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Project

Investigation of G-protein coupled receptors conformational dynamics via single-molecule fluorescence microscopy

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that orchestrate critical physiological processes such as neurotransmission, inflammation, taste, vision, and others. GPCRs recognize a large variety of extracellular ligands and are targets for ca. 30% of all approved drugs. In this study, we used single-molecule Förster resonance energy transfer (smFRET) to track conformational changes in the A2A adenosine receptor, an important target of drugs against insomnia, chronic pain, depression, Parkinson’s disease, and cancer. Our measurements revealed agonist-induced sub-millisecond conformational dynamics in the A2A receptor reconstituted in lipid nanodiscs. We also showed that synthetic derivatives of the solvatochromic GFP chromophore can be used as environment-sensitive fluorescent labels to monitor ligand-induced conformational changes in GPCRs. Using these dyes, we showed that an allosteric modulator of the A2A adenosine receptor, HMA, stabilizes a receptor’s conformation distinct from those of apo, or agonist-bound receptors. Finally, we use single-molecule localization microscopy to observe the inter-molecular organization of seven-transmembrane proteins in human cells. We showed that Channelrhodopsin-2, a protein widely used in optogenetics, forms stable dimers in the plasma membrane. We proved that inter-subunit disulfide bonds that stabilize the dimers are not essential for their formation. Our study showcases how advanced fluorescence microscopy facilitates investigations of the structure and function of membrane proteins.

Date:25 May 2020 →  23 Aug 2023
Keywords:GPCR, FRET, single-molecule fluorescence spectroscopy
Disciplines:Proteins, Molecular biophysics, Spectroscopic methods, Structural biology
Project type:PhD project