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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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Comparison between proline 15, 20 and 30 and experiment.Efficiency histograms averaging over fast dye motions only (Fig. 10) are shown for three different polyproline-lengths (red, dashed-dotted, solid, dashed), and corresponding measured efficiency distributions (black). For polyproline-30, inclusion of double-cis isomers (see Text S1) only slightly changes the efficiency histogram (bold dashed line).
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pone-0019791-g011: Comparison between proline 15, 20 and 30 and experiment.Efficiency histograms averaging over fast dye motions only (Fig. 10) are shown for three different polyproline-lengths (red, dashed-dotted, solid, dashed), and corresponding measured efficiency distributions (black). For polyproline-30, inclusion of double-cis isomers (see Text S1) only slightly changes the efficiency histogram (bold dashed line).

Mentions: Next, we compare efficiency distributions for different dye-labeled proline lengths. Figure 11 shows the calculated efficiency distributions (burst averaging over fast dye motions only) from simulations with proline lengths 15, 20, and 30 (solid lines) as well as measured efficiencies for lengths of 14, 20, 27, and 33 (dashed). The general length effect, increase in efficiency for shorter prolines and vice versa, is observed.


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)

Comparison between proline 15, 20 and 30 and experiment.Efficiency histograms averaging over fast dye motions only (Fig. 10) are shown for three different polyproline-lengths (red, dashed-dotted, solid, dashed), and corresponding measured efficiency distributions (black). For polyproline-30, inclusion of double-cis isomers (see Text S1) only slightly changes the efficiency histogram (bold dashed line).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0019791-g011: Comparison between proline 15, 20 and 30 and experiment.Efficiency histograms averaging over fast dye motions only (Fig. 10) are shown for three different polyproline-lengths (red, dashed-dotted, solid, dashed), and corresponding measured efficiency distributions (black). For polyproline-30, inclusion of double-cis isomers (see Text S1) only slightly changes the efficiency histogram (bold dashed line).
Mentions: Next, we compare efficiency distributions for different dye-labeled proline lengths. Figure 11 shows the calculated efficiency distributions (burst averaging over fast dye motions only) from simulations with proline lengths 15, 20, and 30 (solid lines) as well as measured efficiencies for lengths of 14, 20, 27, and 33 (dashed). The general length effect, increase in efficiency for shorter prolines and vice versa, is observed.

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