MOLECULAR INSIGHTS INTO PHYTOCHROME PHOTOACTIVATION AND SIGNALING

对光敏色素光激活和信号转导的分子洞察

• 批准号: 1329956
• 负责人: Richard Vierstra
• 金额: $ 115万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1329956&HistoricalAwards=false
• 关键词: MOLECULAR; INSIGHTS; INTO; PHYTOCHROME; PHOTOACTIVATION

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.

智力优点复杂的光感受器阵列协调生物体对其周围光环境的反应。 其中一个有影响力的组是光敏色素 (Phys)，这是一组庞大且多样化的光可逆色素蛋白，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。尽管它们在农业和病理学上具有重要意义，并且具有进化保守性，但在分子水平上尚未完全了解 Phy 型光感受器如何在 Pr 和 Pfr 之间进行光转换，也没有完全了解这种转换如何向生物体报告周围的光。 在过去的几年中，通过确定几个微生物代表的光传感模块的3D结构模型，在理解Phys如何在原子水平上工作方面取得了重大突破。 这些结构揭示了胆色素的结构以及它如何被固定在其结合袋中，确定了植物Phys从细菌祖细胞进化的可能途径，并确定了可能对信号传导至关重要的几种独特的结构特征。在之前 NSF 资助的研究中，通过核磁共振 (NMR) 光谱生成了来自两个不同物理场的发色团口袋的第一对基态和光活化态结构。这些结构的比较让我们第一次了解光如何触发光感知。其中包括光驱动的胆碱旋转，随后重新排列许多发色团/氨基酸接触。 最终，这些改变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。 该项目将以这些结构研究为基础来回答关键问题。 完整的 Phy 二聚体的结构是什么？胆碱的旋转以及结合袋内的结构变化如何改变 Phy 信号传导？ 植物 Phy 的结构是什么？如何利用这些信息来设计 Phy 信号以实现农业效益？这项工作的重要意义在于开发了能够产生大量组装光感受器的重组系统，以及最终产生了植物 Phy 的衍射质量晶体的进展。 具体来说，该研究计划将：(1) 开发更完整的微生物 Phy 结构及其信号输出模块，(2) 定义结、脊柱和发夹特征如何促进 Phy 信号传导，(3) 确定独特的蓝细菌色素亚家族如何独特地检测光谱的其他部分，(4) 生成植物光敏模块的 X 射线晶体结构 Phy 和 (5) 结合使用 NMR 光谱、X 射线晶体学和单粒子电子显微镜方法来生成具有或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。培训还将涉及威斯康星州青年学徒计划赞助的高中生。