Estimating protein-protein interaction affinity in living cells using quantitative Förster resonance energy transfer measurements

Huanmian Chen; Henry L. Puhl III; Stephen R. Ikeda

doi:10.1117/1.2799171

1 September 2007 Estimating protein-protein interaction affinity in living cells using quantitative Förster resonance energy transfer measurements

Huanmian Chen, Henry L. Puhl III, Stephen R. Ikeda

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Journal of Biomedical Optics, Vol. 12, Issue 5, 054011 (September 2007). https://doi.org/10.1117/1.2799171

Abstract

We have previously demonstrated that Förster 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 (K_d) and the intrinsic FRET efficiency (E_max) of an interacting donor-acceptor pair. Assuming bimolecular interaction, the predicted FRET efficiency is a function of donor concentration, acceptor concentration, K_d, and E_max. We estimate K_d and E_max 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 K_d and E_max values. Applying an F-test, the 95% confidence contour of K_d and E_max 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 K_d 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.

©(2007) Society of Photo-Optical Instrumentation Engineers (SPIE)

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Huanmian Chen, Henry L. Puhl III, and Stephen R. Ikeda "Estimating protein-protein interaction affinity in living cells using quantitative Förster resonance energy transfer measurements," Journal of Biomedical Optics 12(5), 054011 (1 September 2007). https://doi.org/10.1117/1.2799171

Published: 1 September 2007

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