Molecular Insights into Phytochrome Photoactivation and Signaling

智力优势一组复杂的光感受器协调大多数生物对周围光环境的反应。 其中最有影响力的是光敏色素（Phys），这是一组庞大而多样的光可逆色蛋白，它们使用胆色素进行光检测。 这些胆蛋白通过两种相对稳定的构象状态感测红光（R）和远红光（FR），R-吸收Pr形式通常代表基态，FR-吸收Pfr形式通常代表活化态。 通过Pr和Pfr之间的光相互转换，Phys充当光调节开关。 Phy型光感受器首先在高等植物中发现，它们能够触发对农业生产力至关重要的许多光反应。 最近，它们在包括细菌和真菌在内的各种微生物中被发现。 尽管它们在农业上的重要性和进化上的保守性，但在分子水平上仍然没有完全理解Phy型光感受器如何在Pr和Pfr之间进行光转换，也没有完全理解这种开关如何告诉生物体周围的光。 一个重大的突破是成功地确定了第一个3-D结构的发色团结合模块Pr的X射线晶体学使用的Phy从变形杆菌Deinococcus radiodurans。 这个结构显示了胆色素的结构，以及它是如何在其结合口袋中的摇篮，确定了一个8字结，稳定了口袋，发现了一个迄今未知的姐妹Phys之间的二聚化结构域，并揭示了植物Phys如何从它们的微生物祖先中产生。 在之前的NSF资助的研究中，取得了进展，在确定第一对Pr和Pfr溶液结构的发色团口袋的核磁共振（NMR）光谱使用Phy从耐热蓝藻聚球藻OSB。 这些结构的比较提供了第一次瞥见Phys如何在基态和激活态之间进行光转换。 与预期相反，发现在Pr到Pfr光转换期间，A吡咯环而不是胆色素的D环旋转。 这种翻转诱导多肽内的结构重排，然后似乎改变Phy二聚体内相邻输出结构域之间的接触，以最终调节信号传导。 这个更新项目的智力价值是建立在这些结构研究的基础上，以回答关键问题，包括：这是一个环旋转的中心，所有物理的光转换？完整的Phy二聚体的结构是什么？如何旋转的色素，其次是结合口袋内的结构变化改变Phy信号？ 这项工作的重要性是重组系统的开发，产生大量的组装光感受器，和一组新的物理之间的蓝光和绿光吸收形式，这应该有助于分析的光活化状态的光转换的研究。 具体而言，该研究计划将：（1）使用NMR光谱学和X射线晶体学的组合来为A环在光转化期间的旋转提供进一步的支持，（2）使用X射线晶体学来开发Phys的更完整的结构，（3）利用单粒子电子显微镜来确定Phy二聚体作为Pr和Pfr的结构，以及（4）使用生物化学方法来进一步理解Phy二聚体中光驱动的构象变化如何调节信号传导。更广泛的影响这项研究将提供一个必要的框架，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它们的光环境，这可能对理解微生物生态系统、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力的新策略产生重要影响。 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、本科生和少数民族学生提供生物研究中基于现代分子和结构的方法的培训。

Intellectual MeritA complex array of photoreceptors coordinates the response of most organisms to their surrounding light environment. One of the most influential is the phytochromes (Phys), a large and diverse group of photoreversible chromoproteins that use a bilin pigment for light detection. These 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. Phy-type photoreceptors were first discovered in higher plants by their ability to trigger numerous photoresponses critical for agricultural productivity. More recently, they were found in various microorganisms including bacteria and fungi. Despite their agricultural importance and evolutionary conservation, it is still not fully understood at the molecular level how Phy-type photoreceptors photoconvert between Pr and Pfr nor how this switch tells organisms about the light around them. A major breakthrough was the success in determining the first 3-D structure of the chromophore-binding module as Pr by x-ray crystallography using a Phy from the proteobacterium Deinococcus radiodurans. This structure showed the configuration of the bilin pigment and how it is cradled within its binding pocket, identified a figure-of-eight knot that stabilizes the pocket, discovered a heretofore unknown dimerization domain between sister Phys, and revealed how plant Phys arose from their microbial ancestors. During the prior NSF-funded studies, progress was made in determining the first paired Pr and Pfr solution structures of the chromophore pocket by nuclear magnetic resonance (NMR) spectroscopy using a Phy from the thermotolerant cyanobacterium Synechococcus OSB. Comparison of these structures provided the first glimpse into how Phys photoconvert between their ground and activated states. Contrary to expectations, the A pyrrole ring and not the D ring of the bilin pigment was discovered to rotate during Pr to Pfr photoconversion. This flip induces structural rearrangements within the polypeptide, which then appear to alter the contact between adjacent output domains within the Phy dimer to ultimately modulate signaling. The intellectual merits of this renewal project are to build upon these structural studies to answer key questions, including: is this A ring rotation central to the photoconversion of all Phys? What is the structure of a complete Phy dimer? How does rotation of the pigment followed by structural changes within the binding pocket alter Phy signaling? Significant to this work are the development of recombinant systems that produce large amounts of assembled photoreceptors, and the study of a novel set of Phys that photoconvert between blue- and green-light absorbing forms which should aid in the analysis of the photoactivated state. Specifically, this research plan will: (1) use a combination of NMR spectroscopy and x-ray crystallography to provide further support for the rotation of the A ring during photoconversion, (2) use x-ray crystallography to develop more complete structures of Phys, (3) exploit single particle electron microscopy to determine the architecture of the Phy dimer as Pr and Pfr, and (4) use biochemical methods to further understand how light-driven conformational changes in the Phy dimer regulate signaling. Broader ImpactThis 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 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.