| Prof. Dr. Med. Sc. Ago Rinken|
Institute of Chemistry, University of Tartu, Ravila 14a, 50411 Tartu, Estonia firstname.lastname@example.org
Characterization of ligand binding to membrane proteins by using fluorescence anisotropy and TIRF microscopy-based assays
The utilization of budded baculoviruses that express membrane proteins of interest on their surfaces has opened new possibilities for the characterization of ligand binding . The fluorescence anisotropy (FA) -based assay allows on-line monitoring of ligand binding in solution and obtain valuable kinetic data, which enables to solve even complex binding mechanisms . However, there are still numerous systems where FA assays are not applicable due to ligands’ size, binding affinity, or fluorophores’ properties. Therefore the total internal reflection fluorescence (TIRF) microscopy-based method was developed to study additional aspects of ligand binding. The novel method combines membrane protein display in budded baculovirus particles and the immobilization of these particles to a functionalized coverslip. To scale the system for more routine ligand binding assays, we developed both open-source multiwell systems and image analysis software SPOTNIC (http://gpcr.ut.ee/aparecium.html) for flexible assay design. The immobilization selectivity was determined, and ligand-binding assays were validated using budded baculovirus particles displaying neuropeptide Y Y1 receptors and high-affinity TAMRA labeled fluorescent ligand UR-MC026. The TIRFM based system could be extended to various advanced assays involving super-resolution methods, enabling the investigation and description of the ligand-binding processes of membrane proteins at the single-molecule level.
- Link R., Veiksina S., Tahk M.J., Laasfeld T., Paiste P., Kopanchuk S., Rinken A. (2020) The constitutive activity of melanocortin 4 receptors is allosterically modulated by zinc and copper ions. J. Neurochem. 153, 346-361. https://doi.org/10.1111/JNC.14933
- Rinken A., Lavogina D., Kopanchuk S. (2018) Assays with Detection of Fluorescence Anisotropy: Challenges and Possibilities to Characterize Ligand Binding to GPCRs. Trends Pharmacol. Sci. 39, 187-199. http://dx.doi.org/10.1016/j.tips.2017.10.004