More Like this

1. Unraveling multi-state molecular dynamics in single-molecule FRET experiments. II. Quantitative analysis of multi-state kinetic networks

   https://doi.org/10.1063/5.0095754  

   Opanasyuk, Oleg; Barth, Anders; Peulen, Thomas-Otavio; Felekyan, Suren; Kalinin, Stanislav; Sanabria, Hugo; Seidel, Claus_A_M (July 2022, The Journal of Chemical Physics)

   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. 

   more » « less
2. Unraveling multi-state molecular dynamics in single-molecule FRET experiments. I. Theory of FRET-lines

   https://doi.org/10.1063/5.0089134  

   Barth, Anders; Opanasyuk, Oleg; Peulen, Thomas-Otavio; Felekyan, Suren; Kalinin, Stanislav; Sanabria, Hugo; Seidel, Claus A. (April 2022, The Journal of Chemical Physics)

   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. 

   more » « less
3. FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices

   https://doi.org/10.7554/eLife.60416  

   Lerner, Eitan; Barth, Anders; Hendrix, Jelle; Ambrose, Benjamin; Birkedal, Victoria; Blanchard, Scott C; Börner, Richard; Sung Chung, Hoi; Cordes, Thorben; Craggs, Timothy D; et al (March 2021, eLife)

   null (Ed.)

   Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current ‘state of the art’ from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of ‘soft recommendations’ about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage ‘open science’ practices. 

   more » « less
4. Learned Reconstruction of Protein Folding Trajectories from Noisy Single-Molecule Time Series

   Topel, Maximilian; Ejaz, Ayesha; Squires, Allison; Ferguson, Andrew L. (January 2023, Journal of chemical theory and computation)

   Single-molecule Förster resonance energy transfer (smFRET) is an experimental methodology to track the real-time dynamics of molecules using fluorescent probes to follow one or more intramolecular distances. These distances provide a low-dimensional representation of the full atomistic dynamics. Under mild technical conditions, Takens’ Delay Embedding Theorem guarantees that the full three-dimensional atomistic dynamics of a system are diffeomorphic (i.e., related by a smooth and invertible transformation) to a time-delayed embedding of one or more scalar observables. Appealing to these theoretical guarantees, we employ manifold learning, artificial neural networks, and statistical mechanics to learn from molecular simulation training data the a priori unknown transformation between the atomic coordinates and delay-embedded intramolecular distances accessible to smFRET. This learned transformation may then be used to reconstruct atomistic coordinates from smFRET time series data. We term this approach Single-molecule TAkens Reconstruction (STAR). We have previously applied STAR to reconstruct molecular configurations of a C24H50 polymer chain and the mini-protein Chignolin with accuracies better than 0.2 nm from simulated smFRET data under noise free and high time resolution conditions. In the present work, we investigate the role of signal-to-noise ratio, data volume, and time resolution in simulated smFRET data to assess the performance of STAR under conditions more representative of experimental realities. We show that STAR can reconstruct the Chignolin and Villin mini-proteins to accuracies of 0.12 and 0.42 nm, respectively, and place bounds on these conditions for accurate reconstructions. These results demonstrate that it is possible to reconstruct dynamical trajectories of protein folding from time series in noisy, time binned, experimentally measurable observables and lay the foundations for the application of STAR to real experimental data. 

   more » « less
5. Ultrafast strong-field dissociation of vinyl bromide: An attosecond transient absorption spectroscopy and non-adiabatic molecular dynamics study

   https://doi.org/10.1063/4.0000102  

   Rott, Florian; Reduzzi, Maurizio; Schnappinger, Thomas; Kobayashi, Yuki; Chang, Kristina_F; Timmers, Henry; Neumark, Daniel_M; Vivie-Riedle, Regina_de; Leone, Stephen_R (June 2021, Structural Dynamics)

   Attosecond extreme ultraviolet (XUV) and soft x-ray sources provide powerful new tools for studying ultrafast molecular dynamics with atomic, state, and charge specificity. In this report, we employ attosecond transient absorption spectroscopy (ATAS) to follow strong-field-initiated dynamics in vinyl bromide. Probing the Br M edge allows one to assess the competing processes in neutral and ionized molecular species. Using ab initio non-adiabatic molecular dynamics, we simulate the neutral and cationic dynamics resulting from the interaction of the molecule with the strong field. Based on the dynamics results, the corresponding time-dependent XUV transient absorption spectra are calculated by applying high-level multi-reference methods. The state-resolved analysis obtained through the simulated dynamics and related spectral contributions enables a detailed and quantitative comparison with the experimental data. The main outcome of the interaction with the strong field is unambiguously the population of the first three cationic states, D1, D2, and D3. The first two show exclusively vibrational dynamics while the D3 state is characterized by an ultrafast dissociation of the molecule via C–Br bond rupture within 100 fs in 50% of the analyzed trajectories. The combination of the three simulated ionic transient absorption spectra is in excellent agreement with the experimental results. This work establishes ATAS in combination with high-level multi-reference simulations as a spectroscopic technique capable of resolving coupled non-adiabatic electronic-nuclear dynamics in photoexcited molecules with sub-femtosecond resolution. 

   more » « less