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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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FRET efficiency of trans and cis isomers.Comparison between measured FRET efficiency histograms (black) and histograms computed from the simulations (blue: ensemble, all-trans and cis01 ... cis20). Red dots denote the respective mean values. The simple model sketched on top and defined in the text describes the general trend (green line) that isomers with a cis-bond close to the termini show lower efficiencies, whereas those with cis-bonds close to the polymer center tend to yield higher efficiencies. The dashed green lines estimate the spread of the average efficiencies of the cis simulations mirroring the spread found for the all-trans simulations (). For illustration purposes, the photons of the individual cis were not discarded when generated below  as described in the Methods Section. The high efficiencies observed for cis-6 to cis-12 result from dyes in contact and are quenched in the experiment.
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pone-0019791-g009: FRET efficiency of trans and cis isomers.Comparison between measured FRET efficiency histograms (black) and histograms computed from the simulations (blue: ensemble, all-trans and cis01 ... cis20). Red dots denote the respective mean values. The simple model sketched on top and defined in the text describes the general trend (green line) that isomers with a cis-bond close to the termini show lower efficiencies, whereas those with cis-bonds close to the polymer center tend to yield higher efficiencies. The dashed green lines estimate the spread of the average efficiencies of the cis simulations mirroring the spread found for the all-trans simulations (). For illustration purposes, the photons of the individual cis were not discarded when generated below as described in the Methods Section. The high efficiencies observed for cis-6 to cis-12 result from dyes in contact and are quenched in the experiment.

Mentions: Figure 9 shows FRET efficiency distributions and averages for the all-trans and cis polyproline-20 chains in comparison with experiment. As expected, the average efficiencies of the cis-chains are larger than that of the all-trans isomer, due to the reduced distance of the terminal prolines. The largest reduction is seen for cis-bonds in central positions, thus attributing measured high efficiencies to those isomers. This behavior can be captured in a simple model (Fig. 9, top), in which the cis-isomer is described by a kink angle between the two stiff parts of the molecule, with distances and between the cis-bond and the respective termini, and being the all-trans distance between the two termini. , was determined from the all-trans mean efficiency using Eq. 2 and split up on and for each cis isomer according to the cis-bond position. To account for the distance change due to the linker and the observed dye conformations (Fig. 7), an offset and was allowed for as an additional fit parameter. After fitting to the model to the average cis-efficiencies using Eq. 2, an angle of , and and offset was obtained. The resulting model is shown as green line in Fig. 9 and has to be compared to the mean efficiency values (red dots). The dashed line shows an offset of in efficiency space as error estimate. The offset towards Alexa 594 agrees with the deviation of the average dye-to-dye distance from the proline length () within the accuracy of this simple model.


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)

FRET efficiency of trans and cis isomers.Comparison between measured FRET efficiency histograms (black) and histograms computed from the simulations (blue: ensemble, all-trans and cis01 ... cis20). Red dots denote the respective mean values. The simple model sketched on top and defined in the text describes the general trend (green line) that isomers with a cis-bond close to the termini show lower efficiencies, whereas those with cis-bonds close to the polymer center tend to yield higher efficiencies. The dashed green lines estimate the spread of the average efficiencies of the cis simulations mirroring the spread found for the all-trans simulations (). For illustration purposes, the photons of the individual cis were not discarded when generated below  as described in the Methods Section. The high efficiencies observed for cis-6 to cis-12 result from dyes in contact and are quenched in the experiment.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0019791-g009: FRET efficiency of trans and cis isomers.Comparison between measured FRET efficiency histograms (black) and histograms computed from the simulations (blue: ensemble, all-trans and cis01 ... cis20). Red dots denote the respective mean values. The simple model sketched on top and defined in the text describes the general trend (green line) that isomers with a cis-bond close to the termini show lower efficiencies, whereas those with cis-bonds close to the polymer center tend to yield higher efficiencies. The dashed green lines estimate the spread of the average efficiencies of the cis simulations mirroring the spread found for the all-trans simulations (). For illustration purposes, the photons of the individual cis were not discarded when generated below as described in the Methods Section. The high efficiencies observed for cis-6 to cis-12 result from dyes in contact and are quenched in the experiment.
Mentions: Figure 9 shows FRET efficiency distributions and averages for the all-trans and cis polyproline-20 chains in comparison with experiment. As expected, the average efficiencies of the cis-chains are larger than that of the all-trans isomer, due to the reduced distance of the terminal prolines. The largest reduction is seen for cis-bonds in central positions, thus attributing measured high efficiencies to those isomers. This behavior can be captured in a simple model (Fig. 9, top), in which the cis-isomer is described by a kink angle between the two stiff parts of the molecule, with distances and between the cis-bond and the respective termini, and being the all-trans distance between the two termini. , was determined from the all-trans mean efficiency using Eq. 2 and split up on and for each cis isomer according to the cis-bond position. To account for the distance change due to the linker and the observed dye conformations (Fig. 7), an offset and was allowed for as an additional fit parameter. After fitting to the model to the average cis-efficiencies using Eq. 2, an angle of , and and offset was obtained. The resulting model is shown as green line in Fig. 9 and has to be compared to the mean efficiency values (red dots). The dashed line shows an offset of in efficiency space as error estimate. The offset towards Alexa 594 agrees with the deviation of the average dye-to-dye distance from the proline length () within the accuracy of this simple model.

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