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

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.