Department of Bioengineering, Bourns College of Engineering, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA.
Biotechnol Bioeng. 2012 Nov;109(11):2875-83. doi: 10.1002/bit.24564. Epub 2012 Jun 18.
The dissociation constant, K(d) , is an important parameter for characterizing protein-protein interaction affinities. SUMOylation is one of the important protein post-translational modifications and it involves a multi-step enzymatic cascade reaction, resulting in peptide activation and substrate conjugation. Multiple covalent and non-covalent protein-protein interactions are involved in this cascade. Techniques involving Förster resonance energy transfer (FRET) have been widely used in biological studies in vitro and in vivo, and they are very powerful tools for elucidating protein interactions in many regulatory cascades. In our previous studies, we reported the attempt to develop a new method for the determination of the K(d) by FRET assay using the interaction of SUMO1 and its E2 ligase, Ubc9 as a test system. However, the generality and specifications of this new method have not been fully determined. Here we report a systematic approach for determining the dissociation constant (K(d) ) in the SUMOylation cascade and for further sensitivity and accuracy testing by the FRET technology. From a FRET donor to acceptor concentration ratio range of 4-40, the K(d) s of SUMO1 and Ubc9 consistently agree well with values from surface plasmon resonance and isothermal titration calorimetry. These results demonstrate the high sensitivity and accuracy of the FRET-based K(d) determination approach. This technology, therefore, can be used in general for protein-protein interaction dissociation constant determination.
离解常数（K(d)）是表征蛋白质-蛋白质相互作用亲和力的重要参数。SUMOylation 是一种重要的蛋白质翻译后修饰，它涉及多步酶级联反应，导致肽的激活和底物的连接。在这个级联反应中涉及多种共价和非共价蛋白质-蛋白质相互作用。涉及Förster 共振能量转移（FRET）的技术已广泛应用于体外和体内的生物学研究，是阐明许多调节级联中蛋白质相互作用的非常有力的工具。在我们之前的研究中，我们报告了尝试开发一种新的方法，通过使用 SUMO1 与其 E2 连接酶 Ubc9 的相互作用作为测试系统，通过 FRET 测定法来确定 K(d)。然而，这种新方法的通用性和规格尚未完全确定。在这里，我们报告了一种系统的方法，用于通过 FRET 技术确定 SUMOylation 级联中的离解常数（K(d)），并进一步进行灵敏度和准确性测试。从 FRET 供体到受体浓度比为 4-40 的范围，SUMO1 和 Ubc9 的 K(d) 值与表面等离子体共振和等温滴定量热法的值一致，非常吻合。这些结果表明基于 FRET 的 K(d)测定方法具有很高的灵敏度和准确性。因此，该技术通常可用于确定蛋白质-蛋白质相互作用的离解常数。
