Molecular Mechanisms of Signaling in E. coli Chemotaxis

描述（由申请人提供）：细胞使用信号转导途径监测其环境并对变化做出适当的反应。这个过程的一个中心特征是信号分子在活性和非活性形式之间的循环。例如，在许多系统中，瞬态蛋白质磷酸化被用作外部条件的动态内部表示。因此，对磷酸化在蛋白质之间转移的机制和调控以及磷酸化对蛋白质活性的影响的基本理解是广泛关注的。这项应用背后的长期目标是确定细菌信号转导的分子机制。

DESCRIPTION (provided by applicant): Cells use signal transduction pathways to monitor their environment and implement appropriate responses to change. A central feature of this process is the cycling of signal molecules between active and inactive forms. For example, transient protein phosphorylation is used as a dynamic internal representation of external conditions in many systems. A fundamental understanding of the mechanisms and regulation of phosphoryl group transfer among proteins, as well as the impact of phosphorylation on protein activity, is thus of broad interest. The long term objective behind this application is to define the molecular mechanisms of signal transduction in bacteria. This proposal takes advantage of a particularly well understood signaling pathway, the two-component regulatory system that governs chemotaxis by Escherichia coli. The CheY and CheB response regulators are activated by self-catalyzed transfer of phosphoryl groups from either small molecules or the CheA sensor kinase, and inactivated by self-catalyzed dephosphorylation. CheY-P also releases phosphoryl groups with the assistance of the CheZ protein. The purpose of this proposal is to develop a comprehensive understanding of the phosphoryl group transactions that occur during chemotactic signal transduction, with the additional intention of clarifying which features are generally applicable to other two-component regulatory systems. Towards that end, Specific Aims 1 and 2 explore the factors that determine the rates and specificity of the reactions catalyzed by representative response regulators, including CheY and CheB. Specific Aims 3 and 4 build on information derived from the recently determined structure of a CheY/CheZ complex to clarify many aspects of CheZ function. An integrated biochemical, genetic, and physical approach is planned. A variety of both established and new biochemical assays will be used to characterize the reactions of wildtype and mutant signaling proteins in vitro. Numerous informative CheY and CheZ mutants are already in hand, and screening strategies to isolate more are described. In appropriate cases, complete structures will be determined by X-ray crystallography. Regulatory systems highly analogous to chemotaxis but far less well understood control expression of virulence factors by many bacterial pathogens. The detailed mechanistic understanding of two-component regulatory systems that will result from the proposed research may be relevant to designing new classes of therapeutic agents that interfere with microbial virulence signaling pathways. Fundamental insights applicable to other biological signaling systems are also anticipated.