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Theory and Analysis of Single-Molecule FRET Experiments

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Protein Folding

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2376))

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Abstract

Inter-dye distances and conformational dynamics can be studied using single-molecule FRET measurements. We consider two approaches to analyze sequences of photons with recorded photon colors and arrival times. The first approach is based on FRET efficiency histograms obtained from binned photon sequences. The experimental histograms are compared with the theoretical histograms obtained using the joint distribution of acceptor and donor photons or the Gaussian approximation. In the second approach, a photon sequence is analyzed without binning. The parameters of a model describing conformational dynamics are found by maximizing the appropriate likelihood function. The first approach is simpler, while the second one is more accurate, especially when the population of species is small and transition rates are fast. The likelihood-based analysis as well as the recoloring method has the advantage that diffusion of molecules through the laser focus can be rigorously handled.

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Acknowledgments

We thank Attila Szabo for many helpful discussions and comments. This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health.

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Authors and Affiliations

  1. Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA

    Irina V. Gopich & Hoi Sung Chung

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  1. Irina V. Gopich
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Correspondence to Irina V. Gopich .

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  1. Department of Bioengineering and Center for Cellular and Biomolecular Machines, University of California, Merced, Merced, CA, USA

    Victor Muñoz

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Gopich, I.V., Chung, H.S. (2022). Theory and Analysis of Single-Molecule FRET Experiments. In: Muñoz, V. (eds) Protein Folding. Methods in Molecular Biology, vol 2376. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1716-8_14

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