Molecular mechanisms of signaling systems responsive to light, redox and chemical environment

Crane小组研究响应或涉及光化学和氧化还原的信号转导系统 化学反应。我们的总体目标是了解细菌的趋化行为和真核生物的昼夜节律。 通过研究作为基础的大分子络合物，在分子反应性水平上的节律 跨膜信号、运动性和基因表达。趋化性长期以来一直是 研究跨膜信号、细胞内信息传递和细胞运动。此外，许多 导致疾病的人类病原体，如霍乱、胃癌和莱姆病，依赖于趋化作用来 建立并维持感染。趋化性感觉器表现出显著的敏感性、动态性 射程和分子记忆。化学受体、组氨酸激酶(CHEA)和偶联蛋白组装成 大分子阵列，其中组分之间的远程协同作用产生高度特异的 适应不断变化的条件的反应。这一提议继续努力了解受体：激酶 组装，化学受体构象信号，最终，CHEA的调节。CHEA输出调制 自然界完美的纳米机器--鞭毛马达。电机内部开关综合体的架构 将得到改进，以更好地了解扭矩生成和方向切换。一个特别的焦点将是 致病螺旋体，它们在各自的细胞末端表现出不对称的鞭毛旋转。第二 生物钟系统由细胞自主的计时装置组成，这些计时装置将新陈代谢速度调整到每天的周期。 生物钟由转录-翻译反馈环(TTFL)组成，在TTFL中有抑制蛋白 抑制自身基因的转录激活因子。光通过刺激生物钟的相位 直接照射在TTFL上的光传感器。在人类中，异常的时钟功能会导致精神疾病(睡眠 紊乱、抑郁和躁狂)、细胞生长失调(癌症)和代谢缺陷(糖尿病和 肥胖)。本项目建议对关键的光传感器和抑制器进行结构和机理研究。 真菌(粗枝脉孢菌)和苍蝇(黑腹果蝇)的代表性生物钟的活动。一个 免费提供一整套生物物理技术，包括X射线结晶学、小角X射线散射、 光学光谱学、低温电子显微镜和脉冲偶极ESR光谱学(PDS)将应用于 完成这些目标。利用蛋白质工程获取关键实体的生化重组 将与细胞分析和生物体研究相结合，以便将物理特性与 生物功能。对于PDS，引入基于氮氧化物、黄素、 核苷酸和金属离子将被用于测量体外和体内的结构和动力学。 计算设计和分子动力学将被用来测试和巩固模型。总体而言，该计划 旨在通过协同应用提供对传感和响应的分子水平的理解 化学和生物物理方法。

The Crane group studies signal transduction systems that respond to or involve photochemistry and redox chemistry. Our overall goal is to understand the behaviors of bacterial chemotaxis and eukaryotic circadian rhythms at the level of molecular reactivity through the study of macromolecular complexes that underlie transmembrane signaling, motility, and gene expression. Chemotaxis has long served as a key system for studying transmembrane signaling, intracellular information transfer, and cell locomotion. Furthermore, many human pathogens that cause diseases, such as cholera, gastric cancer, and Lyme, rely on chemotaxis to establish and sustain infection. The sensory apparatus of chemotaxis displays remarkable sensitivity, dynamic range, and molecular memory. Chemoreceptors, histidine kinases (CheA), and coupling proteins assemble into large molecular arrays, wherein long-range cooperative interactions among components produce highly specific responses that adapt to changing conditions. This proposal continues efforts to understand receptor:kinase assembly, chemoreceptor conformational signaling, and ultimately, CheA regulation. CheA output modulates Nature's consummate nanomachine – the flagella motor. The architecture of the switch complex within the motor will be refined to better understand torque generation and direction switching. A particular focus will be the pathogenic spirochetes, which exhibit asymmetric flagella rotation at their respective cell ends. The second system, circadian clocks, comprises of cell-autonomous timing devices that pace metabolism to the diurnal cycle. Clocks are composed of transcriptional-translational feedback loops (TTFLs) within which repressor proteins inhibit the transcriptional activators of their own genes. Light entrains the clock phase by stimulating photosensors that impinge directly on the TTFLs. In humans, aberrant clock function causes mental illness (sleep disorders, depression, and mania), cell growth deregulation (cancer), and metabolic defects (diabetes and obesity). This project proposes structural and mechanistic investigations of the key light sensor and repressor activities in representative clocks from fungi (Neurospora crassa) and flies (Drosophila melanogaster). A complimentary set of biophysical techniques, including X-ray crystallography, small-angle X-ray scattering, optical spectroscopy, cryo-electron microscopy, and pulse dipolar ESR spectroscopy (PDS), will be applied to accomplish these goals. Biochemical reconstitution that leverages protein engineering to procure key entities will be combined with cellular assays and organismal studies in order to correlate physical properties with biological function. For PDS, new methods for incorporating spin probes that are based on nitroxides, flavins, nucleotides, and metal ions will be deployed for measuring structure and dynamics both in vitro and in vivo. Computational design and molecular dynamics will be used to test and consolidate models. Overall, this program aims to provide a molecular-level understanding for sensing and response through the synergistic application of chemical and biophysical methods.