Imaging of G protein-coupled receptors in solid-supported planar membranes at the single molecule level
Odorant receptors are an excellent example of natural superiority in binding specific, small and hydrophobic molecules, and therefore 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 based on a spot analysis. 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 first 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 within this class of lipid membranes. Further studies on the receptor mobility and ligand-receptor interaction properties of membrane embedded GPCRs in different membrane classes including synthetic polymer membrane architectures will be pursued.
Record created on 2008-02-26, modified on 2016-08-08