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

Iwan Märki; Marcel Leutenegger; Matthias Geissbuehler; Rudolf Robelek; Eva-Kathrin Sinner; Theo Lasser

doi:10.1117/12.763088

21 February 2008 Imaging of G protein-coupled receptors in solid-supported planar membranes at the single molecule level

Iwan Märki, Marcel Leutenegger, Matthias Geissbuehler, Rudolf Robelek, Eva-Kathrin Sinner, Theo Lasser

Author Affiliations +

Proceedings Volume 6862, Single Molecule Spectroscopy and Imaging; 68620X (2008) https://doi.org/10.1117/12.763088
Event: SPIE BiOS, 2008, San Jose, California, United States

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.

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Iwan Märki, Marcel Leutenegger, Matthias Geissbuehler, Rudolf Robelek, Eva-Kathrin Sinner, and Theo Lasser "Imaging of G protein-coupled receptors in solid-supported planar membranes at the single molecule level", Proc. SPIE 6862, Single Molecule Spectroscopy and Imaging, 68620X (21 February 2008); https://doi.org/10.1117/12.763088

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