10 research outputs found
Precision and accuracy of single-molecule FRET measurements - a multi-laboratory benchmark study
Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods
Publisher Correction: Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study
A new approach to the analyses of fluorescence depolarisation experiments in the presence of electronic energy transport
A new and general procedure is described for a detailed analysis of time-resolved fluorescence depolarisation data in the presence of electronic energy migration. An isotropic ensemble of bifluorophoric molecules (D1-R-D2) has been studied to demonstrate its utility. Intramolecular donor-donor energy migration occurs between the two donor groups (D), which are covalently connected to a rigid linker group (R). These groups undergo restricted reorientational motions with respect to the R group. The analysis of depolarisation data basically involves the search for best-fit parameters which describe the local reorienting motions, the interfluorophore D1-D2 distance, as well as the mutual orientations of the donors. For this, the analysis is partly performed in the Fourier domain and the best-fit parameters are determined by using an approach based on a Genetic Algorithm. The energy migration process has been described by using Monte Carlo simulations and an extended Förster theory. It is found that this theory provides the least time-consuming computational method. Since one-photon and two-photon excited fluorescence experiments can be applied for energy migration studies, a general and unified theoretical formulation is given. To exemplify the developed quantitative approach the depolarisation of the fluorescence in the presence of electronic energy migration within a bis-(9-anthrylmethylphosphonate) bisteroid molecule has been studied by time-resolved two-photon excited fluorescence depolarisation experiments. To solely obtain information about local reorientations of the 9-anthrylmethyl group, also the mono-(9-anthrylmethylphosphonate) bisteroid was studied, which enabled modelling of the ordering potential of the donor. Values of the two-photon absorption tensor components were obtained. To describe the discrepancy between the measured values of the initial anisotropy and fundamental anisotropy predicted by theory the distribution of absorption tensor caused by fast processes have been introduced. An angular parameter of absorption tensor was determined. Reasonable values of the distance between the 9-anthrylmethyl groups, as well as for their mutual orientation were obtained
A new approach to the analyses of fluorescence depolarisation experiments in the presence of electronic energy transport
A new and general procedure is described for a detailed analysis of time-resolved fluorescence depolarisation data in the presence of electronic energy migration. An isotropic ensemble of bifluorophoric molecules (D1-R-D2) has been studied to demonstrate its utility. Intramolecular donor-donor energy migration occurs between the two donor groups (D), which are covalently connected to a rigid linker group (R). These groups undergo restricted reorientational motions with respect to the R group. The analysis of depolarisation data basically involves the search for best-fit parameters which describe the local reorienting motions, the interfluorophore D1-D2 distance, as well as the mutual orientations of the donors. For this, the analysis is partly performed in the Fourier domain and the best-fit parameters are determined by using an approach based on a Genetic Algorithm. The energy migration process has been described by using Monte Carlo simulations and an extended Förster theory. It is found that this theory provides the least time-consuming computational method. Since one-photon and two-photon excited fluorescence experiments can be applied for energy migration studies, a general and unified theoretical formulation is given. To exemplify the developed quantitative approach the depolarisation of the fluorescence in the presence of electronic energy migration within a bis-(9-anthrylmethylphosphonate) bisteroid molecule has been studied by time-resolved two-photon excited fluorescence depolarisation experiments. To solely obtain information about local reorientations of the 9-anthrylmethyl group, also the mono-(9-anthrylmethylphosphonate) bisteroid was studied, which enabled modelling of the ordering potential of the donor. Values of the two-photon absorption tensor components were obtained. To describe the discrepancy between the measured values of the initial anisotropy and fundamental anisotropy predicted by theory the distribution of absorption tensor caused by fast processes have been introduced. An angular parameter of absorption tensor was determined. Reasonable values of the distance between the 9-anthrylmethyl groups, as well as for their mutual orientation were obtained
On the analyses of fluorescence depolarisation data in the presence of electronic energy migration : Part I. Theory and general description
A new and general procedure is described for a detailed analysis of time-resolved fluorescence depolarisation data in the presence of electronic energy migration. An isotropic ensemble of bifluorophoric molecules (D1-R-D2) has been studied to demonstrate its utility. Intramolecular donor-donor energy migration occurs between the two donor groups (D), which are covalently connected to a rigid linker group (R). These groups undergo restricted reorientational motions with respect to the R group. The analysis of depolarisation data basically involves the search for best-fit parameters which describe the local reorienting motions, the intermolecular D1-D2 distance, as well as the mutual orientations of the donors. For this, the analysis is partly performed in the Fourier domain and the best-fit parameters are determined by using an approach based on a Genetic Algorithm. The energy migration process has been described by using Monte Carlo simulations and an extended Förster theory (EFT). It is found that the EFT provides the least time-consuming computational method. Since one-photon and two-photon excited fluorescence experiments can be applied for energy migration studies, a general and unified theoretical formulation is given
Combining Graphical and Analytical Methods with Molecular Simulations To Analyze Time-Resolved FRET Measurements of Labeled Macromolecules Accurately
Förster resonance energy transfer
(FRET) measurements from
a donor, D, to an acceptor, A, fluorophore are frequently used <i>in vitro</i> and in live cells to reveal information on the
structure and dynamics of DA labeled macromolecules. Accurate descriptions
of FRET measurements by molecular models are complicated because the
fluorophores are usually coupled to the macromolecule via flexible
long linkers allowing for diffusional exchange between multiple states
with different fluorescence properties caused by distinct environmental
quenching, dye mobilities, and variable DA distances. It is often
assumed for the analysis of fluorescence intensity decays that DA
distances and D quenching are uncorrelated (homogeneous quenching
by FRET) and that the exchange between distinct fluorophore states
is slow (quasistatic). This allows us to introduce the FRET-induced
donor decay, ε<sub>D</sub>(<i>t</i>), a function solely
depending on the species fraction distribution of the rate constants
of energy transfer by FRET, for a convenient joint analysis of fluorescence
decays of FRET and reference samples by integrated graphical and analytical
procedures. Additionally, we developed a simulation toolkit to model
dye diffusion, fluorescence quenching by the protein surface, and
FRET. A benchmark study with simulated fluorescence decays of 500
protein structures demonstrates that the quasistatic homogeneous model
works very well and recovers for single conformations the average
DA distances with an accuracy of < 2%. For more complex
cases, where proteins adopt multiple conformations with significantly
different dye environments (heterogeneous case), we introduce a general
analysis framework and evaluate its power in resolving heterogeneities
in DA distances. The developed fast simulation methods, relying on
Brownian dynamics of a coarse-grained dye in its sterically accessible
volume, allow us to incorporate structural information in the decay
analysis for heterogeneous cases by relating dye states with protein
conformations to pave the way for fluorescence and FRET-based dynamic
structural biology. Finally, we present theories and simulations to
assess the accuracy and precision of steady-state and time-resolved
FRET measurements in resolving DA distances on the single-molecule
and ensemble level and provide a rigorous framework for estimating
approximation, systematic, and statistical errors
On the analyses of fluorescence depolarisation data in the presence of electronic energy migration. Part I: Theory and general description
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Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.
Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods