Pusch Stefan, Dissmeyer Nico, Schnittger Arp
German Cancer Research Center (DKFZ), Ruprecht-Karls Universität Heidelberg, Heidelberg, Germany.
Methods Mol Biol. 2011;779:245-57. doi: 10.1007/978-1-61779-264-9_14.
Enzyme-substrate interactions are weak and occur only transiently and thus, a faithful analysis of these interactions typically requires elaborated biochemical methodology. The bimolecular-fluorescence complementation (BiFC) assay, also referred to as split YFP assay, is a powerful and straightforward tool to test protein-protein interactions. This system is commonly used due to many advantages and especially due to its simple ease of use. BIFC relies on the reconstitution of an N-terminal and C-terminal half of YFP into a functional, i.e., fluorescent protein. Noteworthy, the dissociation constant of the two YFP halves is much lower than the association constant leading to a stabilization of the protein-protein interaction to be monitored. Whereas this property is sometimes critical, it also increases the sensitivity of the detection system by stabilizing transient interactions. Here, we exploit this property to detect and monitor interaction between a kinase and its substrate. In particular, we characterize with the BiFC system kinase-variants that show an altered substrate binding.
酶与底物的相互作用很弱，只是短暂发生，因此，要对这些相互作用进行可靠的分析通常需要复杂的生化方法。双分子荧光互补（BiFC）分析，也称为分裂黄色荧光蛋白（YFP）分析，是一种用于检测蛋白质-蛋白质相互作用的强大而直接的工具。由于具有许多优点，特别是使用简单方便，该系统被广泛应用。BiFC依赖于将YFP的N端和C端半段重新组装成一个功能性的，即荧光蛋白。值得注意的是，两个YFP半段的解离常数远低于缔合常数，从而使待监测的蛋白质-蛋白质相互作用得以稳定。虽然这一特性有时至关重要，但它也通过稳定瞬时相互作用提高了检测系统的灵敏度。在这里，我们利用这一特性来检测和监测激酶与其底物之间的相互作用。特别是，我们用BiFC系统对显示底物结合改变的激酶变体进行了表征。
