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Previous studies of the N-terminal PDZ tandem from PSD-95 produced divergent models and failed to identify interdomain contacts stabilizing the structure. We used ensemble and single-molecule FRET along with replica-exchange molecular dynamics to fully characterize the energy landscape. Simulations and experiments identified two conformations: an open-like conformation with a small contact interface stabilized by salt bridges, and a closed-like conformation with a larger contact interface stabilized by surface-exposed hydrophobic residues. Both interfaces were confirmed experimentally. Proximity of interdomain contacts to the binding pockets may explain the observed coupling between conformation and binding. The low-energy barrier between conformations allows submillisecond dynamics, which were time-averaged in previous NMR and FRET studies. Moreover, the small contact interfaces were likely overridden by lattice contacts as crystal structures were rarely sampled in simulations. Our hybrid approach can identify transient interdomain interactions, which are abundant in multidomain proteins yet often obscured by dynamic averaging.

 

Abstract 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 scaffold protein PSD-95 links postsynaptic receptors to sites of presynaptic neurotransmitter release. Flexible linkers between folded domains in PSD-95 enable a dynamic supertertiary structure. Interdomain interactions within the PSG supramodule, formed by P DZ3, S H3, and G uanylate Kinase domains, regulate PSD-95 activity. Here we combined discrete molecular dynamics and single molecule Förster resonance energy transfer (FRET) to characterize the PSG supramodule, with time resolution spanning picoseconds to seconds. We used a FRET network to measure distances in full-length PSD-95 and model the conformational ensemble. We found that PDZ3 samples two conformational basins, which we confirmed with disulfide mapping. To understand effects on activity, we measured binding of the synaptic adhesion protein neuroligin. We found that PSD-95 bound neuroligin well at physiological pH while truncated PDZ3 bound poorly. Our hybrid structural models reveal how the supertertiary context of PDZ3 enables recognition of this critical synaptic ligand. 

Single-molecule Förster Resonance Energy Transfer (smFRET) experiments are ideally suited to resolve the structural dynamics of biomolecules. A significant challenge to date is capturing and quantifying the exchange between multiple conformational states, mainly when these dynamics occur on the sub-millisecond timescale. Many methods for quantitative analysis are challenged if more than two states are involved, and the appropriate choice of the number of states in the kinetic network is difficult. An additional complication arises if dynamically active molecules coexist with pseudo-static molecules in similar conformational states with undistinguishable Förster Resonance Energy Transfer (FRET) efficiencies. To address these problems, we developed a quantitative integrative analysis framework that combines the information from FRET-lines that relate average fluorescence lifetimes and intensities in two-dimensional burst frequency histograms, fluorescence decays obtained by time-correlated single-photon-counting, photon distribution analysis of the intensities, and fluorescence correlation spectroscopy. Individually, these methodologies provide ambiguous results for the characterization of dynamics in complex kinetic networks. However, the global analysis approach enables accurate determination of the number of states, their kinetic connectivity, the transition rate constants, and species fractions. To challenge the potential of smFRET experiments for studying multi-state kinetic networks, we apply our integrative framework using a set of synthetic data for three-state systems with different kinetic connectivity and exchange rates. Our methodology paves the way toward an integrated analysis of multiparameter smFRET experiments that spans all dimensions of the experimental data. Finally, we propose a workflow for the analysis and show examples that demonstrate the usefulness of this toolkit for dynamic structural biology. 

Conformational dynamics of biomolecules are of fundamental importance for their function. Single-molecule studies of Förster Resonance Energy Transfer (smFRET) between a tethered donor and acceptor dye pair are a powerful tool to investigate the structure and dynamics of labeled molecules. However, capturing and quantifying conformational dynamics in intensity-based smFRET experiments remains challenging when the dynamics occur on the sub-millisecond timescale. The method of multiparameter fluorescence detection addresses this challenge by simultaneously registering fluorescence intensities and lifetimes of the donor and acceptor. Together, two FRET observables, the donor fluorescence lifetime τ D and the intensity-based FRET efficiency E, inform on the width of the FRET efficiency distribution as a characteristic fingerprint for conformational dynamics. We present a general framework for analyzing dynamics that relates average fluorescence lifetimes and intensities in two-dimensional burst frequency histograms. We present parametric relations of these observables for interpreting the location of FRET populations in E–τ D diagrams, called FRET-lines. To facilitate the analysis of complex exchange equilibria, FRET-lines serve as reference curves for a graphical interpretation of experimental data to (i) identify conformational states, (ii) resolve their dynamic connectivity, (iii) compare different kinetic models, and (iv) infer polymer properties of unfolded or intrinsically disordered proteins. For a simplified graphical analysis of complex kinetic networks, we derive a moment-based representation of the experimental data that decouples the motion of the fluorescence labels from the conformational dynamics of the biomolecule. Importantly, FRET-lines facilitate exploring complex dynamic models via easily computed experimental observables. We provide extensive computational tools to facilitate applying FRET-lines. 

Thiamine pyrophosphate (TPP) riboswitches regulate thiamine metabolism by inhibiting the translation of enzymes essential to thiamine synthesis pathways upon binding to thiamine pyrophosphate in cells across all domains of life. Recent work on the Arabidopsis thaliana TPP riboswitch suggests a multistep TPP binding process involving multiple riboswitch configurational ensembles and Mg 2+ dependence underlies the mechanism of TPP recognition and subsequent transition to the expression-inhibiting state of the aptamer domain followed by changes in the expression platform. However, details of the relationship between TPP riboswitch conformational changes and interactions with TPP and Mg 2+ in the aptamer domain constituting this mechanism are unknown. Therefore, we integrated single-molecule multiparameter fluorescence and force spectroscopy with atomistic molecular dynamics simulations and found that conformational transitions within the aptamer domain's sensor helices associated with TPP and Mg 2+ ligand binding occurred between at least five different ensembles on timescales ranging from µs to ms. These dynamics are orders of magnitude faster than the 10 sec-timescale folding kinetics associated with expression-state switching in the switch helix. Together, our results show that a TPP and Mg 2+ dependent mechanism determines dynamic configurational state ensemble switching of the aptamer domain's sensor helices that regulate the switch helix's stability, which ultimately may lead to the expression-inhibiting state of the riboswitch. Additionally, we propose that two pathways exist for ligand recognition and that this mechanism underlies a kinetic rheostat-like behavior of the Arabidopsis thaliana TPP riboswitch.