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

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子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光感受器的一个主要家族。它们在红色吸收（Pr）和远红色吸收（Pfr）状态之间进行可逆的光转换，从而最终将光信号转换为介导随后细胞反应的独特生物信号。本研究以铜绿假单胞菌和红球藻的代表性细菌光敏色素（BphP）为研究对象，探讨Pr/Pfr光转化和信号转导的分子机制。沼泽BphPs由三个N-末端光敏结构域（PAS GAF和PHY）和一个C-末端组氨酸激酶（HK）效应结构域组成，其中HK活性由光敏核心结构域（PCD）检测到的光信号调节。在以前的建议期间，我们已经确定了几个晶体结构的PCD在Pr和Pfr状态分别。我们还捕获了三个早期的反应中间体，通过启动和以下的PFR到Pr的光反应，在光敏PaBphP晶体使用温度扫描cryo-crystallography。总之，这些反应产物和中间状态的晶体结构提供了结构洞察BphPs的初始分子事件后，感测红光和随后的构象变化的发色团和相邻的蛋白质基质。在下一阶段（1），我们计划将我们的晶体学研究扩展到全长（FL）BphPs，其中包括C-末端组氨酸激酶（HK）效应结构域。我们的目标是确定三级和四级结构元件，负责传输光诱导的局部构象变化的发色团结合口袋的远程HK产生的生物信号。 (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的光束线科学家合作，研究各种时间尺度（从ps到ms）上的反应中间体。(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，我们将对人工光感受器进行晶体学研究，以将光敏性传递给其他光惰性系统，如离子通道。

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.