MOLECULAR STUDIES OF CHEMORECEPTION

活细胞的一个基本特征是能够对特定的， 外部化学信号 就像真核细胞 激素或神经递质，细菌含有特定的 受体，暴露在细胞表面，识别相关的 化合物. 这些细胞是趋化性的结果， 感觉反应系统的功能，将受体连接到 鞭毛 该实验室的长期目标是促进 一个详细的分子生物学描述的趋化 系统 一类重要的跨膜受体 含有两个亲水结构域， 疏水的跨膜序列。 受体 胰岛素、表皮生长因子和细菌化学引诱物 都是这个组织的重要成员 这些受体蛋白 含有多个共价修饰位点， 感觉适应或“脱敏”。 了解 任何这些受体的功能机制都需要 关于配体如何结合或 蛋白质上一个位置的共价修饰可以影响蛋白质的 活动的一个遥远的领域，如何改变在不同的网站上 一种蛋白质可以协同或拮抗地相互作用， 信息通过域之间的膜传递 由链接的最西边连接。 正是考虑到这些目标， 我们建议继续研究细菌的趋水性。 该提案涉及生理、遗传和生化 跨膜受体蛋白的研究进展 参与大肠杆菌和其他细菌的趋化性。 这些蛋白质，称为转换器，是核心的， 趋化行为的兴奋和适应阶段。 适应包括转换器的共价修饰， 特别是蛋白质多个谷氨酰羧基甲基化 残留位点。 其中一些谷氨酰残基 因为谷氨酰胺被酶促脱酰胺， 谷氨酸盐 多重甲基化的功能意义 和脱酰胺将通过表征突变体 蛋白质，其中修饰的残基已被改变， 利多卡因定向定点诱变。 的 在修饰时具有取代的突变蛋白质的集合 地点和其他地方将被净化的程序开发， 上一个资助期并进行光谱检查， 从生物化学上寻找与 他们的功能状态。 比较生物化学 将通过确定核苷酸 来自盐生盐杆菌的基因序列， 通过与E.大肠杆菌转导基因和类似基因 将使用一种方法来识别、隔离和排序传感器 各种细菌的基因。 一个广泛的项目 将启动旨在生产换能器晶体或 适合于X射线衍射分析的换能器段。 转导蛋白共享一个共同结合位点的迹象 对于一个小分子将继续进行，希望能确定一个 功能相关的生化活性。

A basic feature of living cells is the ability to respond to specific, external chemical signals. Like eukaryotic cells that respond to hormones or neurotransmitters, bacteria contain specific receptors, exposed on the cell surface, that recognize relevant compounds. These cells are chemotactic as the result of the functioning of a sensory-response system that links receptors to flagella. The long-term goal of this laboratory is to contribute to a detailed molecular biological description of the chemotactic system. One important class of transmembrane receptors contains two hydrophilic domains connected by a minimal amount of hydrophobic, membrane-spanning sequence. Receptors for insulin, epidermal growth factor and bacterial chemoattractants are prominent members of this group. These receptor proteins contain multiple sites for covalent modification, implicated in sensory adaptation or "desensitization". Understanding the mechanisms by which any of these receptors function will require answers to fundamental questions about how ligand binding or covalent modification at one place on a protein can affect the activity of a distant domain, how alterations at different sites on a protein can interact cooperatively or antagonistically, and how information is passed across a membrane between domains connected by the narrowest of links. It is with these goals in mind that we propose to continue our investigations of bacterial taxis. The proposal involves physiological, genetic and biochemcial approaches to the study of transmembrane receptor protein involved in chemotaxis by Escherichia coli and other bacteria. These proteins, called transducers, are central to both the excitation and adaptation phases of chemotactic behavior. Adaptation involves covalent modification of transducers, specifically protein carboxyl methylation at multiple glutamyl residue sites. Some of those glutamyl residues are present because glutamines are enzymatically deamidated to create glutamates. The functional significance of multiple methylation and deamidation will be examined by characterizing mutant proteins in which modified residues have been altered by oligonucleotide-directed, site-specific mutagenesis. The collection of mutant proteins with substitutions at modification sites and elsewhere will be purified by a procedure developed in the previous grant period and examined spectroscopically and biochemically for conformational differences that correlate with their functional states. A program of comparative biochemistry of transducers will be initiated by determining the nucleotide sequences of genes from Halobacterium halobium that were isolated by homology with E. coli transducer genes and similar approach will be used to identify, isolate and sequence transducer genes from a variety of bacterial species. An extensive project will be initiated aimed at producing crystals of transducers or transducer segments suitable for analysis by X-ray diffraction. Indications that transducer proteins share a common binding site for a small molecule will be pursued with the hope of identifying a functionally relevant biochemical activity.