Kentner David, Sourjik Victor
Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Heidelberg, Germany.
Mol Syst Biol. 2009;5:238. doi: 10.1038/msb.2008.77. Epub 2009 Jan 20.
Protein-protein interactions play key roles in virtually all cellular processes, often forming complex regulatory networks. A powerful tool to study interactions in vivo is fluorescence resonance energy transfer (FRET), which is based on the distance-dependent energy transfer from an excited donor to an acceptor fluorophore. Here, we used FRET to systematically map all protein interactions in the chemotaxis signaling pathway in Escherichia coli, one of the most studied models of signal transduction, and to determine stimulation-induced changes in the pathway. Our FRET analysis identified 19 positive FRET pairs out of the 28 possible protein combinations, with 9 pairs being responsive to chemotactic stimulation. Six stimulation-dependent and five stimulation-independent interactions were direct, whereas other interactions were apparently mediated by scaffolding proteins. Characterization of stimulation-induced responses revealed an additional regulation through activity dependence of interactions involving the adaptation enzyme CheB, and showed complex rearrangement of chemosensory receptors. Our study illustrates how FRET can be efficiently employed to study dynamic protein networks in vivo.
蛋白质-蛋白质相互作用在几乎所有细胞过程中都起着关键作用，常常形成复杂的调控网络。一种用于研究体内相互作用的强大工具是荧光共振能量转移（FRET），它基于从激发供体到受体荧光团的距离依赖性能量转移。在这里，我们使用FRET系统地绘制了大肠杆菌趋化信号通路中的所有蛋白质相互作用，大肠杆菌是信号转导研究最多的模型之一，并确定了该通路中刺激诱导的变化。我们的FRET分析在28种可能的蛋白质组合中鉴定出19对正向FRET对，其中9对响应趋化刺激。6种刺激依赖性和5种刺激非依赖性相互作用是直接的，而其他相互作用显然是由支架蛋白介导的。对刺激诱导反应的表征揭示了通过涉及适应酶CheB的相互作用的活性依赖性进行的额外调控，并显示了化学感受受体的复杂重排。我们的研究说明了如何有效地利用FRET来研究体内动态蛋白质网络。
