STRUCTURE/FUNCTION OF RESPONSE REGULATOR PROTEINS

描述：所有细胞中的刺激-反应偶联都涉及信号传导。 将信息从受体传递到靶点的转导途径 影响最终反应的分子。 在细菌中， 调节系统利用保守的磷酸转移信号策略 包括两个保守的蛋白质组分，一个组氨酸蛋白激酶和一个 响应调节器 反应调节蛋白通常由以下组成： 两个结构域，一个保守的N-末端调节结构域和一个可变的 C-末端效应域。 调节域催化 磷酰基从组氨酸蛋白激酶转移到自身 作为一个磷酸化激活的开关来控制的活动， 相关的效应域。 结构和功能表征 这些蛋白质的分子基础对于理解 信号转导 更具体地，这样的信息可以帮助当前的 制药公司努力开发抗微生物剂， 这些蛋白质。 近年来，许多方面的结构和 保守的调节结构域的生物化学活性已经被 阐明。 然而，短寿命的磷酸化状态的 这些结构域阻碍了磷酸化如何改变 调节结构域的构象以及这些构象变化 导致效应子结构域活性的激活。 拟议 研究的重点是使用多域解决这些问题 反应调节因子切布、OmpR和DrrA作为模型蛋白。 有四 具体目标： 1. 反应激活机制的确定 监管部门 磷酸化蛋白质的不可水解类似物将是 通过修饰独特的半胱氨酸残基构建。 的影响 这些对分子内和分子间相互作用的修饰将 使用活性测定、有限蛋白水解、荧光 测量和最终的X射线晶体学来确定 三维结构。 2. OmpR转录家族对DNA结合的表征 因素 转录因子OmpR和OmpR的DNA结合活性 DrrA，最大缓解亚家族的代表性成员 调节剂，将表征和这些蛋白质的晶体结构 将被追踪。 3. 反应调节剂与 辅助蛋白 更多的结构研究将集中在 反应调节剂与辅助蛋白的相互作用， 确定相似的分子表面是否用于蛋白质-蛋白质 不同反应调节器的相互作用。 4. 组氨酸蛋白激酶的结构分析。 的x射线晶体 热稳定组氨酸蛋白质胞质区的结构 激酶、T. Maritima HpkA将被追踪。

DESCRIPTION: Stimulus-response coupling in all cells involves signal transduction pathways that carry information from receptors to the target molecules that effect the final responses. In bacteria, a large number of regulatory systems utilize a conserved phosphotransfer signaling strategy involving two conserved protein components, a histidine protein kinase and a response regulator. Response regulator proteins are typically composed of two domains, a conserved N-terminal regulatory domain and a variable C-terminal effector domain. The regulatory domain which catalyzes the transfer of phosphoryl groups to itself from the histidine protein kinase functions as a phosphorylation-activated switch to control the activity of the associated effector domain. Structural and functional characterization of these proteins is important for understanding the molecular basis of signal transduction. More specifically, such information may aid current pharmaceutical efforts to develop anti-microbial agents targeted against these proteins. During recent years, many aspects of the structure and biochemical activities of the conserved regulatory domain have been elucidated. However, the short lifetimes of the phosphorylated states of these domains has hindered investigation of how phosphorylation alters the conformation of the regulatory domain and how these conformational changes lead to activation of the activity of the effector domain. The proposed research focuses on addressing these questions using the muli-domain response regulators CheB, OmpR and DrrA as model proteins. There are four Specific Aims: 1. Determination of the mechanism of activation of response regulators. Non-hydrolyzable analogs of the phosphorylated proteins will be constructed by modification of unique cysteine residues. The effects of these modifications on intra- and intermolecular interactions will be characterized using activity assays, limited proteolysis, fluorescence measurements and ultimately X-ray crystallography to determine the three-dimensional structures. 2. Characterization of DNA binding by the OmpR family of transcription factors. The DNA-binding activities of the transcription factors OmpR and DrrA, representative members of the largest subfamily of response regulators, will be characterized and crystal structures of these proteins bound to DNA will be pursued. 3. Structural characterization of response regulator interactions with auxiliary proteins. Additional structural studies will focus on the interactions of response regulators with auxiliary proteins with the goal of determining whether similar molecular surfaces are used for protein-protein interactions in different response regulators. 4. Structural analysis of a histidine protein kinase. The X-ray crystal structure of the cytoplasmic region of a thermostable histidine protein kinase, T. maritima HpkA, will be pursued.