Protein-Protein Affinity Determination by Quantitative FRET Quenching

Abstract

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.

Figure 1

Schematic of the principle of donor quenching for protein interaction affinity determination by FRET assay. (A) Diagram of fluorescence emissions of FRET pair, CyPet and YPet, when tagged with interactive partners, SUMO1 and Ubc9, respectively. The fluorescence emission of donor CyPet-SUMO1 decreases when it binds acceptor Ypet-Ubc9. (B) Quantitative analysis of fluorescence signals. The decrease of CyPet emission is proportional the concentration of bound donor/acceptor complex.

Figure 2

Fluorescence emission from the mixture of CyPet-SUMO1 and YPet-Ubc9. (A) Spectrum changes of the mixture of CyPet-SUMO1 and YPet-Ubc9 proteins at increasing amount of acceptor when excited at 414 nm. The CyPet-SUMO1 concentration is fixed at 1.5 μM, and YPet-Ubc9 concentrations range from 0 to 4 μM. (B) Emission intensity of CyPet-SUMO1 at 475 nm (Ex = 414 nm) decreases at different concentrations of CyPet-SUMO1 with increasing YPet-Ubc9 concentrations. ● 0.5 μM of CyPet-SUMO1, ■ 1 μM of CyPet-SUMO1, and ▲ 1.5μM of CyPet-SUMO1. (C) The emissions of CyPet and YPet when excited at 441 nm.

Figure 3

Curve fitting for K_d determination. (A) Decreased emission of Em₄₇₅ was calculated by subtracting the emission intensity of CyPet-SUMO1 in the presence of YPet-Ubc9 from the emission in the absence of YPet-Ubc9. The value of decrease was then fitted with total YPet-Ubc9 concentration to derive the value of K_d according the formula Y = n/2 (A + X + K_d − √((A + X + K_d)^2−4AX)) (see text for details). (B) Linearity of maximum quenching signal of CyPet-SUMO1 by Ypet-Ubc9 at different concentrations was determined.

Figure 4

Comparison of K_d values obtained from two approaches by the quenched donor emission or acceptor emission. (A) K_d values determined by either FRET quenching or emission at different concentrations of CyPet-SUMO1 were compared. (B) K_d values after global optimization of either FRET quenching or emission datasets. (C) Bootstrap analysis of both K_d values revealed no significant difference.