STATIC AND TIME-RESOLVED CRYSTALLOGRAPHIC STUDIES ON BACTERIOPHYTOCHROMES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 光敏色素是一个广泛分布的光感受器家族，其使用线性四吡咯分子（胆色素）作为发色团来感知光，主要在红色和远红色区域。它们通过两种热稳定的光谱不同形式Pr和Pfr之间的可逆光转换来调节生理反应谱。 首先在植物中发现，光敏色素也在蓝细菌，紫色细菌，非光合细菌，甚至真菌中发现。 植物光敏色素是许多重要发育过程的关键调节因子，如叶绿素合成、种子萌发、成花诱导和避荫，其中它们的原核祖先被发现参与控制光系统和光收获复合物的合成、趋光性、线性四吡咯胆色素发色团中C15-C16双键附近的Z到E异构化被认为是Pr和Pfr形式之间可逆光转换的主要光事件，但对Pr/Pfr可逆光转化的分子和机理细节以及正向和反向过程中的中间结构知之甚少。 我们将利用静态和时间分辨的X射线晶体学技术来研究几个细菌光敏色素系统，如R. palustris、A.来自根瘤菌的PaBphP和来自铜绿假单胞菌的PaBphP，具有不同的结构域组合。 我们的具体目标是：1.揭示Pr/Pfr光开关的分子基础; 2.使用捕获和时间分辨技术提取Pr/Pfr光转换过程中的结构中间体; 3.了解N端光敏结构域（PLD、GAF和PHY结构域）接收的光信号如何被转换成C端效应组氨酸激酶中的化学信号; 4.利用定点诱变和蛋白质工程设计具有确定和调节的光化学性质的嵌合蛋白质。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Phytochromes are a widespread family of photoreceptors that use linear tetrapyrrole molecules (bilins) as chromophores to sense light, primarily in the red and far-red region. They regulate a spectrum of physiological responses via reversible photoconversion between two thermo-stable, spectrally distinct forms, Pr and Pfr. First discovered in plants, phytochromes are also identified in cyanobacteria, purple bacteria, non-photosynthetic bacteria, and even fungi. Plant phytochromes are key regulators in many important developmental processes such as chlorophyll synthesis, seed germination, floral induction and shade avoidance, where their prokaryotic ancestors are found to be involved in key processes that control synthesis of photosystems and light harvest complexes, phototaxis, etc. The Z to E isomerization around C15-C16 double bond in linear tetrapyrrole bilin chromophore is believed to be the primary photoevent in the reversible photoconversion between Pr and Pfr forms, but little is known about molecular and mechanistic details of Pr/Pfr reversible photoconversion as well as intermediate structures in both forward and reverse processes. We will take advantage of both static and time-resolved X-ray crystallographic techniques to explore several bateriophytochrome systems, such as RpBphP2, RpBphP3, RpBphP5 from R. palustris, AtBphP2 from A. tumefaciens, and PaBphP from P. aeruginosa, with different domain combinations. Our specific aims are: 1. to reveal molecular basis of Pr/Pfr photoswitching; 2. to extract structural intermediates during Pr/Pfr photoconversion using both trapping and time-resolved techniques; 3. to understand how the light signal recieved by the N-terminal photosensory domains (PLD, GAF and PHY domains) is transduced into a chemical signal in the C-terminal effector histidine kinase; 4. to design chimeric proteins with defined and modulated photochemical properties using site-directed mutagensis and protein engineering.