Rademacher Nils, Kunde Stella-Amrei, Kalscheuer Vera M, Shoichet Sarah A
Neuroscience Research Center and Cluster of Excellence NeuroCure, Charité-Universitätsmedizin, 10117 Berlin, Germany.
Chem Biol. 2013 Aug 22;20(8):1044-54. doi: 10.1016/j.chembiol.2013.06.016.
To examine the scaffolding properties of PSD-95, we have taken advantage of established ligand/PDZ domain interactions and developed a cell-based assay for investigating protein complex formation. This assay enables quantitative analysis of PDZ domain-mediated protein clustering using bimolecular fluorescence complementation (BiFC). Two nonfluorescent halves of EYFP were fused to C-terminal PDZ ligand sequences to generate probes that sense for PDZ domain binding grooves of adjacent (interacting) molecules. When these probes are brought into proximity by the PDZ domains of a multiprotein scaffold, a functional fluorescent EYFP molecule can be detected. We have used this system to examine the properties of selected PSD-95 variants and thereby delineated regions of importance for PSD-95 complex formation. Further analysis led to the finding that PSD-95 multimerization is PDZ domain-mediated and promoted by ligand binding.
为了研究PSD-95的支架特性，我们利用了已建立的配体/PDZ结构域相互作用，并开发了一种基于细胞的检测方法来研究蛋白质复合物的形成。该检测方法能够使用双分子荧光互补（BiFC）对PDZ结构域介导的蛋白质聚集进行定量分析。将EYFP的两个无荧光的半部分与C端PDZ配体序列融合，以生成能够检测相邻（相互作用）分子的PDZ结构域结合凹槽的探针。当这些探针通过多蛋白支架的PDZ结构域靠近时，就可以检测到功能性的荧光EYFP分子。我们使用这个系统来检测选定的PSD-95变体的特性，从而确定了对PSD-95复合物形成重要的区域。进一步的分析发现，PSD-95多聚化是由PDZ结构域介导的，并由配体结合促进。
