MOLECULAR STUDIES OF CHEMORECEPTION

介导大肠杆菌趋化性的受体是最好的 以细菌化学感受器大家族的成员为特征 以及更大的原核和真核感觉受体家族。 这项建议的重点是感官转导，这些 化学感受器，重点是跨膜和分子间 发信号。 这项工作将生物化学， 生物物理学、遗传学和结构学方法， 在化学感受器Trg上投射。 在当前的融资期内， PI创建、表征并利用了Trg变体的集合， 每个多肽链一个或两个半胱氨酸，主要位于一个或两个半胱氨酸中， 两个跨膜片段。 将广泛利用这一点 在实验中收集突变受体， 跨膜组织和信号传导的核心问题。 的 该建议的主要目的是：（1）确定螺旋相互作用 （2）测量跨膜结构域之间的距离; 跨膜片段中的位置，或特定的 位置和膜表面;和（3）定义分子 涉及跨膜构象变化的细节 信号 其中一些研究将包括使用电子 顺磁共振光谱，并将在合作完成 和韦恩·哈贝尔医生在一起 在其他研究中， 化学受体和配体占据的结合蛋白将被 使用表面等离子体共振研究。 “少数人” 化学受体Trg在配体诱导的信号传导中有效， 缺陷的稳态激活的CheA信号激酶。 的 这种信号传导行为的基础将通过生理学， 基因和生物化学研究，希望了解 在细菌中， 化学感受系统 此外，遗传和生物化学 将使用方法来描述三者之间的相互作用 化学感受器结构域，并且努力将针对获得 受体-CheW-CheA的颗粒和二维晶体 适合于通过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.