Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking. Here, we report the results of a blind benchmark study assessing eleven analysis tools used to infer kinetic rate constants from smFRET trajectories. We test them against simulated and experimental data containing the most prominent difficulties encountered in analyzing smFRET experiments: different noise levels, varied model complexity, non-equilibrium dynamics, and kinetic heterogeneity. Our results highlight the current strengths and limitations in inferring kinetic information from smFRET trajectories. In addition, we formulate concrete recommendations and identify key targets for future developments, aimed to advance our understanding of biomolecular dynamics through quantitative experiment-derived models.

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.

The Major Facilitator Superfamily (MFS) is the largest family of secondary active membrane transporters and is found in all domains of Life. MFS proteins are known to adopt different conformational states, yet details on the interconversion rates are crucially needed to understand or target their transport mechanism. Here, we studied the proton/multidrug antiporter LmrP as a model system for antibiotic resistance development in bacteria. The conformational cycle of LmrP is triggered by the protonation of a network of specific amino acids, yet the role of the transported substrate in these transitions has been puzzling. To measure LmrP structure in real-time, we performed solution-based single-molecule Förster resonance energy transfer (smFRET) using a confocal microscope with direct alternating donor/acceptor excitation and multiparameter (intensity, lifetime, anisotropy) detection. Lowering pH from 8 to 5 triggered an overall conformational transition, corroborating that detergent solubilization allows studying the LmrP transport cycle using smFRET. Using a newly developed linear 3-state photon distribution analysis (PDA) model, we show that the apo protein interconverted between two structures at low rate (>>10 ms dwell time) at the cytosolic side while it interconverts dynamically BRIEF SUMMARYWe studied the conformational cycle of LmrP, a model for multidrug efflux pumps, using single-molecule Förster resonance energy transfer (smFRET). By following changes in FRET signal between different sets of positions, we specifically investigated how substrate binding modulates structural conversions between inward-open and outward-open states. Using a newly developed probabilistic analysis for describing sequential interconversion kinetics, we show that the apo protein slowly interconverts between defined states at the cytosolic and at the extracellular sides. Binding of the model substrate Hoechst33342 leads to an increase in conformational interconversions at the intracellular side while the extracellular side shows a drastic decrease in conversion, indicating a kinetic uncoupling between both sides. Remarkably, binding of roxithromycin, while also increasing interconversion on the intracellular side, did not slow the extracellular conversions. This indicates that multidrug pumps have evolved substrate-dependent transport mechanisms than enable transport of structurally diverse collection of substrates.