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.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 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。光敏色素是植物、真菌和细菌中负责红光感知的主要光感受器家族。它们在红光吸收(PR)和远红光吸收(PFR)状态之间进行可逆光转换，从而最终将光信号转换为不同的生物信号，从而调节随后的细胞反应。本研究旨在利用铜绿假单胞菌和沼泽假单胞菌的代表性细菌光敏色素(BphP)来研究光敏色素中Pr/PfR光转化和信号转导的分子机制。BphP由三个N端的光感受域(PAS、GAF和PHY)和一个C端的组氨酸激酶(HK)效应域组成，其中HK的活性受光敏核心域(PCD)检测到的光信号的调节。在前面的建议期中，我们已经确定了PCD的几种晶体结构，分别处于Pr和Pfr态。我们还利用温度扫描低温结晶学在光活性PaBphP晶体中通过引发和跟踪PfR到Pr的光反应捕获了三个早期反应中间体。综上所述，这些反应物产物和中间态的晶体结构提供了对BphPs在检测到红光时的初始分子事件以及随后发色团和邻近蛋白质基质中的构象变化的结构洞察。在下一阶段(1)，我们计划将我们的结晶学研究扩展到全长(FL)BphP，其中包括一个C末端的组氨酸激酶(HK)效应结构域。我们的目标是识别负责将光诱导的发色团结合口袋中的局部构象变化传递到遥远的HK以产生生物信号的三级和四级结构元件。(2)我们将进一步探索基于劳厄和单色衍射的时间分辨实验策略，与BioCARS的束线科学家合作，在不同的时间尺度(从ps到ms)研究反应中间体。(3)如果结合我们正在进行的蛋白质设计工作，出现合适的项目/晶体，以将光敏感传递到离子通道等其他光惰性系统，我们将对人造光感受器进行结晶学研究。