Molecular Studies of Chemoreception

描述(由申请人提供)： 在大肠杆菌中介导趋化作用的受体是细菌感觉受体大家族和更大的“双组分”受体家族的范例。这一建议侧重于化学感受器及其形成的信号复合体的作用机制和结构。这一重点与NIGMS基础研究的使命是一致的，该使命增加了对生命过程的理解，并为医学进步奠定了基础。研究将侧重于大肠杆菌化学受体，并利用生化、突变、生物物理、结构和建模方法。一些方法涉及与其他研究小组的合作，其中一些方法利用了插入纳米盘中的化学受体的优势，纳米盘是一种被蛋白质环包围的脂双层的水溶性塞子，对操纵受体是有效的。具体目标是解决了解化学感受器的核心问题。1)基本受体组织和功能单位的结构、同源二聚体和与构象信号有关的结构变化。受体的结构和由配体占据和/或适应性修饰产生的变化将使用定点自旋标记和电子顺磁共振光谱、高分辨率电子显微镜、X射线衍射、单分子Fürster共振能量转移(FRET)和分子模拟加上粗粒度分子动力学来表征。结果将确定这一关键单位的结构，并确定受体分子内和跨膜信号的基本机制。2)核心信号复合体的功能特性，它是激酶激活和控制的基本单位。将通过功能分析对使用纳米盘插入的受体分离的纯化的、激活激酶的核心复合体进行全面的表征。结果将定义核心单元在多大程度上执行信令复合体的每个功能。将探讨纯化的核心复合体中异源受体之间的功能相互作用，解决变构偶联的基本问题，并提供对信号整合和受体间相互作用的见解。3)核心信号复合体的结构特性。纯化的络合物将通过分析活性和保留组分来表征稳定性，通过分析性超速离心和高效尺寸排除层析来表征大小和均一性，并通过先进的电子显微镜和分子建模技术来表征三维结构。结果将提供有关核心信令单元结构的关键信息。4)适应性改造的非常规动力学。受体甲基化和去甲基化的非常规动力学将使用暂态动力学策略和高级拟合软件进行研究。结果将确定反应参数和机制，提供有关化学感受器感觉适应机制的基本信息。总体而言，这项拟议的研究将增加对跨膜受体信号传递的理解。这种信号存在于许多生命过程中，与人类健康和疾病有关。

DESCRIPTION (provided by applicant): Receptors that mediate chemotaxis in Escherichia coli are paradigms for the large family of bacterial sensory receptors and a larger family of "two-component" receptors. This proposal focuses on functional mechanisms and structures of chemoreceptors and the signaling complexes they form. This focus is consistent with the NIGMS mission of basic research that increases understanding of life processes and lays the foundation for medical advances. Studies will emphasize E. coli chemoreceptors and utilize biochemical, mutational, biophysical, structural and modeling approaches. Some approaches involve collaborative efforts with other research groups and several exploit advantages of chemoreceptors inserted in Nanodiscs, water-soluble plugs of lipid bilayer surrounded by a protein annulus, which are effective for manipulating receptors. Specific aims address issues central to understanding chemoreceptors. 1) Structure of the fundamental receptor organizational and functional unit, the homodimer and structural changes linked to conformational signaling. Receptor structure and changes generated by ligand occupancy and/or adaptational modification will be characterized using site-directed spin labeling and electron paramagnetic resonance spectroscopy, high- resolution electron microscopy, X-ray diffraction, single-molecule F¿rster resonance energy transfer (FRET) and molecular modeling plus coarse-grained molecular dynamics. Results will define the structure of this crucial unit and identify fundamental mechanisms of receptor intra-molecular and transmembrane signaling. 2) Functional properties of core signaling complexes, the fundamental unit of kinase activation and control. Comprehensive characterization by functional assays will be performed on purified, kinase-activating core complexes isolated using Nanodisc-inserted receptors. Results will define the extent to which each function of signaling complexes is performed by the core unit. Functional interactions between heterologous receptors in purified core complexes will be probed, addressing basic issues of allosteric coupling and providing insights into signal integration and inter-receptor interactions. 3) Structural properties of core signaling complexes. Purified complexes will be characterized for stability by assaying activity and retention of components, for size and homogeneity by analytical ultracentrifugation and high-performance, size-exclusion chromatography, and for three-dimensional organization by advanced techniques of electron microscopy and molecular modeling. Results will provide crucial information about the structure of core signaling units. 4) Non-conventional kinetics of adaptational modification. Non-conventional kinetics of receptor methylation and demethylation will be studied using strategies of transient-state kinetics and advanced fitting software. Results will identify reaction parameters and mechanisms, providing fundamental information about mechanisms of chemoreceptor sensory adaptation. Overall, the proposed research will increase understanding of signaling by transmembrane receptors. Such signaling, present in many life processes, is relevant to human health and disease.