Molecular Studies of Chemoreception

描述(申请人提供)：介导大肠杆菌趋化作用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中特征最好的成员。这一建议侧重于这些受体的感觉转导，重点放在结构、信号以及与其他感觉组件和更高阶组装的相互作用上。拟议的工作结合了生化、生物物理、遗传和结构方法。几个方面包括与著名生物物理学家的持续合作，以及与分子模型师的互动合作。在过去的8年里，我们广泛地使用了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，我们获得的迹象表明，化学感受器的跨膜区是显著动态和松散包装的。我们将通过一系列生化、生物物理和建模方法来调查这种意想不到的观察结果。化学感受器跨膜螺旋的疏水边界只有模糊的定义，就像大多数膜蛋白一样。由于这个问题对结构和信号都有影响，我们将使用多种策略来解决它。我们使用膜上半胱氨酸对之间的诊断交联，在检测化学感受器跨膜信号的构象变化方面被证明是显着的信息。我们建议扩展和利用这一策略，以了解更多关于配体结合诱导的构象信号，并识别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受体的羧基末端结合了相同的作用部位。然而，这些相互作用增强适应性酶活性的机制似乎截然不同。我们建议对此进行详细调查。不同的化学感受器在大肠杆菌中是不相同的。在目前的资助期间，我们通过描述“低丰度”受体Trg的作用模式，定义了受体相互作用和合作的最好的记录例子。我们建议继续对该受体的结构和功能特征进行表征，并利用高丰度受体对低丰度受体的适应性辅助现象作为研究一个诱人的概念的工具，即受体聚集、合作和相互作用是趋化机制的基础。我们将确定趋化成分的细胞剂量，以确定信号复杂化学计量学的限制。我们的多方面、多学科的方法有望揭示跨膜蛋白用来传递、传递和整合感觉信息的结构、相互作用和构象变化的新信息。

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.