Molecular Insights into Phytochrome Photoactivation and Signaling

光敏色素光活化和信号传导的分子洞察

• 批准号: 1022010
• 负责人: Richard Vierstra
• 金额: $ 86.54万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2014-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1022010&HistoricalAwards=false
• 关键词: Molecular; Insights; into; Phytochrome; Photoactivation

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.

智力MeritA复杂的光感受器阵列协调大多数生物体对周围光环境的反应。其中最具影响力的是光敏色素（phyys），这是一大类多样化的光可逆色蛋白，它们使用十亿蛋白色素进行光检测。这些胆囊蛋白通过两种相对稳定的构象状态感知红光(R)和远红光（FR），一种吸收R的Pr形式通常代表基态，一种吸收FR的Pfr形式通常代表激活态。通过在Pr和Pfr之间的光相互转换，物理充当光调节开关。物理型光感受器最初是在高等植物中发现的，因为它们能够触发对农业生产力至关重要的许多光反应。最近，在包括细菌和真菌在内的各种微生物中发现了它们。尽管它们具有重要的农业意义和进化保护作用，但在分子水平上仍然没有完全理解物理型光感受器如何在Pr和Pfr之间进行光转换，以及这种开关如何告诉生物体周围的光。一项重大突破是利用来自耐辐射球菌变形杆菌的Phy，通过x射线晶体学成功地确定了发色团结合模块Pr的第一个三维结构。这个结构显示了十亿素的结构以及它是如何在它的结合口袋里被固定的，确定了一个稳定口袋的8字形结，发现了姐妹Phys之间迄今未知的二聚化结构域，并揭示了植物Phys是如何从它们的微生物祖先中产生的。在之前的nsf资助的研究中，利用来自耐热蓝藻聚球菌OSB的Phy，通过核磁共振（NMR）光谱确定发色团口袋的第一对Pr和Pfr溶液结构取得了进展。通过对这些结构的比较，我们可以第一次了解到物理粒子是如何在基态和激活态之间进行光转换的。与预期相反，在Pr到Pfr的光转化过程中，发现了A吡咯环而不是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二聚体内相邻输出域之间的接触，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：A环旋转是所有物理的光转换的中心吗？一个完整的Phy二聚体的结构是什么？色素旋转后结合袋内的结构变化是如何改变Phy信号的？对这项工作有重要意义的是重组系统的发展，产生大量的组装光感受器，以及一组新的物理的研究，在蓝光和绿光吸收形式之间进行光转换，这将有助于光激活状态的分析。具体而言，本研究计划将：(1)利用核磁共振光谱和x射线晶体学的结合为光转化过程中a环的旋转提供进一步的支持；(2)利用x射线晶体学开发更完整的Phys结构；(3)利用单粒子电子显微镜确定Phy二聚体作为Pr和Pfr的结构；(4)利用生化方法进一步了解Phy二聚体的光驱动构象变化如何调节信号传导。更广泛的影响本研究将为更好地理解Phy超家族的结构、功能和进化提供一个基本框架。预期的结果将最终有助于阐明微生物和植物如何感知其光环境，这可能对理解微生物生态系统，重要微生物病原体的控制以及开发提高粮食和生物燃料作物生产力的新策略具有重要影响。此外，该项目将通过合作安排，加强科学基础设施，培训博士后、研究生、本科生和少数民族学生，学习现代分子和基于结构的生物研究方法。