1. Fast three-color single-molecule FRET using statistical inference
- Author
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Hoi Sung Chung, Jae-Yeol Kim, Janghyun Yoo, John M. Louis, and Irina V. Gopich
- Subjects
0301 basic medicine ,Protein Folding ,Photon ,Time Factors ,Protein Conformation ,Science ,General Physics and Astronomy ,Color ,Intrinsically disordered proteins ,General Biochemistry, Genetics and Molecular Biology ,Article ,Quantitative Biology::Subcellular Processes ,03 medical and health sciences ,Single-molecule biophysics ,Fluorescence Resonance Energy Transfer ,Spectroscopy ,lcsh:Science ,Physics ,Physics::Biological Physics ,Photons ,Quantitative Biology::Biomolecules ,Multidisciplinary ,Microscopy, Confocal ,Photobleaching ,030102 biochemistry & molecular biology ,Proteins ,General Chemistry ,Single-molecule FRET ,Models, Theoretical ,Kinetics ,030104 developmental biology ,Förster resonance energy transfer ,Protein folding ,lcsh:Q ,Biological system ,Excitation ,Algorithms ,Protein Binding - Abstract
We describe theory, experiments, and analyses of three-color Förster resonance energy transfer (FRET) spectroscopy for probing sub-millisecond conformational dynamics of protein folding and binding of disordered proteins. We devise a scheme that uses single continuous-wave laser excitation of the donor instead of alternating excitation of the donor and one of the acceptors. This scheme alleviates photophysical problems of acceptors such as rapid photobleaching, which is crucial for high time resolution experiments with elevated illumination intensity. Our method exploits the molecular species with one of the acceptors absent or photobleached, from which two-color FRET data is collected in the same experiment. We show that three FRET efficiencies and kinetic parameters can be determined without alternating excitation from a global maximum likelihood analysis of two-color and three-color photon trajectories. We implement co-parallelization of CPU-GPU processing, which leads to a significant reduction of the likelihood calculation time for efficient parameter determination., Three-colour FRET is a powerful tool to study macromolecular conformational dynamics, but is temporally limited due to the experimental complexity. Here the authors develop experimental and analytical methods for probing submillisecond-time scale dynamics using single continuous-wave excitation.
- Published
- 2020