Combining Graphical and Analytical Methods with MolecularSimulations To Analyze Time-Resolved FRET Measurements of LabeledMacromolecules Accurately

摘要

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, εD(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 analyticalprocedures. Additionally, we developed a simulation toolkit to modeldye diffusion, fluorescence quenching by the protein surface, andFRET. A benchmark study with simulated fluorescence decays of 500protein structures demonstrates that the quasistatic homogeneous modelworks very well and recovers for single conformations the averageDA distances with an accuracy of < 2%. For more complexcases, where proteins adopt multiple conformations with significantlydifferent dye environments (heterogeneous case), we introduce a generalanalysis framework and evaluate its power in resolving heterogeneitiesin DA distances. The developed fast simulation methods, relying onBrownian dynamics of a coarse-grained dye in its sterically accessiblevolume, allow us to incorporate structural information in the decayanalysis for heterogeneous cases by relating dye states with proteinconformations to pave the way for fluorescence and FRET-based dynamicstructural biology. Finally, we present theories and simulations toassess the accuracy and precision of steady-state and time-resolvedFRET measurements in resolving DA distances on the single-moleculeand ensemble level and provide a rigorous framework for estimatingapproximation, systematic, and statistical errors.