Abstract
The activation mechanisms of GPCRs, where an external signal is propagated across the membrane through conformational rearrangements, have been extensively studied over the last decades by various biochemical, structural and biophysical methods. These have led to the conclusion that GPCR activation cannot be sufficiently explained by a simple on/off transition from an inactive to a distinct active state. Instead, it is rather a highly dynamic process where the equilibrium between multiple coexisting conformational states is altered by interacting molecules such as proteins, lipids, ions and others. Therefore, methods to monitor these conformational changes, preferentially at the single molecule level, are needed. Here, using single molecule Förster resonance energy transfer (smFRET) we are studying the structural dynamics that occur during activation of metabotropic glutamate receptors (mGluRs) in response to ligands. We have previously shown that isolated ligand binding domains oscillate between an active/open and an inactive/closed state in a time range of ∼100 µs and that orthosteric ligands shift the equilibrium depending on their efficacy. We have now extended these observations to detergent-solubilized full-length receptor dimers. We use allosteric modulators to decipher the role of the 7 transmembrane domain, and explore the allosteric communication between the various mGluR functional domains.