MOLECULAR STUDIES OF CHEMORECEPTION

介导大肠杆菌趋化作用的受体是最好的 具有细菌化学感受器大家族成员的特征 以及一个更大的原核和真核感觉受体家族。 这项提议关注的是这些感官传导 化学受体，重点是跨膜和分子间 发信号。这项拟议的工作结合了生化、 生物物理、遗传和结构方法，集中在许多 化学受体Trg.上的投射。在本供资期间， PI创建、表征和利用了Trg变体的集合 每个多肽链上有一个或两个半胱氨酸，主要放置在一个或两个 两段都是跨膜的。将广泛使用这一技术 在实验中收集突变受体，旨在解决 跨膜组织和信号传递的中心问题。这个 该提案的主要目的是：(1)确定螺旋相互作用 在跨膜域中；(2)测量成对的 在跨膜节段中的位置，或在特定的 位置和膜表面；以及(3)定义分子 跨膜相关构象变化的细节 发信号。其中几项研究将包括电子的使用。 顺磁共振波谱学和将在合作中完成 和韦恩·哈贝尔医生。在其他研究中，相互作用 化学受体和配体占据的结合蛋白将是 利用表面等离子激元共振进行了研究。“少数人” 化学受体Trg在配体诱导的信号转导中有效，但 在稳态激活CHEA信号通路方面存在缺陷。这个 这一信号行为的基础将由生理学、 基因和生化研究，希望了解 细菌中鲜为人知的信号放大现象 化学传感系统。此外，遗传和生化 将使用各种方法来描述三者之间的相互作用 化学感受器结构域，并将努力获得 受体-Chew-Chea的颗粒和二维晶体 适合用电子显微镜进行结构分析的络合物。

The receptors that mediate chemotaxis by Escherichia coli are the best characterized members of a large family of bacterial chemoreceptors and a larger family of prokaryotic and eukaryotic sensory receptors. This proposal focuses on sensory transduction by these chemoreceptors, with emphasis on transmembrane and intermolecular signaling. The proposed work is a combination of biochemical, biophysical, genetic and structural approaches, concentrating in many projects on the chemoreceptor Trg. In the current funding period the PI created, characterized and utilized a collection of Trg variants with one or two cysteines per polypeptide chain, placed primarily in one or both transmembrane segments. Extensive use will be made of this collection of mutant receptors in experiments designed to address central issues of transmembrane organization and signaling. The principal aims of the proposal are to: (1) determine helical interactions in the transmembrane domain; (2) measure distances between pairs of positions in the transmembrane segments, or between a specific position and the membrane surface; and (3) define the molecular details of the conformational change involved in transmembrane signaling. Several of these studies will include the use of electron paramagnetic resonance spectroscopy and will be done in collaboration with Dr. Wayne Hubbell. In other studies the interaction of chemoreceptor and ligand-occupied binding proteins will be investigated using surface plasmon resonance. The "minority" chemoreceptor Trg is effective in ligand-induced signaling but defective in steady-state activation of the CheA signaling kinase. The basis of this signaling behavior will be investigated by physiological, genetic and biochemical studies with the hope of learning about the little-understood phenomenon of signal amplification in the bacterial chemosensory system. In addition, genetic and biochemical approaches will be used to characterize interactions between the three chemoreceptor domains, and efforts will be directed toward obtaining particles and two-dimensional crystal of receptor-CheW-CheA complexes suitable for structural analysis by electron microscopy.