Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

48-POS Board 24 GENETIC CODE EXPANSION AND BIOORTHOGONAL CHEMISTRIES FOR STUDYING CONFORMATIONAL CHANGES OF GPCRS USING SINGLE MOLECULE FRET Glauce Moreno Barbosa 1 ; Rémy Char 1 ; Tala Khazaal 1 ; Caroline Clerté 1 ; Emmanuel Margeat 1 ; Robert B. Quast 1 ; 1 Centre de Biologie Structurale CNRS UMR 5048 - UM - INSERM U 1054, 34090, France Single molecule Förster Resonance Energy Transfer (smFRET) has emerged as a powerful technique for probing conformational dynamics of G protein-coupled receptors (GPCR) at the nanoscale. Yet, its broad application remains fundamentally constrained by the challenge of achieving precise, chemically orthogonal fluorophore attachment. Conventional labeling strategies, including cysteine-maleimide and lysine-NHS ester conjugation, frequently suffer from limited site selectivity or restricted applicability to complex multidomain systems such as for instance GPCRs. To overcome these limitations, we have exploited the power of genetic code expansion to incorporate reactive non-canonical amino acids (ncAAs) in mammalian cells in combination with biorthogonal click chemistries in recent years, in order to dissect the conformational dynamics of metabotropic glutamate (mGlu) receptors using two- and three-color smFRET [1-3]. mGlu receptors are multidomain GPCRs that assemble in homo- and heterodimers from 8 different subtypes to regulate neuronal excitability and synaptic transmission in the brain. Here, I will present our recent progress on using genetic code expansion and biorthogonal click chemistries to study the asymmetric activation mechanisms of mGlu receptor homo- and heterodimers, our efforts to establish minimally invasive and site specific triple labeling using distinct ncAAs and reactions to correlated individual domain movements through three-color smFRET, as well as preliminary data towards site-specific labeling on cells for live-cell smFRET measurements. [1] Quast RB*, Cao AM*, Fatemi F, Rondard P, Pin JP, Margeat E. Nat Commun. 2021, https://doi.org/10.1038/s41467-021-25620-5 [2] Quast RB*, Lecat-Guillet N*, Liu H, Bourrier E, Møller TC, Rovira X, Soldevila S, Lamarque L, Trinquet E, Liu J, Pin JP, Rondard P, Margeat E. Sci. Adv. 2023, https://doi.org/10.1126/sciadv.adf1378 [3] Bonhomme L, Bilgen E, Clerté C, Pin JP, Rondard P, Margeat E, Lamb DC, Quast RB. JACS 2025, https://doi.org/10.1021/jacs.4c18364

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