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Protein-Protein Affinity Determination by Quantitative FRET Quenching.

  • Author(s): Jiang, Ling
  • Xiong, Zhehao
  • Song, Yang
  • Lu, Yanrong
  • Chen, Younan
  • Schultz, Jerome S
  • Li, Jun
  • Liao, Jiayu
  • et al.
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.

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