title: A method to quantify FRET stoichiometry with phasor plot analysis and acceptor lifetime ingrowth.
abstract: FRET is widely used for the study of protein-protein interactions in biological samples. However, it is difficult to quantify both the FRET efficiency (E) and the affinity (Kd) of the molecular interaction from intermolecular FRET signals in samples of unknown stoichiometry. Here, we present a method for the simultaneous quantification of the complete set of interaction parameters, including fractions of bound donors and acceptors, local protein concentrations, and dissociation constants, in each image pixel. The method makes use of fluorescence lifetime information from both donor and acceptor molecules and takes advantage of the linear properties of the phasor plot approach. We demonstrate the capability of our method in vitro in a microfluidic device and also in cells, via the determination of the binding affinity between tagged versions of glutathione and glutathione S-transferase, and via the determination of competitor concentration. The potential of the method is explored with simulations.
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: WeiYue
authors.initials: W
authors.lastname: Chen
authors.affiliation: The Wellcome Trust Medical Research Council Institute of Metabolic Science and National Institute for Health Research, Cambridge Biomedical Research Centre, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Edward
authors.initials: E
authors.lastname: Avezov
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Simon C
authors.initials: SC
authors.lastname: Schlachter
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Gielen
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Romain F
authors.initials: RF
authors.lastname: Laine
authors.affiliation: The Wellcome Trust Medical Research Council Institute of Metabolic Science and National Institute for Health Research, Cambridge Biomedical Research Centre, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Heather P
authors.initials: HP
authors.lastname: Harding
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Florian
authors.initials: F
authors.lastname: Hollfelder
authors.affiliation: The Wellcome Trust Medical Research Council Institute of Metabolic Science and National Institute for Health Research, Cambridge Biomedical Research Centre, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Ron
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, United Kingdom. Electronic address: cfk23@cam.ac.uk.
authors.forename: Clemens F
authors.initials: CF
authors.lastname: Kaminski
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D044085:Microfluidics / Q000379:methods*
chemical_list: D008164:Luminescent Proteins