STUDYING NATURAL AND DESIGNED RED-LIGHT PHOTORECEPTORS BY STATIC AND DYNAMIC

通过静态和动态研究天然和设计的红光感光器

• 批准号: 8363682
• 负责人: JOHN Keith MOFFAT
• 金额: $ 6.69万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363682
• 关键词: Beryllium; Binding; Biological; C-terminal; Circadian Rhythms; Collaborations; Crystallography; Development; Event; Family; Funding; Grant; Ion Channel; Length; Life; Light; Mediating; Molecular; N-terminal; National Center for Research Resources; Organism; Perception; Phosphotransferases; Photophobia; Photoreceptors; Physiological Processes; Phytochrome; Principal Investigator; Proteins; Pseudomonas aeruginosa; Reaction; Research; Research Infrastructure; Resources; Scanning; Scientist; Signal Transduction; Source; Structure; System; Temperature; Time; United States National Institutes of Health; base; beamline; chromophore; cost; design; insight; plant fungi; protein-histidine kinase; response; structural biology

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Light is a fundamental signal that regulates important physiological processes such as development and circadian rhythm in living organisms. Phytochromes form a major family of photoreceptors responsible for red light perception in plants fungi and bacteria1. They undergo reversible photoconversion between red-absorbing (Pr) and far-red-absorbing (Pfr) states thereby ultimately converting a light signal into a distinct biological signal that mediates subsequent cellular responses. Our research aims to understand the molecular mechanisms of Pr/Pfr photoconversion and signal transduction in phytochromes using representative bacteriophytochromes (BphP) from P. aeruginosa and R. palustris. BphPs consist of three N-terminal photosensory domains (PAS GAF and PHY) and a C-terminal histidine kinase (HK) effector domain in which the HK activity is regulated by light signals detected by the photosensory core domains (PCD). During previous proposal periods we have determined several crystal structures of PCD in the Pr and Pfr states respectively. We have also captured three early reaction intermediates by initiating and following the Pfr-to-Pr photoreaction in photoactive PaBphP crystals using temperature-scanning cryo-crystallography. Taken together these crystal structures of reactant product and intermediate states provide structural insight into initial molecular events of BphPs upon sensing red-light and subsequent conformational changes in the chromophore and adjacent protein matrix. During the next period (1) we plan to extend our crystallographic studies to full-length (FL) BphPs which include a C-terminal hisitidine kinase (HK) effector domain. We aim to identify tertiary and quaternary structural elements that are responsible for transmitting light-induced local conformational changes in the chromophore-binding pocket to the remote HK to generate a biological signal. (2) We will further explore time-resolved experimental strategies based on both Laue and monochromatic diffraction to study reaction intermediates on various time-scales (from ps to ms) in collaboration with beamline scientists at BioCARS. (3) We will carry out crystallographic studies on artificial photoreceptors if suitable projects/crystals arise in couple with our ongoing protein design efforts to convey light-sensitivity to otherwise light-inert systems such as ion channels.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 光是调节生物体发育和昼夜节律等重要生理过程的基本信号。光敏色素是光感受器的一个主要家族，负责植物真菌和细菌的红光感知1。它们在红光吸收（Pr）和远红光吸收（Pfr）状态之间进行可逆的光转换，从而最终将光信号转化为介导后续细胞反应的独特生物信号。我们的研究旨在利用来自铜绿假单胞菌和沼泽红藻的代表性细菌光敏色素 (BphP) 来了解光敏色素中 Pr/Pfr 光转换和信号转导的分子机制。 BphP 由三个 N 端光传感结构域（PAS GAF 和 PHY）和一个 C 端组氨酸激酶 (HK) 效应结构域组成，其中 HK 活性由光传感核心结构域 (PCD) 检测到的光信号调节。在之前的提案期间，我们已经分别确定了 Pr 和 Pfr 态 PCD 的几种晶体结构。我们还通过使用温度扫描冷冻晶体学在光活性 PaBphP 晶体中引发和跟踪 Pfr-to-Pr 光反应，捕获了三种早期反应中间体。总而言之，反应物产物和中间状态的这些晶体结构提供了对 BphP 在感测红光时的初始分子事件以及发色团和相邻蛋白质基质中随后的构象变化的结构洞察。在下一阶段 (1)，我们计划将晶体学研究扩展到全长 (FL) BphP，其中包括 C 末端组氨酸激酶 (HK) 效应结构域。我们的目标是识别三级和四级结构元件，这些元件负责将光诱导的发色团结合袋中的局部构象变化传输到远程 HK 以产生生物信号。 （2）我们将与BioCARS的光束线科学家合作，进一步探索基于劳厄和单色衍射的时间分辨实验策略，以研究不同时间尺度（从皮秒到毫秒）的反应中间体。 (3) 如果合适的项目/晶体与我们正在进行的蛋白质设计工作相结合，将光敏感性传递给离子通道等其他光惰性系统，我们将对人造光感受器进行晶体学研究。