Dataset Information

Theory of the energy transfer efficiency and fluorescence lifetime distribution in single-molecule FRET.

ABSTRACT: In single-molecule FRET experiments with pulsed lasers, not only the colors of the photons but also the fluorescence lifetimes can be monitored. Although these quantities appear to be random, they are modulated by conformational dynamics. In order to extract information about such dynamics, we develop the theory of the joint distribution of FRET efficiencies and fluorescence lifetimes determined from bins (or bursts) of photons. Our starting point is a rigorous formal expression for the distribution of the numbers of donor and acceptor photons and donor lifetimes in a bin that treats the influence of conformational dynamics on all timescales. This formula leads to an analytic result for a two-state system interconverting on a timescale slower than the interphoton time and to an efficient simulation algorithm for multistate dynamics. The shape of the joint distribution contains more information about conformational dynamics than the FRET efficiency histogram alone. In favorable cases, the connectivity of the underlying conformational states can be determined directly by simple inspection of the projection of the joint distribution on the efficiency-lifetime plane.

SUBMITTER: Gopich IV

PROVIDER: S-EPMC3356627 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Theory of the energy transfer efficiency and fluorescence lifetime distribution in single-molecule FRET.

Gopich Irina V IV Szabo Attila A

Proceedings of the National Academy of Sciences of the United States of America 20120501 20

In single-molecule FRET experiments with pulsed lasers, not only the colors of the photons but also the fluorescence lifetimes can be monitored. Although these quantities appear to be random, they are modulated by conformational dynamics. In order to extract information about such dynamics, we develop the theory of the joint distribution of FRET efficiencies and fluorescence lifetimes determined from bins (or bursts) of photons. Our starting point is a rigorous formal expression for the distribu ...[more]

PMID: 22550169

Similar Datasets

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.

Dataset Information

Theory of the energy transfer efficiency and fluorescence lifetime distribution in single-molecule FRET.

Publications

Theory of the energy transfer efficiency and fluorescence lifetime distribution in single-molecule FRET.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets