MOLECULAR STUDIES OF CHEMORECEPTION

介导大肠杆菌趋化性的化学感受器是 最好的表征成员的一个大家庭的细菌感官 受体。原核生物的受体在组织和功能上 具有某些真核生物激素受体的特征。该提案重点 通过化学感受器的感觉传导，特别强调 跨膜信号传导。拟议的工作是一个组合， 生物化学、遗传学和结构学方法， 项目，化学感受器Trg。广泛的位点特异性突变 创建了一个编码含半胱氨酸Trg蛋白的基因家族， 每个在两个中的52个位置之一处具有单个半胱氨酸， 受体的跨膜片段。这个家庭将用于（1） 确定跨膜结构域的详细组织，使用 氧化交联，巯基试剂的可及性， 评估半胱氨酰取代的功能后果，或 半胱氨酰残基的修饰，（2）开发用于 在体内和体外检测跨膜结构域中的构象变化， 体外并应用程序搜索与以下因素相关的变化： 跨膜信号传导，和（3）限定跨膜信号传导的边界。 域，并研究与信号相关的可能运动。 （2）和（3）的方面将涉及与韦恩哈贝尔博士的合作， 使用与巯基连接的电子顺磁共振探针 试剂合作者亚历山大·巴尔纳科夫博士现已加入 组，获得的二维晶体的Trg适合于结构 通过电子显微镜分析。这些阵列的优化和分析 是拟开展工作的一个重要重点。最苛刻的方面 该项目将在一流的设施进行结构 劳伦斯伯克利实验室的电子显微镜分析， Drs.罗伯特·格莱泽和肯尼斯·唐宁提供了设施， 专业知识该分析有可能提供大量的 关于完整受体的结构信息，特别是困难的 跨膜结构域政府亦会继续努力， 适用于X射线衍射的三维晶体。两个项目 将利用DNA操作和遗传分析来研究 化学感受器的分子内信号至关重要，特别是在 跨膜方面。融合蛋白将用于研究 对配对残基或片段的功能要求 跨膜结构域有效的筛选方法，包括 体内信号传导状态或融合质子，其中信号传导可以 通过生产β-半乳糖苷酶检测将用于追踪 信号通路的突变分析。最后一个项目研究 Trg的配体结合结构域通过表征与 多肽配体和分离的结构域的纯化， 结晶和随后的X射线衍射。后一个项目将 与Sung-Hou Kim博士合作 这种多方面、多学科的方法有望揭示新的 关于结构和构象变化的信息， 跨膜受体用于识别和传递感觉信息。

The chemoreceptors that mediate chemotaxis by Escherichia coli are the best characterized members of a large family of bacterial sensory receptors. The prokaryotic receptors share organizational and functional features with certain eukaryotic hormone receptors. This proposal focuses on sensory transduction through chemoreceptors, with particular emphasis on transmembrane signalling. The proposed work is a combination of biochemical, genetic and structural approaches, utilizing, in many projects, the chemoreceptor Trg. Extensive site-specific mutagenesis has created a family of genes coding for cysteine-containing Trg proteins, each with a single cysteine at one of 52 positions in the two transmembrane segments of the receptor. This family will be used to (1) determine the detailed organization of the transmembrane domain, using oxidative crosslinking, accessibility to sulfhydryl reagents and assessment of the functional consequences of cysteinyl substitutions or modification of the cysteinyl residues, (2) develop procedures for detecting conformational change in the transmembrane domain in vivo and in vitro and apply the procedures to search for changes associated with transmembrane signalling, and (3) define the borders of the transmembrane domain and investigate possible movements associated with signalling. Aspects of (2) and (3) will involve collaboration with Dr. Wayne Hubbell, using electron-paramagnetic-resonance probes linked to sulfhydryl reagents. Dr. Alexander Barnakov, a collaborator who has now joined the group, obtained two-dimensional crystals of Trg suitable for structural analysis by electron microscopy. Optimization and analysis of these arrays is an important emphasis of the proposed work. The most demanding aspects of this project will be performed at the superb facility for structural analysis by electron microscopy at the Lawrence Berkeley Laboratory, where Drs. Robert Glaeser and Kenneth Downing have offered facilities and expertise. The analysis has the potential of providing substantial structural information about intact receptors, particularly the difficult transmembrane domain. There will also be continuing efforts to obtain the three-dimensional crystals suitable for x-ray diffraction. Two projects will utilize DNA manipulation and genetic analysis to investigate features of chemoreceptors crucial for intramolecular signalling, particularly in its transmembrane aspects. Fusion proteins will be used investigate functional requirements for paired residues or segments in the transmembrane domain. Effective screening methods involving assays of signalling state in vivo or a fusion proton in which signalling can be detected by production of beta-galactosidase will be used to trace the signalling pathway by mutational analysis. A final project studies the ligand-binding domain of Trg by characterization of association with polypeptide ligands and purification of the isolated domain for crystallization and subsequent x-ray diffraction. The latter project will be in collaboration with Dr. Sung-Hou Kim. This multi-faceted, multi-disciplinary approach promises to reveal new information about the structures and conformational changes that transmembrane receptors use to transduce and transmit sensory information.