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Structural heterogeneity and quantitative FRET efficiency distributions of polyprolines through a hybrid atomistic simulation and Monte Carlo approach.

Hoefling M, Lima N, Haenni D, Seidel CA, Schuler B, Grubmüller H - PLoS ONE (2011)

Bottom Line: Using polyproline chains with attached Alexa 488 and Alexa 594 dyes as a test system, we demonstrate the feasibility of this approach by direct comparison to experimental data.We identified cis-isomers and different static local environments as sources of the experimentally observed heterogeneity.Reconstructions of distance distributions from experimental data at different levels of theory demonstrate how the respective underlying assumptions and approximations affect the obtained accuracy.

View Article: PubMed Central - PubMed

Affiliation: Theoretical and Computational Biophysics Department, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

ABSTRACT
Förster Resonance Energy Transfer (FRET) experiments probe molecular distances via distance dependent energy transfer from an excited donor dye to an acceptor dye. Single molecule experiments not only probe average distances, but also distance distributions or even fluctuations, and thus provide a powerful tool to study biomolecular structure and dynamics. However, the measured energy transfer efficiency depends not only on the distance between the dyes, but also on their mutual orientation, which is typically inaccessible to experiments. Thus, assumptions on the orientation distributions and averages are usually made, limiting the accuracy of the distance distributions extracted from FRET experiments. Here, we demonstrate that by combining single molecule FRET experiments with the mutual dye orientation statistics obtained from Molecular Dynamics (MD) simulations, improved estimates of distances and distributions are obtained. From the simulated time-dependent mutual orientations, FRET efficiencies are calculated and the full statistics of individual photon absorption, energy transfer, and photon emission events is obtained from subsequent Monte Carlo (MC) simulations of the FRET kinetics. All recorded emission events are collected to bursts from which efficiency distributions are calculated in close resemblance to the actual FRET experiment, taking shot noise fully into account. Using polyproline chains with attached Alexa 488 and Alexa 594 dyes as a test system, we demonstrate the feasibility of this approach by direct comparison to experimental data. We identified cis-isomers and different static local environments as sources of the experimentally observed heterogeneity. Reconstructions of distance distributions from experimental data at different levels of theory demonstrate how the respective underlying assumptions and approximations affect the obtained accuracy. Our results show that dye fluctuations obtained from MD simulations, combined with MC single photon kinetics, provide a versatile tool to improve the accuracy of distance distributions that can be extracted from measured single molecule FRET efficiencies.

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Photon generation by Monte Carlo.FRET transfer rate coefficient vs. time, calculated from a molecular dynamics simulation (box, left part). A random starting point on the trajectory is chosen at which the donor dye is assumed to be excited by a photon (blue). Then, for each time step the MC process on the right side is evaluated according to the corresponding probabilities until de-excitation occurs. Four de-excitation pathways are considered, thermal de-excitation of donor or acceptor (dark-blue) and respective photon emissions (donor: green; acceptor: red). The ratio of the collected donor and acceptor photons is used to calculate a FRET transfer efficiency.
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pone-0019791-g004: Photon generation by Monte Carlo.FRET transfer rate coefficient vs. time, calculated from a molecular dynamics simulation (box, left part). A random starting point on the trajectory is chosen at which the donor dye is assumed to be excited by a photon (blue). Then, for each time step the MC process on the right side is evaluated according to the corresponding probabilities until de-excitation occurs. Four de-excitation pathways are considered, thermal de-excitation of donor or acceptor (dark-blue) and respective photon emissions (donor: green; acceptor: red). The ratio of the collected donor and acceptor photons is used to calculate a FRET transfer efficiency.

Mentions: For direct comparison with smFRET burst counts, we developed a Monte Carlo (MC) procedure to calculate single burst FRET efficiencies from . In the experiments, the arrival times of individual photons from single molecules were recorded. Accordingly, and following the kinetics scheme Eq. 3, multiple individual photons were generated in a Monte Carlo process (Fig. 4). For each photon, we proceeded as follows.


