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

Yuriy Alexandrov*, Dino Solar Nikolic, Christopher Dunsby, Paul M.W. French

*Corresponding author for this work

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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.
Original languageEnglish
Article numbere201700366
JournalJournal of Biophotonics
Issue number7
Number of pages17
Publication statusPublished - 2018

Bibliographical note

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.


  • Förster resonant energy transfer (FRET)
  • Biosensors
  • Fluorescence
  • Fluorescent proteins
  • Molecular dynamics
  • Protein-protein interactions

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