Single-molecule fluorescence-based realtime structural biology of the adenosine A2A G-protein coupled receptor in microfabricated cells (R-12270)

G-protein coupled receptors (GPCRs) are the main target for drug discovery, yet only a marginal fraction of GPCR structures have been solved. This is mainly because mimicking a natural membrane context compatible with structural investigations is extremely challenging. I hypothesize that reconstituting GPCRs in microfluidic devices containing pore-spanning lipid membranes allows investigating their structure in a realtime manner compatible with high-throughput screening. I will use the adenosine A2A receptor as a model system. The A2AR plays important roles in the brain, the immune system and in different human diseases. I will reconstitute A2AR in free-standing lipid membranes using highly ordered porous substrates inside a microfluidic device. Moreover, owing to their small geometry, many single A2AR molecules can be investigated in parallel, significantly increasing throughput. To measure the A2AR structure in realtime, I will use single-molecule Förster resonance energy transfer (smFRET), a method that can measure the 1-10 nanometer distances between two fluorescent probes with subnanometer resolution, allowing fast and sensitive imaging of protein movement in realtime. After validating my methodology, I subject the A2AR to a detailed investigation of the role of the electric membrane potential and of a(nta)gonists and allosteric modulators. My project provides a solid basis for future screening assays targeting G-protein coupled receptors.

Keywords:adenosin A2A receptor, confocal microscopy, conformational dynamics, electrochemical membrane potential, Förster resonance energy transfer, GPCR, lipid bilayer, liposomes, microarrays, microfabrication, microfluidics, protein conformation, single molecule spectroscopy, structure-based drug discovery, TIRF microscopy

Disciplines:Flow chemistry, Biophotonics, Molecular biophysics, Structural biology, Compound screening