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A blind benchmark of analysis tools to infer kinetic rate constants from single-molecule FRET trajectories.

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.

SUBMITTER: Gotz M

PROVIDER: S-EPMC9474500 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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A blind benchmark of analysis tools to infer kinetic rate constants from single-molecule FRET trajectories.

Götz Markus M Barth Anders A Bohr Søren S-R SS Börner Richard R Chen Jixin J Cordes Thorben T Erie Dorothy A DA Gebhardt Christian C Hadzic Mélodie C A S MCAS Hamilton George L GL Hatzakis Nikos S NS Hugel Thorsten T Kisley Lydia L Lamb Don C DC de Lannoy Carlos C Mahn Chelsea C Dunukara Dushani D de Ridder Dick D Sanabria Hugo H Schimpf Julia J Seidel Claus A M CAM Sigel Roland K O RKO Sletfjerding Magnus Berg MB Thomsen Johannes J Vollmar Leonie L Wanninger Simon S Weninger Keith R KR Xu Pengning P Schmid Sonja S

Nature communications 20220914 1

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 ...[more]

PMID: 36104339

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Project description:Recently developed statistical methods by Gopich and Szabo were used to extract folding and unfolding rate coefficients from single-molecule Förster resonance energy transfer (FRET) data for proteins with kinetics too fast to measure waiting time distributions. Two types of experiments and two different analyses were performed. In one experiment bursts of photons were collected from donor and acceptor fluorophores attached to a 73-residue protein, α(3)D, freely diffusing through the illuminated volume of a confocal microscope system. In the second, the protein was immobilized by linkage to a surface, and photons were collected until one of the fluorophores bleached. Folding and unfolding rate coefficients and mean FRET efficiencies for the folded and unfolded subpopulations were obtained from a photon by photon analysis of the trajectories using a maximum likelihood method. The ability of the method to describe the data in terms of a two-state model was checked by recoloring the photon trajectories with the extracted parameters and comparing the calculated FRET efficiency histograms with the measured histograms. The sum of the rate coefficients for the two-state model agreed to within 30% with the relaxation rate obtained from the decay of the donor-acceptor cross-correlation function, confirming the high accuracy of the method. Interestingly, apparently reliable rate coefficients could be extracted using the maximum likelihood method, even at low (<10%) population of the minor component where the cross-correlation function was too noisy to obtain any useful information. The rate coefficients and mean FRET efficiencies were also obtained in an approximate procedure by simply fitting the FRET efficiency histograms, calculated by binning the donor and acceptor photons, with a sum of three-Gaussian functions. The kinetics are exposed in these histograms by the growth of a FRET efficiency peak at values intermediate between the folded and unfolded peaks as the bin size increases, a phenomenon with similarities to NMR exchange broadening. When comparable populations of folded and unfolded molecules are present, this method yields rate coefficients in very good agreement with those obtained with the maximum likelihood method. As a first step toward characterizing transition paths, the Viterbi algorithm was used to locate the most probable transition points in the photon trajectories.

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A blind benchmark of analysis tools to infer kinetic rate constants from single-molecule FRET trajectories.

Publications

A blind benchmark of analysis tools to infer kinetic rate constants from single-molecule FRET trajectories.

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