Structural heterogeneity and quantitative FRET efficiency distributions of polyprolines through a hybrid atomistic simulation and Monte Carlo approach.

Hoefling M, Lima N, Haenni D, Seidel CA, Schuler B, Grubmüller H - PLoS ONE (2011)

Photon generation by Monte Carlo.FRET transfer rate coefficient vs. time, calculated from a molecular dynamics simulation (box, left part). A random starting point on the trajectory is chosen at which the donor dye is assumed to be excited by a photon (blue). Then, for each time step the MC process on the right side is evaluated according to the corresponding probabilities until de-excitation occurs. Four de-excitation pathways are considered, thermal de-excitation of donor or acceptor (dark-blue) and respective photon emissions (donor: green; acceptor: red). The ratio of the collected donor and acceptor photons is used to calculate a FRET transfer efficiency.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101224&req=5

pone-0019791-g004: Photon generation by Monte Carlo.FRET transfer rate coefficient vs. time, calculated from a molecular dynamics simulation (box, left part). A random starting point on the trajectory is chosen at which the donor dye is assumed to be excited by a photon (blue). Then, for each time step the MC process on the right side is evaluated according to the corresponding probabilities until de-excitation occurs. Four de-excitation pathways are considered, thermal de-excitation of donor or acceptor (dark-blue) and respective photon emissions (donor: green; acceptor: red). The ratio of the collected donor and acceptor photons is used to calculate a FRET transfer efficiency.
Mentions: For direct comparison with smFRET burst counts, we developed a Monte Carlo (MC) procedure to calculate single burst FRET efficiencies from . In the experiments, the arrival times of individual photons from single molecules were recorded. Accordingly, and following the kinetics scheme Eq. 3, multiple individual photons were generated in a Monte Carlo process (Fig. 4). For each photon, we proceeded as follows.

Bottom Line: Using polyproline chains with attached Alexa 488 and Alexa 594 dyes as a test system, we demonstrate the feasibility of this approach by direct comparison to experimental data.We identified cis-isomers and different static local environments as sources of the experimentally observed heterogeneity.Reconstructions of distance distributions from experimental data at different levels of theory demonstrate how the respective underlying assumptions and approximations affect the obtained accuracy.

View Article: PubMed Central - PubMed

Affiliation: Theoretical and Computational Biophysics Department, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

ABSTRACT
Förster Resonance Energy Transfer (FRET) experiments probe molecular distances via distance dependent energy transfer from an excited donor dye to an acceptor dye. Single molecule experiments not only probe average distances, but also distance distributions or even fluctuations, and thus provide a powerful tool to study biomolecular structure and dynamics. However, the measured energy transfer efficiency depends not only on the distance between the dyes, but also on their mutual orientation, which is typically inaccessible to experiments. Thus, assumptions on the orientation distributions and averages are usually made, limiting the accuracy of the distance distributions extracted from FRET experiments. Here, we demonstrate that by combining single molecule FRET experiments with the mutual dye orientation statistics obtained from Molecular Dynamics (MD) simulations, improved estimates of distances and distributions are obtained. From the simulated time-dependent mutual orientations, FRET efficiencies are calculated and the full statistics of individual photon absorption, energy transfer, and photon emission events is obtained from subsequent Monte Carlo (MC) simulations of the FRET kinetics. All recorded emission events are collected to bursts from which efficiency distributions are calculated in close resemblance to the actual FRET experiment, taking shot noise fully into account. Using polyproline chains with attached Alexa 488 and Alexa 594 dyes as a test system, we demonstrate the feasibility of this approach by direct comparison to experimental data. We identified cis-isomers and different static local environments as sources of the experimentally observed heterogeneity. Reconstructions of distance distributions from experimental data at different levels of theory demonstrate how the respective underlying assumptions and approximations affect the obtained accuracy. Our results show that dye fluctuations obtained from MD simulations, combined with MC single photon kinetics, provide a versatile tool to improve the accuracy of distance distributions that can be extracted from measured single molecule FRET efficiencies.

Show MeSH
Related in: MedlinePlus