PROJECT C2

Ligand receptor interaction is often simply seen as a binding event and neglects the conformational dynamic of the target protein and its influence on ligand binding or the efficacy of the ligand. The central question within this project will be to address conformational dynamic of a receptor and monitor ligand binding with high kinetic resolution in living cells at the same time. We will use a fluorescence resonance energy transfer (FRET) based approach to address this question. Previously we have developed a generalized procedure which allows us to label G protein-coupled receptors at the C-terminus and third intracellular loop with two fluorophores. This approach allows us to monitor conformational changes of a receptor which occur upon ligand binding by intramolecular FRET. In particular it allows us to measure ligand on/off-rates and receptor activation/deactivation in real-time and will permit to obtain ligand residence time information and ligand efficacy data. We will combine such measurements with mutational analysis of the receptor protein to provide molecular insight into mechanisms of dynamic ligand binding, ligand efficacy, and the underlying influence on ligand residence time and vice versa.