MOLECULAR INSIGHTS INTO PHYTOCHROME PHOTOACTIVATION AND SIGNALING

智能Merita复合光感受器阵列协调有机体对周围光环境的反应。一个有影响力的群体是光敏色素(Phys)，这是一大群不同的光可逆染色蛋白，使用胆色素进行光检测。这些二聚体胆蛋白通过两种相对稳定的构象来感知红色(R)和远红光(FR)，一种是吸收R的Pr形式，通常代表基态，另一种是吸收FR的PFR形式，通常代表激活状态。通过Pr和Pfr之间的光相互转换，Phys充当光调节开关。尽管它们在农业和病理上的重要性以及进化上的保守性，但在分子水平上还没有完全了解Phy型光感受器如何在Pr和Pfr之间进行光转换，也不知道这种转换是如何向生物体报告它们周围的光的。在过去的几年里，通过确定几个微生物代表的光敏模块的三维结构模型，在理解物理如何在原子水平上工作方面取得了重大突破。这些结构揭示了BLIN色素的结构以及它是如何被放置在其结合口袋中的，确定了植物物理从细菌前体进化的可能途径，并确定了几个可能对信号转导至关重要的独特结构特征。在之前的NSF资助的研究中，来自两个不同物理的发色团口袋的第一对基态和光激活态结构是通过核磁共振(核磁共振)光谱产生的。对这些结构的比较首次让我们得以一窥光是如何触发光的感知的。包括光驱动的比林旋转，然后是许多发色团/氨基酸接触的重排。最终，这些变化必须将光能转化为机械运动，从而重组输出模块并影响其信号潜力。该项目将在这些结构研究的基础上回答关键问题。一个完整的Phy二聚体的结构是什么？BLIN的旋转以及结合口袋内的结构变化是如何改变Phy信号的？植物Phy的结构是什么？如何利用这些信息来设计Phy信号以造福农业？对这项工作具有重要意义的是产生大量组装的光受体的重组系统的开发，以及最终产生植物Phy的衍射性晶体的进展。具体地说，这项研究计划将：(1)开发微生物Phy及其信号输出模块的更完整的结构；(2)确定结、刺和发夹特征如何有助于Phy信号传递；(3)确定独特的蓝藻碘色素亚家族如何唯一地检测光谱的其他部分；(4)产生植物Phy感光模块的X射线晶体结构；以及(5)结合使用核磁共振光谱、X射线结晶学和单粒子电子显微镜方法来生成具有和不具有信号伙伴的完整植物Phy二聚体的模型。更广泛的影响：这项研究将提供一个基本的框架，以更好地了解Phy超家族的结构、功能和进化。预期的结果最终将有助于阐明微生物和植物如何感知其光环境，这可能对了解微生物生态系统、控制重要的微生物病原体以及开发提高粮食和生物燃料作物生产率的新战略具有重要影响。此外，该项目将通过合作安排加强科学基础设施，为博士后、研究生、本科生和少数族裔学生提供现代分子和结构生物学研究方法方面的培训。培训还将包括由威斯康星州青年学徒计划赞助的高中生。

Intellectual MeritA complex array of photoreceptors coordinates the response of organisms to their surrounding light environment. One influential set is the phytochromes (Phys), a large and diverse group of photoreversible chromoproteins that use a bilin pigment for light detection. These dimeric biliproteins sense red (R) and far-red light (FR) through two relatively stable conformational states, an R-absorbing Pr form that typically represents the ground state, and an FR-absorbing Pfr form that typically represents the activated state. By photointerconverting between Pr and Pfr, Phys act as light-regulated switches. Despite their agricultural and pathological importance and evolutionary conservation, it is not fully understood at the molecular level how Phy-type photoreceptors photoconvert between Pr and Pfr nor how this switch reports to organisms about the light around them. In the past few years, major breakthroughs have been made in understanding how Phys work at the atomic level through the determination of 3-D structural models of the photosensing module from several microbial representatives. These structures revealed the configuration of the bilin pigment and how it is cradled within its binding pocket, identified a likely route whereby plant Phys evolved from bacterial progenitors, and identified several unique structural features likely critical to signaling. During prior NSF-funded studies, the first paired ground and photoactivated state structures of the chromophore pocket from two divergent Phys were generated by nuclear magnetic resonance (NMR) spectroscopy. Comparisons of these structures provided the first glimpse into how light triggers light perception. Included are light-driven rotation of the bilin followed by rearrangement of numerous chromophore/amino acid contacts. Ultimately, these alterations must convert light energy into mechanical motion that reorganizes the output module and affect its signaling potential. This project will build upon these structural studies to answer key questions. What is the structure of a complete Phy dimer? How does rotation of the bilin followed by structural changes within the binding pocket alter Phy signaling? What is the structure of a plant Phy and how can this information be used to engineer Phy signaling for agricultural benefit? Significant to this work are the development of recombinant systems that produce large amounts of assembled photoreceptors and advances that have culminated in the generation of diffraction quality crystals of a plant Phy. Specifically, this research plan will: (1) develop more complete structures of a microbial Phy with its signal output module, (2) define how the knot, spine, and hairpin features contribute to Phy signaling, (3) determine how the distinctive cyanobacteriochrome subfamily uniquely detects other portions of the light spectrum, (4) generate an X-ray crystallographic structure of the photosensing module of a plant Phy, and (5) use a combination of NMR spectroscopic, X-ray crystallographic, and single particle electron microscopic approaches to generate models of a complete plant Phy dimer with and without signaling partners. Broader Impacts: This research will provide an essential framework to better understand the structure, function, and evolution of the Phy superfamily. The anticipated results will ultimately help elucidate how microorganisms and plants sense their light environment, which could have important ramifications for understanding microbial ecosystems, the control of important microbial pathogens, and for the development of new strategies to improve the productivity of food and biofuel crops. In addition, the project will enhance scientific infrastructure via a cooperative arrangement for the training of postdoctoral, graduate, undergraduate, and minority students in modern molecular and structure-based approaches in biological research. Training will also involve high school students sponsored by the Wisconsin Youth Apprenticeship Program.