Romy Thomas

All Publications

• Ligand-specific activation trajectories dictate GPCR signalling in cells. Nature Thomas, R., Jacoby, P. S., De Faveri, C., Derieux, C., Liebing, A. D., Melkes, B., Martini, H. J., Bermúdez, M., Stäubert, C., Lohse, M. J., Coin, I., Bock, A. 2026

  Abstract

  G-protein-coupled receptors (GPCRs) are key mediators of cell communication and represent the most important class of drug targets1,2. Biophysical studies with purified GPCRs in vitro have suggested that they exist in an equilibrium of distinct inactive and active states, which is modulated by ligands in an efficacy-dependent manner3-11. However, how efficacy is encoded and whether multiple receptor states occur in living cells remain unclear. Here we use genetic code expansion12 and bioorthogonal labelling13-16 to generate a panel of fluorescence-based biosensors for a prototypical GPCR, the M2 muscarinic acetylcholine receptor (M2R). These biosensors enable real-time monitoring of agonist-promoted conformational changes across the receptor's extracellular surface in intact cells. We demonstrate that different agonists produce equilibria of at least four distinct active states of the G-protein-bound M2R, each with a different ability to activate G proteins. The formation of these M2R-G-protein complexes occurs over 0.2-5 s along trajectories that involve both common and ligand-specific conformational changes and appear to determine G-protein selectivity. These observations reveal the molecular nature of ligand efficacy in intact cells. Selectively exploiting such different GPCR activation trajectories and conformational equilibria may open new avenues for GPCR drug discovery.

  View details for DOI 10.1038/s41586-025-09963-3

  View details for PubMedID 41535472

  View details for PubMedCentralID 6535337

• Determination of G-protein-coupled receptor oligomerization by molecular brightness analyses in single cells. Nature protocols Işbilir, A., Serfling, R., Möller, J., Thomas, R., De Faveri, C., Zabel, U., Scarselli, M., Beck-Sickinger, A. G., Bock, A., Coin, I., Lohse, M. J., Annibale, P. 2021; 16 (3): 1419-1451

  Abstract

  Oligomerization of membrane proteins has received intense research interest because of their importance in cellular signaling and the large pharmacological and clinical potential this offers. Fluorescence imaging methods are emerging as a valid tool to quantify membrane protein oligomerization at high spatial and temporal resolution. Here, we provide a detailed protocol for an image-based method to determine the number and oligomerization state of fluorescently labeled prototypical G-protein-coupled receptors (GPCRs) on the basis of small out-of-equilibrium fluctuations in fluorescence (i.e., molecular brightness) in single cells. The protocol provides a step-by-step procedure that includes instructions for (i) a flexible labeling strategy for the protein of interest (using fluorescent proteins, small self-labeling tags or bio-orthogonal labeling) and the appropriate controls, (ii) performing temporal and spatial brightness image acquisition on a confocal microscope and (iii) analyzing and interpreting the data, excluding clusters and intensity hot-spots commonly observed in receptor distributions. Although specifically tailored for GPCRs, this protocol can be applied to diverse classes of membrane proteins of interest. The complete protocol can be implemented in 1 month.

  View details for DOI 10.1038/s41596-020-00458-1

  View details for PubMedID 33514946

  View details for PubMedCentralID 6535337