4.1 Single-molecule FRET (smFRET)
SmFRET detects energy transfer between individual molecules of donor and acceptor fluorophores and provides both structural and dynamic insights into intramolecular conformational changes and intermolecular interactions (Figure 2g). In the last decade, smFRET has been extensively used for studies of GPCR signalling (Quast & Margeat, 2019). SmFRET was used in multiple studies to determine the dynamics of interactions of GPCRs, particularly β2AR and A2A adenosine receptors, with different ligands and subsequent activation of G proteins (Fernandes et al., 2021; Gregorio et al., 2017; Lamichhane et al., 2015). Furthermore, smFRET allowed the detection of submillisecond conformational dynamics of the A2A adenosine receptor (Maslov et al., 2023). In live cells, smFRET was used to determine the abundance of dimers and their spatiotemporal dynamics for the µ-opioid receptor, secretin receptor, and mGluR2 (Asher et al., 2021). Furthermore, smFRET uncovered the mechanisms of β-arrestin1 autoinhibition, binding to GPCRs, and activation (Asher et al., 2022). Recent technical advances have enabled the use of genetic code expansion and click chemistry for the introduction of labels for smFRET (Han et al., 2021) as well as four-colour smFRET measurements in live cells (Sotolongo Bellon et al., 2022). The limitations of smFRET include the requirement for two labels, probe photobleaching, sophisticated labelling procedures, a limited range of usable protein concentrations, and complex data analysis. Future developments of smFRET, particularly in combination with SPT and SRN techniques, such as MINFLUX (Cole et al., 2023), live-cell imaging, and specific direct labelling with small fluorophores (Banerjee et al., 2022), will allow the study of GPCR signalling dynamics in systems closer to natural with even higher temporal resolution.