show Author Affiliations
Thomas Heitkamp, Friedrich Schiller Univ. Jena (Germany)
Reinhard Grisshammer, National Cancer Institute, National Institutes of Health (United States)
Michael Börsch, Friedrich Schiller Univ. Jena (Germany)

Published in SPIE Proceedings Vol. 10498:
Multiphoton Microscopy in the Biomedical Sciences XVIII
Ammasi Periasamy; Peter T. C. So; Karsten König; Xiaoliang S. Xie, Editor(s)

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