Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Tuesday Speaker Abstracts

LOOKING AT METABOTROPIC GLUTAMATE RECEPTORS DYNAMICS WITH SINGLE MOLECULE FRET Emmanuel Margeat 1 ; 1 Centre de Biologie Structurale, Univ. Montpellier, CNRS, INSERM, Montpellier, France Metabotropic glutamate receptors (mGluR) regulate neuronal excitability and synaptic transmission by sensing L-glutamate – the major excitatory neurotransmitter in the central nervous system. Their crucial role for synaptic function makes them attractive targets for the treatment of numerous neurological and psychiatric diseases including for instance anxiety, depression, schizophrenia and addiction. To explore mGluR activation, we use single molecule FRET, that allows to screen the conformations explored by single protein complexes, with high temporal and spatial resolution. 3 color single-molecule FRET extends the conformational analysis to the measurements of 3 distances simultaneously, and therefore to the observation of correlated movements. However, the site-specific labeling of a biomolecular complex with 3 single molecule-compatible fluorophores remains challenging. We established a series of 2-color and 3-color smFRET sensors, through incorporation of up to two orthogonally reactive non-canonical amino-acids (ncAA) , and/or the addition of a SNAP self-labeling tag. These sensors report on the initial steps of mGluR2 activation, including the reorientation of the upper and the lower lobes of the Venus flytrap domain (VFT) or the cysteine rich domain (CRD) in an intersubunit fashion, the VFT closure in an intrasubunit fashion, and the correlation between these movements. We then used 2- and 3-color single molecule FRET to explore ligand induced conformational changes on mGlu2 homodimers. We show that agonist-binding efficiently depopulates the inactive state, leading to an equilibrium of receptors switching between the active and a newly identified intermediate state. Only the addition of a synthetic allosteric modulator, or of the G-protein, leads to a full stabilization of the activated receptor. We also extend this approach to the study of mglu2-containg heterodimers, offering new insights on the allosteric control of one protomer by the other, and on the asymmetric interaction with the G-proteins.

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