The complex pharmacology of G-protein-coupled receptors (GPCRs) is defined by their multi-state conformational dynamics. Single-molecule Förster Resonance Energy Transfer (smFRET) is well-suited to quantify dynamics for individual protein molecules, however, its application to GPCRs is challenging; therefore, smFRET has been limited to studies of interreceptor interactions in cellular membranes and receptors in detergent environments. Here, we performed smFRET experiments on functionally active human A2A adenosine receptor (A2AAR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of A2AAR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A2AAR, explaining the receptor’s constitutive activity. For the agonist-bound A2AAR, we detected faster (390±80 μs) ligand efficacy-dependent dynamics. This work establishes a general smFRET platform for GPCR investigations that can potentially be used for drug screening and/or mechanism-of-action studies.

Cells continuously sense external forces from their microenvironment, the extracellular matrix (ECM). In turn, they generate contractile forces, which stiffen and remodel this matrix. Although this bidirectional mechanical exchange is crucial for many cell functions, it remains poorly understood. Key challenges are that the majority of available matrices for such studies, either natural or synthetic, are difficult to control or lack biological relevance. Here, we use a synthetic, yet highly biomimetic hydrogel based on polyisocyanide (PIC) polymers to investigate the effects of the fibrous architecture and the nonlinear mechanics on cell–matrix interactions. Live-cell rheology was combined with advanced microscopy-based approaches to understand the mechanisms behind cell-induced matrix stiffening and plastic remodeling. We demonstrate how cell-mediated fiber remodeling and the propagation of fiber displacements are modulated by adjusting the biological and mechanical properties of this material. Moreover, we validate the biological relevance of our results by demonstrating that cellular tractions in PIC gels develop analogously to those in the natural ECM. This study highlights the potential of PIC gels to disentangle complex bidirectional cell–matrix interactions and to improve the design of materials for mechanobiology studies.

The complex pharmacology of G-protein-coupled receptors (GPCRs) is defined by their multi-state conformational dynamics. Single-molecule Förster Resonance Energy Transfer (smFRET) is well suited to quantify dynamics for individual protein molecules; however, its application to GPCRs is challenging. Therefore, smFRET has been limited to studies of inter-receptor interactions in cellular membranes and receptors in detergent environments. Here, we performed smFRET experiments on functionally active human A2A adenosine receptor (A2AAR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of A2AAR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A2AAR, explaining the receptor’s constitutive activity. For the agonist-bound A2AAR, we detected faster (390 ± 80 µs) ligand efficacy-dependent dynamics. Our work establishes a general smFRET platform for GPCR investigations that can potentially be used for drug screening and/or mechanism-of-action studies.

Förster resonance energy transfer (FRET) between fluorescent proteins has become a common platform for designing genetically encoded biosensors. For live cell imaging, the acceptor-to-donor intensity ratio is most commonly used to readout FRET efficiency, which largely depends on the proximity between donor and acceptor. Here, we introduce an anisotropy-based mode of FRET detection (FADED: FRET-induced Angular Displacement Evaluation via Dim donor), which probes for relative orientation rather than proximity alteration. A key element in this technique is suppression of donor bleed-through, which allows measuring purer sensitized acceptor anisotropy. This is achieved by developing Geuda Sapphire, a low-quantum-yield FRET-competent fluorescent protein donor. As a proof of principle, Ca2+ sensors were designed using calmodulin as a sensing domain, showing sigmoidal dose response to Ca2+. By monitoring the anisotropy, a Ca2+ rise in living HeLa cells is observed upon histamine challenging. We conclude that FADED provides a method for quantifying the angular displacement via FRET.

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