Molecular Studies of Chemoreception

描述（由申请人提供）：介导大肠杆菌趋化性的受体是细菌化学受体大家族和原核和真核感觉受体大家族的最佳表征成员。该建议的重点是这些受体的感觉转导，重点是结构，信号传导和与其他感觉成分的相互作用，以及更高层次的组装。拟议的工作结合了生物化学，生物物理，遗传和结构的方法。几个方面涉及与著名生物药理学家的持续合作以及与分子建模师的互动合作。在过去的8年里，我们广泛使用半胱氨酸取代和巯基化学。这一战略将再次得到广泛利用。在目前的资助期间，我们获得的迹象表明，跨膜结构域的化学感受器是显着的动态和松散的包装。我们将通过一系列生物化学，生物物理和建模方法来研究这一意想不到的观察结果。化学感受器跨膜螺旋的疏水边界仅被模糊地定义，如大多数膜蛋白的情况。因为这个问题对结构和信号都有影响，我们将使用多种策略来追踪它。我们使用的诊断性交联半胱氨酸对之间的膜已被证明是显着的信息在检测化学感受器跨膜信号的构象变化。我们建议扩展和利用这种策略，以了解更多关于配体结合诱导的构象信号传导，并确定适应性修饰驱动的补偿性构象变化。最近，我们发现甲基酯酶与甲基转移酶在化学感受器的羧基端结合相同的相互作用位点。然而，这些相互作用增强适应酶活性的机制似乎完全不同。我们建议对此进行详细调查。E.大肠杆菌是不等价的。在当前的资助期内，我们通过表征“低丰度”受体Trg的作用模式来定义受体相互作用和合作的最佳记录实例。我们建议继续表征这种受体的结构和功能特征，并使用高丰度受体的低丰度受体的适应性援助的现象作为工具，用于调查诱人的概念，即受体集群，合作和相互作用是根本的趋化性机制。我们将确定趋化性成分的细胞剂量，以确定信号复合物化学计量的限制。我们的多方面，多学科的方法有望揭示新的信息的结构，相互作用和构象变化，跨膜蛋白使用的包裹，传输和整合感觉信息。

DESCRIPTION (provided by applicant): The receptors that mediate chemotaxis by Escherichia coli are the best-characterized members of a large family of bacterial chemoreceptors and of a larger family of prokaryotic and eukaryotic sensory receptors. This proposal focuses on sensory transduction by these receptors, with emphasis on structure, signaling and interactions with other sensory components and in higher order assemblies. The proposed work combines biochemical, biophysical, genetic and structural approaches. Several aspects involve continuing collaborations with prominent biophysicists and an interactive cooperation with a molecular modeler. Over the past 8 years, we have made extensive use of cysteine substitutions and sulfhydryl chemistry. This strategy will again be utilized extensively. In the current funding period, we obtained indications that the transmembrane domain of chemoreceptors was notably dynamic and loosely packed. We will investigate this unexpected observation by an array of biochemical, biophysical and modeling approaches. The hydrophobic boundaries of chemoreceptor transmembrane helices are only vaguely defined, as is the case for most membrane proteins. Because the issue has implications for both structure and signaling, we will purse it, using multiple strategies. Our use of diagnostic cross-linking between cysteine pairs in the membrane has proved notably informative in detecting the conformational change of chemoreceptor transmembrane signaling. We propose to extend and exploit this strategy to learn more about conformational signaling induced by ligand binding and to identify the compensatory conformational changes driven by adaptational modifications. Recently, we discovered that the methylesterase bound the same interaction site on the carboxyl-terminus of chemoreceptors as the methyltransferase. However, the mechanisms by which these interactions enhance activity of the adaptational enzymes appear quite different. We propose to investigate this in detail. Different chemoreceptors in E. coli are not equivalent. In the current funding period we defined the best-documented example of receptor interaction and cooperation by characterizing the mode of action of "low-abundance" receptor Trg. We propose to continue characterization of structural and functional features of this receptor, and to use the phenomenon of adaptational assistance of low-abundance receptors by high-abundance receptors as a tool for investigating the tantalizing notion that receptor clustering, cooperation and interaction are fundamental to the mechanisms of chemotaxis. We will determine cellular dosages of chemotaxis components to define the limits of signaling complex stoichiometries. Our multi-faceted, multi-disciplinary approach promises to reveal new information about the structures, interactions and conformational changes that transmembrane proteins use to transduce, transmit and integrate sensory information.