BACTERIAL CHEMOTAXIS PROBED BY PHOTORELASED LIGANDS

通过光致激光配体探测细菌趋化性

• 批准号: 6043568
• 负责人: SHAHID M KHAN
• 金额: $ 16.56万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-02-01 至 2001-01-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6043568
• 关键词: Escherichia coli; chemoattractants; chemotaxis; cilium /flagellum motility; gene mutation; green fluorescent proteins; leucine; phosphorylation; photolysis; receptor binding; receptor expression; sensory signal detection; serine

Escherichia coli chemotaxis has emerged as a paradigm for the elucidation of the computational strategies employed by intracellular signaling phosphorelays to respond to environmental changes. The chemotactic phosphorelay is part of a large famly of histidyl-aspartyl phosphorelays that mediate diverse sensory transduction schemes in prokaryotes and eukaryotes. The chemotactic response, in common with other sensory systems, exhibits high sensitivity over a large chemoeffector concentration range. This sensitivity may be achieved by utilizing dynamic, multicomponent receptor assemblies to generate and process chemotactic signals. The flash photorelease assay, based on computerized motion analysis of responses to photochemically generated chemoeffector concentration jumps, has been developed and established as a time-resolved method for quantitative analysis of chemotactic signal processing. Sensitivity modulation of chemoattractant (aspartate and glucose) responses by stimulus strength and concentration range has been measured. The data sets constraints to possible signal amplification mechanisms. Planned work will build on these advances. Excitation responses of negative stimuli (i.e. leucine) will be determined, as a prelude to competition assays between positive and negative stimuli, designed to probe for receptor-receptor interactions. Expression of receptor signaling domains restores normal motility, but not chemotaxis, in mutant strains; allowing evaluation of the mutations on signaling properties. Overexpression of the central signaling phosphoprotein, CheY, affects signaling kinetics when it is expressed alone but not with CheZ, a catalyst for CheY dephosphorylation. Green fluorescent protein (GFP) - CheY chimeras have been constructed to visualize dynamics of CheY interactions with receptor and motor assemblies by low-light level fluorescent microscopy techniques; to relate in vivo to in vitro binding parameters, and to time-resolve their modulation during signaling triggered by photoreleased chemoeffectors. The knowledge gained will contribute to fundamental understanding of cellular response/defense mechanisms and aid diagnosis of aberrations.

大肠杆菌趋化性已成为阐明的范式 细胞内信号传导磷光所采用的计算策略 响应环境变化。趋化性磷层是A的一部分 大量的组酰基 - 天冬氨酸磷介导，可介导多种感觉 原核生物和真核生物中的转导计划。趋化反应， 与其他感官系统共同，在大型上表现出高灵敏度 Chemoefector浓度范围。这种敏感性可以通过 利用动态的多组分受体组件来生成和处理 趋化信号。基于计算机运动的Flash Photorelease分析 分析对光化学产生的化学效果浓度的响应 跳跃，已开发并建立为一种时间分辨的方法 趋化信号处理的定量分析。灵敏度调制 通过刺激强度和 浓度范围已经测量。数据集约束可能 信号扩增机制。计划的工作将基于这些进步。 负面刺激的激发反应（即亮氨酸）将被确定为 设计正面刺激和负面刺激之间的竞争测定的前奏 探测受体受体相互作用。受体信号的表达 域在突变菌株中恢复正常运动，但不能恢复趋化性。 允许评估信号传导性能的突变。过表达 中心信号磷蛋白Chey会影响信号动力学 单独表达，但不用Chez表示，Chez是Chey Dephospylation的催化剂。 绿色荧光蛋白（GFP） - 已构建Chey Chimeras 通过可视化Chey与受体和运动组件的动力学 低光水平荧光显微镜技术；在体内关联 体外结合参数，并在信号传导期间定时溶解其调制 由Photoreared Chemoefector触发。获得的知识将有助于 对细胞反应/防御机制和援助的基本了解 诊断畸变。