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Observing monomer: dimer transitions of neurotensin receptors 1 in single SMALPs by homoFRET and in an ABELtrap
Author(s): André Dathe; Thomas Heitkamp; Iván Pérez; Hendrik Sielaff; Anika Westphal; Stefanie Reuter; Ralf Mrowka; Michael Börsch
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Paper Abstract

G protein-coupled receptors (GPCRs) are a large superfamily of membrane proteins that are activated by extracellular small molecules or photons. Neurotensin receptor 1 (NTSR1) is a GPCR that is activated by neurotensin, i.e. a 13 amino acid peptide. Binding of neurotensin induces conformational changes in the receptor that trigger the intracellular signaling processes. While recent single-molecule studies have reported a dynamic monomer – dimer equilibrium of NTSR1 in vitro, a biophysical characterization of the oligomerization status of NTSR1 in living mammalian cells is complicated. Here we report on the oligomerization state of the human NTSR1 tagged with mRuby3 by dissolving the plasma membranes of living HEK293T cells into 10 nm-sized soluble lipid nanoparticles by addition of styrene-maleic acid copolymers (SMALPs). Single SMALPs were analyzed one after another in solution by multi-parameter single molecule spectroscopy including brightness, fluorescence lifetime and anisotropy for homoFRET. Brightness analysis was improved using single SMALP detection in a confocal ABELtrap for extended observation times in solution. A bimodal brightness distribution indicated a significant fraction of dimeric NTSR1 in SMALPs or in the plasma membrane, respectively, before addition of neurotensin.

Paper Details

Date Published: 22 February 2019
PDF: 11 pages
Proc. SPIE 10884, Single Molecule Spectroscopy and Superresolution Imaging XII, 108840N (22 February 2019); doi: 10.1117/12.2507546
Show Author Affiliations
André Dathe, Universitätsklinikum Jena (Germany)
Thomas Heitkamp, Universitätsklinikum Jena (Germany)
Iván Pérez, Universitätsklinikum Jena (Germany)
Hendrik Sielaff, Universitätsklinikum Jena (Germany)
Anika Westphal, Universitätsklinikum Jena (Germany)
Stefanie Reuter, Universitätsklinikum Jena (Germany)
Ralf Mrowka, Universitätsklinikum Jena (Germany)
Michael Börsch, Universitätsklinikum Jena (Germany)
Abbe Ctr. of Photonics (Germany)

Published in SPIE Proceedings Vol. 10884:
Single Molecule Spectroscopy and Superresolution Imaging XII
Zygmunt Karol Gryczynski; Ingo Gregor; Felix Koberling, Editor(s)

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