Abstract
The molecular dissociation constant, Kd, 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 Kd determination using a quantitative FRET method. Here we report a new development of Kd determination by measuring the reduction in donor fluorescence due to acceptor quenching in FRET. A new method of Kd determination was developed from the quantitative measurement of donor fluorescence quenching. The estimated Kd 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.
Highlights
The same concentrations of separate CFP-SUMO1 or YFP-Ubc[9] proteins to derive the FRET emission from YFP-Ubc[96]
CyPet and YPet are fluorescent proteins engineered from CFP and YFP, respectively, with 20-fold greater ratiometric FRET signal than their parental FRET pair[12]
The interaction affinity Kd was determined from the FRET acceptor emission signal at 530 nm after eliminations of direct emissions of CyPet-SUMO1 and YPet-Ubc[97]
Summary
The same concentrations of separate CFP-SUMO1 or YFP-Ubc[9] proteins to derive the FRET emission from YFP-Ubc[96]. The FRET emission intensity was fitted with YFP-Ubc[9] concentration to obtain the maximum FRET emission intensity, which is correlated with maximum bindings of two proteins. A similar strategy was applied to Sentrin/SUMO-specific proteases 1 (SENP1) for its kcat/KM determinations[8]. The results from these quantitative FRET analyses are comparable to or more accurate than traditional biophysical or biochemical approaches, such as the surface plasmon resonance (SPR) or Western blot for estimating binding affinity constants. The estimated KdKd of SUMO1-Ubc[9] interaction is in good agreement in general with those determined by the acceptor emission approach and the surface plasmon resonance (SPR). Our method has broader applications regardless whether the acceptor is an emitting fluorophore or a quencher
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