Sourjik Victor, Vaknin Ady, Shimizu Thomas S, Berg Howard C
ZMBH (Center for Molecular Biology Heidelberg), University of Heidelberg, Heidelberg, Germany.
Methods Enzymol. 2007;423:365-91. doi: 10.1016/S0076-6879(07)23017-4.
The two-component pathway in Escherichia coli chemotaxis has become a paradigm for bacterial signal processing. Genetics and biochemistry of the pathway as well as physiological responses have been studied in detail. Despite its relative simplicity, the chemotaxis pathway is renowned for its ability to amplify and integrate weak signals and for its robustness against various kinds of perturbations. All this information inspired multiple attempts at mathematical analysis and computer modeling, but a quantitative understanding of the pathway was hampered by our inability to follow the signal processing in vivo. To address this problem, we developed assays based on fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) that enabled us to monitor activity-dependent protein interactions in real time directly in living cells. Here, we describe quantitative applications of these assays in cell populations and on a single-cell level to study the interaction of the phosphorylated response regulator CheY with its phosphatase CheZ. Since this interaction defines the rate of CheY dephosphorylation, which at steady state equals the rate of CheY phosphorylation, it can be used to characterize intracellular kinase activity and thus to analyze properties of the chemotaxis signaling network.
大肠杆菌趋化作用中的双组分信号转导途径已成为细菌信号处理的范例。人们已对该途径的遗传学、生物化学以及生理反应进行了详细研究。尽管该趋化途径相对简单，但其以能够放大和整合微弱信号以及对各种干扰具有稳健性而闻名。所有这些信息激发了人们多次进行数学分析和计算机建模的尝试，但由于我们无法在体内追踪信号处理过程，对该途径的定量理解受到了阻碍。为了解决这个问题，我们开发了基于荧光共振能量转移（FRET）和生物发光共振能量转移（BRET）的检测方法，使我们能够直接在活细胞中实时监测依赖活性的蛋白质相互作用。在这里，我们描述了这些检测方法在细胞群体和单细胞水平上的定量应用，以研究磷酸化反应调节蛋白CheY与其磷酸酶CheZ之间的相互作用。由于这种相互作用决定了CheY去磷酸化的速率，而在稳态下该速率等于CheY磷酸化的速率，因此它可用于表征细胞内激酶活性，从而分析趋化信号网络的特性。
