title: Estimating protein-protein interaction affinity in living cells using quantitative Förster resonance energy transfer measurements.
abstract: We have previously demonstrated that Forster resonance energy transfer (FRET) efficiency and the relative concentration of donor and acceptor fluorophores can be determined in living cells using three-cube wide-field fluorescence microscopy. Here, we extend the methodology to estimate the effective equilibrium dissociation constant (Kd) and the intrinsic FRET efficiency (Emax) of an interacting donor-acceptor pair. Assuming bimolecular interaction, the predicted FRET efficiency is a function of donor concentration, acceptor concentration, Kd, and Emax. We estimate Kd and Emax by minimizing the sum of the squared error (SSE) between the predicted and measured FRET efficiency. This is accomplished by examining the topology of SSE values for a matrix of hypothetical Kd and Emax values. Applying an F-test, the 95% confidence contour of Kd and Emax is calculated. We test the method by expressing an inducible FRET fusion pair consisting of FKBP12-Cerulean and Frb-Venus in HeLa cells. As the Kd for FKBP12-rapamycin and Frb has been analytically determined, the relative Kd (in fluorescence units) could be calibrated with a value based on protein concentration. The described methodology should be useful for comparing protein-protein interaction affinities in living cells.
authors.affiliation: National Institutes of Health, National Institute on Alcohol Abuse and Alcoholism, Laboratory of Molecular Physiology, Section on Transmitter Signaling, Bethesda, Maryland 20892-9411, USA.
authors.forename: Huanmian
authors.initials: H
authors.lastname: Chen
authors.forename: Henry L
authors.initials: HL
authors.lastname: Puhl
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Ikeda
mesh_terms: D000465:Algorithms; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009363:Neoplasm Proteins / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*
chemical_list: D009363:Neoplasm Proteins