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.
Highlights
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 describe our development of three-color FRET spectroscopy using intense continuous-wave (CW) laser excitation for the investigation of fast molecular processes occurring on μs to ms time scales
In the binding experiment, disordered transactivation domain (TAD) was labeled with two fluorophores site-immobilized, and incubated with nuclear coactivator binding domain (NCBD) labeled with acceptor 2 (A2)
Summary
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. 1234567890():,; Single-molecule Förster resonance energy transfer (FRET) spectroscopy is a very sensitive tool to detect distance changes on a nanometer scale Since its development, it has become an indispensable experimental method in many areas of modern biological sciences[1,2,3,4,5,6]. If conformational changes occur during protein-protein interactions, such as coupled binding and folding of intrinsically disordered proteins (IDPs)[10], by attaching two fluorophores to a disordered protein and the third fluorophore to the second molecule, it is possible to correlate conformational changes of the IDP and its interaction with a binding partner These processes often occur on a fast time scale (μs–ms)[9,11,12,13,14], but the time resolution of typical three-color FRET experiments remains at tens of milliseconds. We developed a fitting routine using CPU-GPU co-parallelization that reduces the computation time significantly, by almost two orders of magnitude compared to the calculation with a single CPU processor (see Supplementary Fig. 2 and “Methods”). (Analysis codes are available at https://github.com/hoisunglab/FRET_3colorCW.) We describe the correction procedures to obtain accurate FRET efficiencies that can be related to the distances between the dyes
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