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.

智力优点复杂的光感受器阵列协调大多数生物体对其周围光环境的反应。 其中最有影响力的是光敏色素 (Phys)，这是一大类多样化的光可逆色素蛋白，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知红光 (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被发现，因为它们能够触发对农业生产力至关重要的大量光响应。 最近，它们在包括细菌和真菌在内的各种微生物中被发现。 尽管它们在农业上很重要并且具有进化保护性，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转换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变形杆菌的 Phy，通过 X 射线晶体学成功确定了发色团结合模块 Pr 的第一个 3-D 结构。 该结构显示了胆素色素的构型以及它如何被固定在其结合口袋中，识别了稳定口袋的八字结，发现了姐妹 Phys 之间迄今为止未知的二聚化结构域，并揭示了植物 Phys 如何从其微生物祖先中产生。 在之前 NSF 资助的研究中，使用来自耐热蓝细菌聚球藻 OSB 的 Phy 通过核磁共振 (NMR) 光谱确定了第一对发色团袋的 Pr 和 Pfr 溶液结构，并取得了进展。 这些结构的比较让我们首次了解 Phys 如何在基态和激活态之间进行光转换。 与预期相反，在 Pr 到 Pfr 光转换过程中，发现 bilin 颜料的 A 吡咯环而不是 D 环发生旋转。 这种翻转会诱导多肽内的结构重排，从而改变 Phy 二聚体内相邻输出结构域之间的接触，最终调节信号传导。 这个更新项目的智力优点是建立在这些结构研究的基础上来回答关键问题，包括：这个A环旋转是所有物理光转换的中心吗？完整的 Phy 二聚体的结构是什么？颜料旋转后结合口袋内的结构变化如何改变 Phy 信号传导？ 这项工作的重要意义在于开发了可产生大量组装光感受器的重组系统，以及研究了一组新颖的物理结构，可在蓝光和绿光吸收形式之间进行光转换，这有助于分析光激活状态。 具体来说，该研究计划将：（1）利用核磁共振波谱和X射线晶体学的结合，为光转换过程中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Pr和Pfr的结构，以及（4）利用生化方法进一步了解光驱动如何 Phy 二聚体的构象变化调节信号传导。更广泛的影响这项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重要的框架。 预期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、控制重要微生物病原体以及制定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，该项目还将通过合作安排对博士后、研究生、本科生和少数民族学生进行基于现代分子和结构的生物研究方法的培训，从而加强科学基础设施。