STATIC AND TIME-RESOLVED CRYSTALLOGRAPHIC STUDIES ON BACTERIOPHYTOCHROMES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 光敏色素是一个广泛存在的光感受器家族，它使用线性四吡咯分子(Bilins)作为发色团来感知光，主要是在红色和远红区。它们通过两种热稳定的、光谱不同的形式Pr和Pfr之间的可逆光转化来调节一系列生理反应。最先在植物中发现的光敏色素也在蓝藻、紫色细菌、非光合细菌甚至真菌中发现。植物光敏色素是叶绿素合成、种子萌发、开花诱导和遮荫等重要发育过程中的关键调控因子，其原核生物祖先参与控制光系统合成、光收获复合体、趋光性等关键过程。线型四氢吡咯比林中C15-C16双键附近的Z到E异构化被认为是Pr和PfR之间可逆光转化的主要光事件，但对Pr/Pfr可逆光转化的分子和机制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X射线结晶学技术来探索几个具有不同结构域组合的光敏色素系统，如沼泽红假单胞菌的RpBphP2、RpBphP3、RpBphP5、根癌农杆菌的AtBphP2和铜绿假单胞菌的PaBphP。我们的具体目标是：1.揭示Pr/PFR光开关的分子基础；2.利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端光敏域(PLD、GAF和PHY结构域)接收的光信号如何转化为C端效应器组氨酸激酶中的化学信号；4.利用定点突变和蛋白质工程设计具有明确和调制光化学性质的嵌合蛋白。

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.