Sergei Kopanchuk
Institute of Chemistry University , Estonia
Over the past 20 years at the Universities of Tartu and Uppsala, he has gained expertise in studying the molecular mechanisms of the functioning of various G protein-coupled receptors, focusing on the dynamics of system behavior. He is involved in the development of various types of assays for drug screening and biosensors. In the last decade, he has built and configured microscopes with different fluorescence imaging modes (confocal, wide-field and TIRF/HILO) for various measurement techniques such as FLIM in time and frequency domain, TGL, FCS and single molecule/particle detection/tracking. In addition, he has enhanced these optical setups with super-resolution options, STED and localization microscopy.
Nanoparticles with surface display of transmembrane proteins as a tool for
drug screening targeting G-protein coupled receptors.
Naturally occurring extracellular bio-nanoparticles (be they viruses, microvesicles, exosomes, etc.) can be tailored to present a wide range of drug targets, including G protein-coupled receptors (GPCRs). These particles provide a novel source of complex membrane proteins that will be maintained in their native conformation in the lipid bilayer derived from cells in which GPCRs are potentially pharmacologically relevant. GPCRs share several common features, including the coupling of their signal transduction via G proteins. For mechanistic studies, it would be of great interest to have the possibility to enrich these particles, in addition to GPCRs, with G proteins and other modulators. For this purpose, we have developed a modified MultiBacMam technology that allows to express with a single baculovirus several proteins in the same mammalian cell with high efficiency. We detected a large amount of Frizzled 10 receptors on extracellular vesicles, which could be the result of the dispatching from filopodia where they are preconcentrated. To clarify this issue, we also developed a baculoviral library that coexpresses different markers together. As example, multicolor labeled CD9, CD63, CD81 and the plasma membrane markers. MultiBacMam technology also allows us to enrich the receptor fraction in the extracellular space through the production of Rous sarcoma virus-like particles or budded baculoviruses. For particle characterization and study of drug-receptor interactions on the particles, we used total internal reflection fluorescence microscopy with single molecule sensitivity.
