Department of Bioengineering, University of California at Riverside, 900 University Avenue, Riverside, CA, 92521, USA.
Biochemistry and Molecular Biology Department, Heilongjiang University of Chinese Medicine, 24 Heping Road, Harbin, 150040, P.R. China.
Sci Rep. 2019 Feb 14;9(1):2050. doi: 10.1038/s41598-018-35535-9.
The molecular dissociation constant, K, 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 determination using a quantitative FRET method. Here we report a new development of K determination by measuring the reduction in donor fluorescence due to acceptor quenching in FRET. A new method of K determination was developed from the quantitative measurement of donor fluorescence quenching. The estimated K 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.
分子离解常数（K）是定量蛋白质-蛋白质或其他分子相互作用亲和力的一个既定参数。最近，我们报道了使用定量 FRET 方法测定 K 的理论基础和实验程序。在这里，我们报告了通过测量由于 FRET 中供体荧光猝灭而导致供体荧光减少来确定 K 的新进展。通过定量测量供体荧光猝灭，开发了一种新的 K 测定方法。基于该方法，SUMO1-Ubc9 相互作用的估计 K 值与其他技术（包括 FRET 受体发射）确定的 K 值非常吻合。因此，受体猝灭法可以作为先前开发的受体激发法的补充。无论受体是可激发荧光团还是猝灭剂，新方法都具有更广泛的应用。因此，这些发展为溶液中蛋白质或其他分子相互作用亲和力的测定提供了完整的方法。
