Protein–Protein Affinity Determination by Quantitative FRET Quenching

The molecular dissociation constant, K_d, is a well-established parameter to quantitate the affinity of protein-protein or other molecular interactions. Recently, we reported the theoretical basis and experimental procedure for K_d determination using a quantitative FRET method. Here we report a new development of K_d determination by measuring the reduction in donor fluorescence due to acceptor quenching in FRET. A new method of K_d determination was developed from the quantitative measurement of donor fluorescence quenching. The estimated K_d values of SUMO1-Ubc9 interaction based on this method are in good agreement with those determined by other technologies, including FRET acceptor emission. Thus, the acceptor-quenched approach can be used as a complement to the previously developed acceptor excitation method. The new methodology has more general applications regardless whether the acceptor is an excitable fluorophore or a quencher. Thus, these developments provide a complete methodology for protein or other molecule interaction affinity determinations in solution.