Gegner J A, Dahlquist F W
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):750-4. doi: 10.1073/pnas.88.3.750.
An essential step in the signal transduction pathway of Escherichia coli is the control of the protein kinase activity of CheA by the chemotaxis receptor proteins. This control requires the participation of the CheW protein. Although the biochemical nature of the coupling between the receptors and the kinase is unknown, it is likely that CheW interacts with the receptors and with CheA. In this communication, we report direct measurement of a physical interaction between CheW and CheA. We utilized the equilibrium column chromatography method of Hummel and Dreyer to show that CheW and CheA exhibit reversible binding with the stoichiometry of two CheW monomers per CheA dimer. CheW was found to exist as monomers and CheA was found to exist as dimers by equilibrium analytical ultracentrifugation. The dissociation constant for the CheW-CheA interaction (in 160 mM KCl/5 mM MgCl2, pH 7.4 at 4 degrees C) was determined to be in the physiologically relevant range of 17 microM. No evidence for cooperativity in the association of CheW with CheA was found.
大肠杆菌信号转导途径中的一个关键步骤是趋化性受体蛋白对CheA蛋白激酶活性的控制。这种控制需要CheW蛋白的参与。尽管受体与激酶之间偶联的生化本质尚不清楚，但CheW可能与受体以及CheA相互作用。在本通讯中，我们报告了对CheW与CheA之间物理相互作用的直接测量。我们利用Hummel和Dreyer的平衡柱色谱法表明，CheW和CheA以每CheA二聚体两个CheW单体的化学计量比表现出可逆结合。通过平衡分析超速离心发现CheW以单体形式存在，CheA以二聚体形式存在。CheW-CheA相互作用的解离常数（在160 mM KCl/5 mM MgCl2、4℃pH 7.4条件下）经测定在17 microM的生理相关范围内。未发现CheW与CheA结合存在协同性的证据。
