Quantifying intracellular equilibrium dissociation constants using single-channel time-resolved FRET

Gloria de Las Heras-Martínez, Josu Andrieu, Banafshé Larijani, Jose Requejo-Isidro

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7 Citations (SciVal)


Quantification of the intracellular equilibrium dissociation constant of the interaction, K d, is challenging due to the variability of the relative concentrations of the interacting proteins in the cell. Fluorescence lifetime imaging microscopy (FLIM) of the donor provides an accurate measurement of the molecular fraction of donor involved in FRET, but the fraction of bound acceptor is also needed to reliably estimate K d. We present a method that exploits the spectroscopic properties of the widely used eGFP – mCherry FRET pair to rigorously determine the intracellular K d based on imaging the fluorescence lifetime of only the donor (single-channel FLIM). We have assessed the effect of incomplete labelling and determined its range of application for different K d using Monte Carlo simulations. We have demonstrated this method estimating the intracellular K d for the homodimerisaton of the oncogenic protein 3-phosphoinositide-dependent kinase 1 (PDK1) in different cell lines and conditions, revealing a competitive mechanism for its regulation. The measured intracellular K d was validated against in-vitro data. This method provides an accurate and generic tool to quantify protein interactions in situ. (Figure presented.).

Original languageEnglish
Article numbere201600272
Number of pages16
JournalJournal of Biophotonics
Issue number1
Early online date9 May 2017
Publication statusPublished - 1 Jan 2018

Bibliographical note

© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


  • FLIM
  • FRET
  • K
  • equilibrium dissociation constant
  • intracellular
  • protein interactions

ASJC Scopus subject areas

  • General Chemistry
  • General Materials Science
  • General Biochemistry,Genetics and Molecular Biology
  • General Engineering
  • General Physics and Astronomy


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