Molecular Mechanisms of Signaling in E. coli Chemotaxis

大肠杆菌趋化性信号转导的分子机制

• 批准号: 7151918
• 负责人: Robert B. Bourret
• 金额: $ 32.41万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7151918
• 关键词: Active Sites; Address; Amino Acid Substitution; Bacteria; Binding; Biochemical; Biochemical Genetics; Biological; Biological Assay; Bypass; Cells; Characteristics; Chemotaxis; Class; Complex; Condition; Coupling; Engineering; Environment; Equilibrium; Escherichia coli; Event; Exhibits; Family; Foundations; Half-Life; Hand; Helix (Snails); Hour; In Vitro; Intention; Ionic Strengths; Laboratories; Measures; Metals; Molecular; Molecular Conformation; Monitor; N-terminal; Output; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Process; Protein Dephosphorylation; Proteins; Purpose; Range; Rate; Reaction; Regulation; Research; Research Personnel; Resolution; Role; Screening procedure; Series; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Molecule; Signaling Protein; Site; Specificity; Structure; Substrate Specificity; Surface; System; Testing; Therapeutic Agents; Time; Virulence; Virulence Factors; X-Ray Crystallography; acetyl phosphate; base; design; ear helix; gain of function; gain of function mutation; in vivo; insight; interest; microbial; mutant; pathogen; phosphoramidate; programs; research study; response; sensor; small molecule

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.

描述(由申请人提供)：细胞使用信号转导途径来监测它们的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跃和不活跃的形式之间循环。例如，在许多系统中，瞬时蛋白质磷酸化被用作外部条件的动态内部表示。因此，对蛋白质间磷酸基转移的机制和调节以及磷酸化对蛋白质活性的影响的基本了解具有广泛的意义。这一应用背后的长期目标是定义细菌中信号转导的分子机制。 这一建议利用了一条特别为人所熟知的信号通路，这是一种由两个成分组成的调控系统，负责管理大肠杆菌的趋化作用。Chey和Cheb反应调节子通过小分子或CHEA传感器激酶的磷酸基的自催化转移而激活，并通过自催化的去磷酸化而失活。Chey-P还在CHEZ蛋白的帮助下释放磷酸基。这项建议的目的是全面了解在趋化信号转导过程中发生的磷酸基交易，并进一步澄清哪些特征通常适用于其他双组分调节系统。为此，具体目标1和2探索决定由具有代表性的反应调节器催化的反应的速率和特异性的因素，包括Chey和Cheb。具体目标3和4建立在从最近确定的CHEY/CHEZ复合体结构中获得的信息基础上，以阐明CHEZ功能的许多方面。 计划采用一种综合的生化、遗传和物理方法。各种现有的和新的生化分析方法将用于表征野生型和突变型信号蛋白的体外反应。许多信息丰富的CHEY和CHEZ突变体已经在手中，并描述了分离更多突变体的筛选策略。在适当的情况下，完整的结构将由X射线结晶学确定。 调控系统与趋化作用高度相似，但远不为人所知，它控制着许多细菌病原体对毒力因子的表达。这项拟议的研究将导致对双组分调控系统的详细机制理解，这可能与设计干扰微生物毒力信号通路的新型治疗剂有关。还预期了适用于其他生物信号系统的基本见解。