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Proceedings Paper

Imaging of G protein-coupled receptors in solid-supported planar membranes at the single molecule level
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Paper Abstract

Odorant receptors are an excellent example of natural superiority in specifically binding specific, small and hydrophobic molecules. They are of particular interest in the development of a sensor platform for G protein-coupled receptors (GPCRs). Odorant receptors (OR5) of Rattus norvegicus were incorporated into model membranes by in vitro synthesis and vectorial incorporation for achieving natural receptor function. The vectorial insertion of OR5 into the planar membrane and their lateral distribution, their interactions and their mobility within the membrane are of great importance for ligand-receptor interaction. We applied total internal reflection fluorescence (TIRF) microscopy and image analysis to assess the insertion and the OR5 distribution as well as the lateral mobility of these receptors at the single molecule level. The vectorial incorporation of OR5 into planar lipid membranes was investigated with TIRF microscopy and image segmentation. With increasing expression time, the OR5 incorporation density and aggregation increased linearly by about 0.02μm-2min-1. The expression and incorporations of single OR5s were completed within about 8 minutes. The mobility of the incorporated receptors was measured with fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photo-bleaching (FRAP). These measurements revealed that the incorporated receptors were immobilized with this class of lipid membranes.

Paper Details

Date Published: 21 February 2008
PDF: 8 pages
Proc. SPIE 6862, Single Molecule Spectroscopy and Imaging, 68620X (21 February 2008); doi: 10.1117/12.763088
Show Author Affiliations
Iwan Märki, Ecole Polytechnique Fédérale de Lausanne (Switzerland)
Marcel Leutenegger, Ecole Polytechnique Fédérale de Lausanne (Switzerland)
Matthias Geissbuehler, Ecole Polytechnique Fédérale de Lausanne (Switzerland)
Rudolf Robelek, Univ. of Regensburg (Germany)
Eva-Kathrin Sinner, Max-Planck-Institut für Polymerforschung (Germany)
Theo Lasser, Ecole Polytechnique Fédérale de Lausanne (Switzerland)


Published in SPIE Proceedings Vol. 6862:
Single Molecule Spectroscopy and Imaging
Jörg Enderlein; Zygmunt K. Gryczynski; Rainer Erdmann, Editor(s)

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