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A flexible approach to assess fluorescence decay functions in complex energy transfer systems.

Roethlein C, Miettinen MS, Ignatova Z - BMC Biophys (2015)

Bottom Line: Moreover, time-correlated photon count measurements bear additional information on the variety of donor surroundings allowing more detailed differentiation between distinct structural geometries which are typically inaccessible to general fitting solutions.This simulation solution assesses the full statistics of time-correlated photon counts and distance distributions of fluorescently labeled biomolecules.This approach is powerful in distinguishing distance distributions in a wide variety of experimental setups, thus providing a versatile tool to accurately distinguish between different structural assemblies in highly complex systems.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry and Biology, University of Potsdam, Potsdam, Germany.

ABSTRACT

Background: Time-correlated Förster resonance energy transfer (FRET) probes molecular distances with greater accuracy than intensity-based calculation of FRET efficiency and provides a powerful tool to study biomolecular structure and dynamics. Moreover, time-correlated photon count measurements bear additional information on the variety of donor surroundings allowing more detailed differentiation between distinct structural geometries which are typically inaccessible to general fitting solutions.

Results: Here we develop a new approach based on Monte Carlo simulations of time-correlated FRET events to estimate the time-correlated single photon counts (TCSPC) histograms in complex systems. This simulation solution assesses the full statistics of time-correlated photon counts and distance distributions of fluorescently labeled biomolecules. The simulations are consistent with the theoretical predictions of the dye behavior in FRET systems with defined dye distances and measurements of randomly distributed dye solutions. We validate the simulation results using a highly heterogeneous aggregation system and explore the conditions to use this tool in complex systems.

Conclusion: This approach is powerful in distinguishing distance distributions in a wide variety of experimental setups, thus providing a versatile tool to accurately distinguish between different structural assemblies in highly complex systems.

No MeSH data available.


Related in: MedlinePlus

Reproduction of theoretically calculated fluorescent decay times. A: Simulation of time-dependent photon counts for single donor environments. The acceptor distances were between 1 and 10 nm. B, C: Simulation of fluorescent decay signals of equal amounts of FRET pairs (from A). Representative curves of simulations with one donor-acceptor distance fixed at 4 nm (B) or 6 nm (C) while for the other donor-acceptor pair the distance was variable and the distances are displayed in the legend. For better representation of the 6-nm experiment the curves are split in two plots. The amount of the two donors in each simulation was equal. No acceptor denotes a simulation experiment with donor only. Simulated values are depicted with symbols and the matching theoretical calculations as lines in the same color.
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Fig2: Reproduction of theoretically calculated fluorescent decay times. A: Simulation of time-dependent photon counts for single donor environments. The acceptor distances were between 1 and 10 nm. B, C: Simulation of fluorescent decay signals of equal amounts of FRET pairs (from A). Representative curves of simulations with one donor-acceptor distance fixed at 4 nm (B) or 6 nm (C) while for the other donor-acceptor pair the distance was variable and the distances are displayed in the legend. For better representation of the 6-nm experiment the curves are split in two plots. The amount of the two donors in each simulation was equal. No acceptor denotes a simulation experiment with donor only. Simulated values are depicted with symbols and the matching theoretical calculations as lines in the same color.

Mentions: To evaluate the predictions made with our code, we first performed simulations with simple dye systems whose fluorescent decay function can be calculated (Eq. 5). We created input files with one (Figure 2A) or two (Figure 2B, and C) distinct donor environments. The acceptor was simulated for each donor environment at defined distances with 0.5 nm step (Figure 2). Notably, for a single donor environment all fluorescent decay functions were predicted accurately and precisely (Figure 2A).Figure 2


A flexible approach to assess fluorescence decay functions in complex energy transfer systems.

Roethlein C, Miettinen MS, Ignatova Z - BMC Biophys (2015)

Reproduction of theoretically calculated fluorescent decay times. A: Simulation of time-dependent photon counts for single donor environments. The acceptor distances were between 1 and 10 nm. B, C: Simulation of fluorescent decay signals of equal amounts of FRET pairs (from A). Representative curves of simulations with one donor-acceptor distance fixed at 4 nm (B) or 6 nm (C) while for the other donor-acceptor pair the distance was variable and the distances are displayed in the legend. For better representation of the 6-nm experiment the curves are split in two plots. The amount of the two donors in each simulation was equal. No acceptor denotes a simulation experiment with donor only. Simulated values are depicted with symbols and the matching theoretical calculations as lines in the same color.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4403788&req=5

Fig2: Reproduction of theoretically calculated fluorescent decay times. A: Simulation of time-dependent photon counts for single donor environments. The acceptor distances were between 1 and 10 nm. B, C: Simulation of fluorescent decay signals of equal amounts of FRET pairs (from A). Representative curves of simulations with one donor-acceptor distance fixed at 4 nm (B) or 6 nm (C) while for the other donor-acceptor pair the distance was variable and the distances are displayed in the legend. For better representation of the 6-nm experiment the curves are split in two plots. The amount of the two donors in each simulation was equal. No acceptor denotes a simulation experiment with donor only. Simulated values are depicted with symbols and the matching theoretical calculations as lines in the same color.
Mentions: To evaluate the predictions made with our code, we first performed simulations with simple dye systems whose fluorescent decay function can be calculated (Eq. 5). We created input files with one (Figure 2A) or two (Figure 2B, and C) distinct donor environments. The acceptor was simulated for each donor environment at defined distances with 0.5 nm step (Figure 2). Notably, for a single donor environment all fluorescent decay functions were predicted accurately and precisely (Figure 2A).Figure 2

Bottom Line: Moreover, time-correlated photon count measurements bear additional information on the variety of donor surroundings allowing more detailed differentiation between distinct structural geometries which are typically inaccessible to general fitting solutions.This simulation solution assesses the full statistics of time-correlated photon counts and distance distributions of fluorescently labeled biomolecules.This approach is powerful in distinguishing distance distributions in a wide variety of experimental setups, thus providing a versatile tool to accurately distinguish between different structural assemblies in highly complex systems.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry and Biology, University of Potsdam, Potsdam, Germany.

ABSTRACT

Background: Time-correlated Förster resonance energy transfer (FRET) probes molecular distances with greater accuracy than intensity-based calculation of FRET efficiency and provides a powerful tool to study biomolecular structure and dynamics. Moreover, time-correlated photon count measurements bear additional information on the variety of donor surroundings allowing more detailed differentiation between distinct structural geometries which are typically inaccessible to general fitting solutions.

Results: Here we develop a new approach based on Monte Carlo simulations of time-correlated FRET events to estimate the time-correlated single photon counts (TCSPC) histograms in complex systems. This simulation solution assesses the full statistics of time-correlated photon counts and distance distributions of fluorescently labeled biomolecules. The simulations are consistent with the theoretical predictions of the dye behavior in FRET systems with defined dye distances and measurements of randomly distributed dye solutions. We validate the simulation results using a highly heterogeneous aggregation system and explore the conditions to use this tool in complex systems.

Conclusion: This approach is powerful in distinguishing distance distributions in a wide variety of experimental setups, thus providing a versatile tool to accurately distinguish between different structural assemblies in highly complex systems.

No MeSH data available.


Related in: MedlinePlus