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Fast single-molecule FRET spectroscopy: theory and experiment.

ABSTRACT: Single-molecule spectroscopy is widely used to study macromolecular dynamics. Although this technique provides unique information that cannot be obtained at the ensemble level, the possibility of studying fast molecular dynamics is limited by the number of photons detected per unit time (photon count rate), which is proportional to the illumination intensity. However, simply increasing the illumination intensity often does not help because of various photophysical and photochemical problems. In this Perspective, we show how to improve the dynamic range of single-molecule fluorescence spectroscopy at a given photon count rate by considering each and every photon and using a maximum likelihood method. For a photon trajectory with recorded photon colors and inter-photon times, the parameters of a model describing molecular dynamics are obtained by maximizing the appropriate likelihood function. We discuss various likelihood functions, their applicability, and the accuracy of the extracted parameters. The maximum likelihood method has been applied to analyze the experiments on fast two-state protein folding and to measure transition path times. Utilizing other information such as fluorescence lifetimes is discussed in the framework of two-dimensional FRET efficiency-lifetime histograms.

SUBMITTER: Chung HS

PROVIDER: S-EPMC4154966 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Fast single-molecule FRET spectroscopy: theory and experiment.

Chung Hoi Sung HS Gopich Irina V IV

Physical chemistry chemical physics : PCCP 20140901 35

Single-molecule spectroscopy is widely used to study macromolecular dynamics. Although this technique provides unique information that cannot be obtained at the ensemble level, the possibility of studying fast molecular dynamics is limited by the number of photons detected per unit time (photon count rate), which is proportional to the illumination intensity. However, simply increasing the illumination intensity often does not help because of various photophysical and photochemical problems. In ...[more]

PMID: 25088495

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Project description:We describe the theory, experiment, and analysis of three-color Förster resonance energy transfer (FRET) spectroscopy for probing conformational dynamics of a fast-folding protein, α3D. In three-color FRET, site-specific labeling of fluorophores is required to avoid ambiguity resulting from various species with different combinations of labeling positions. To this end, we first attached two dyes to a cysteine residue and an unnatural amino acid and then appended a cysteine residue to the C-terminus of the protein by the sortase-mediated ligation for attaching the third dye. To determine all three FRET efficiencies, we used alternating excitation of the donor and acceptor 1 with two picosecond-pulsed lasers. Since the folded and unfolded states are not distinguishable in binned fluorescence trajectories due to fast-folding on a millisecond time scale, we used a maximum likelihood method that analyzes photon trajectories without binning the data. The extracted kinetic parameters agree very well with the previously measured parameters for the same protein with two-color FRET, suggesting that the addition of the third fluorophore does not affect the folding dynamics of the protein. From the extracted fractions of acceptor photon counts, the FRET efficiencies for all three dye pairs were calculated after various corrections. They were compared with the FRET efficiencies obtained from the global analysis of two-color segments collected in the same experiment. The FRET efficiencies of the folded state from the three-color segments agree with those from the two-color segments, whereas the three-color and two-color FRET efficiencies of the unfolded state are different. This happens because fluctuations of all three interdye distances contribute to the FRET efficiency measured in three-color FRET. We show that this difference can be accounted for by using the Gaussian chain model for the unfolded state with the parameters obtained from the analysis of two-color segments. This result shows that three-color FRET provides additional information on the flexibility of molecules that cannot be obtained from a combination of two-color FRET experiments with three dye pairs. Using the delay times of photons from the laser pulse, fluorescence lifetimes were determined using the maximum likelihood analysis. The correlation between FRET efficiencies and lifetimes of the donor, acceptor 1, and acceptor 2 was visualized in two-dimensional FRET efficiency-lifetime histograms. These histograms can be used to demonstrate the presence of conformational dynamics in a protein.

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Fast single-molecule FRET spectroscopy: theory and experiment.

Publications

Fast single-molecule FRET spectroscopy: theory and experiment.

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