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2017 ; 121
(35
): 8211-8241
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Combining Graphical and Analytical Methods with Molecular Simulations To Analyze
Time-Resolved FRET Measurements of Labeled Macromolecules Accurately
#MMPMID28709377
Peulen TO
; Opanasyuk O
; Seidel CAM
J Phys Chem B
2017[Sep]; 121
(35
): 8211-8241
PMID28709377
show ga
Förster resonance energy transfer (FRET) measurements from a donor, D, to an
acceptor, A, fluorophore are frequently used in vitro 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, ?(D)(t), 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.