Quantitative time domain analysis of lifetime‐based Förster resonant energy transfer measurements with fluorescent proteins: Static random isotropic fluorophore orientation distributions

Yuriy Alexandrov,Paul M W French,Dino S Nikolic,Christopher Dunsby

doi:10.1002/jbio.201700366

Abstract

Förster resonant energy transfer (FRET) measurements are widely used to obtain information about molecular interactions and conformations through the dependence of FRET efficiency on the proximity of donor and acceptor fluorophores. Fluorescence lifetime measurements can provide quantitative analysis of FRET efficiency and interacting population fraction. Many FRET experiments exploit the highly specific labelling of genetically expressed fluorescent proteins, applicable in live cells and organisms. Unfortunately, the typical assumption of fast randomization of fluorophore orientations in the analysis of fluorescence lifetime-based FRET readouts is not valid for fluorescent proteins due to their slow rotational mobility compared to their upper state lifetime. Here, previous analysis of effectively static isotropic distributions of fluorophore dipoles on FRET measurements is incorporated into new software for fitting donor emission decay profiles. Calculated FRET parameters, including molar population fractions, are compared for the analysis of simulated and experimental FRET data under the assumption of static and dynamic fluorophores and the intermediate regimes between fully dynamic and static fluorophores, and mixtures within FRET pairs, is explored. Finally, a method to correct the artefact resulting from fitting the emission from static FRET pairs with isotropic angular distributions to the (incorrect) typically assumed dynamic FRET decay model is presented.

Highlights

Förster resonant energy transfer (FRET) [1] is the nonradiative transfer of energy from an excited donorChris Dunsby and Paul M
We demonstrate that our sκ2 simulation of the probability distribution of FRET efficiencies for 3 different values of η agrees well (Figure 1A) with the analytic solution from [3] and we show (Figure 1B) how the mean FRET efficiency varies as a function of η for the simulated sκ2 distribution compared with the theoretical curve for dynamic, randomly orientated donor-acceptor pairs
Because our nonlinear fluorescence decay fitting software tool incorporating the sκ2 model of Eq (15) is not yet able to be applied to image data, we developed a method to “correct” our existing Ras association domain family (RASSF)-MST1 fluorescence lifetime imaging (FLIM) FRET data, which was obtained by global fitting of a double exponential decay model to the experimental data using FLIMfit

Summary

Introduction

Förster resonant energy transfer (FRET) [1] is the nonradiative transfer of energy from an excited donorChris Dunsby and Paul M. Förster resonant energy transfer (FRET) [1] is the nonradiative transfer of energy from an excited donor. Fluorophore to a proximate acceptor fluorophore (or fluorophores) through the short-range dipole-dipole interaction that results in relaxation of the excited singlet state of the former and the promotion to the excited singlet state of the latter. Measurements of fluorescence in spectrofluorometers or imaging systems are widely used for FRET-based. Analysis of molecular interactions or changes in conformation of biosensors. Such molecular dynamics can be quantified through calculations of FRET efficiency and/or the FRETing population fraction. Detecting and/or quantifying biomolecular processes using FRET-based readouts is important for basic research and drug discovery, including for assays of protein-protein interactions

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