Molecular Insights into Phytochrome Photoactivation and Signaling

智能Merita复合光感受器阵列协调大多数生物体对周围光环境的反应。其中最有影响力的是光敏色素(Phys)，这是一大群种类繁多的光可逆染色蛋白，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。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Phy型光感受器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，也没有完全了解这种转换是如何告诉生物关于它们周围的光的。一项重大突破是成功地使用耐辐射变形杆菌的Phy通过X射线结晶学确定了生色团结合模块的第一个三维结构为Pr。这种结构显示了碧林色素的结构以及它是如何被夹在其结合口袋内的，确定了一个稳定口袋的八字结，发现了姐妹物理之间迄今未知的二聚化结构域，并揭示了植物物理是如何从它们的微生物祖先进化而来的。在之前由NSF资助的研究中，通过使用耐热蓝藻聚球藻OSB的Phy来确定发色团口袋的第一对PR和PFR溶液结构方面取得了进展。通过对这些结构的比较，我们第一次得以一窥物理如何在基态和活化态之间进行光转换。与预期相反，在Pr到PfR的光转化过程中，发现比林颜料的A吡咯环而不是D环发生旋转。这种翻转导致多肽内部的结构重排，然后似乎改变了Phy二聚体内相邻输出域之间的接触，最终调制信号。这个更新项目的智力优势是建立在这些结构研究的基础上来回答关键问题，包括：这是所有物理的光转化的中心A环旋转吗？一个完整的Phy二聚体的结构是什么？色素的旋转以及结合口袋内的结构变化如何改变Phy信号？对这项工作具有重要意义的是开发能够产生大量组装的光感受器的重组系统，以及研究一组新的物理，这些物理可以在蓝光和绿光吸收形式之间进行光转换，这应该有助于分析光激活状态。具体地说，这项研究计划将：(1)结合核磁共振光谱和X射线结晶学为光转化过程中A环的旋转提供进一步的支持，(2)使用X射线结晶学来开发更完整的物理结构，(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)使用生化方法进一步了解光驱动的Phy二聚体的构象变化如何调节信号传递。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必要的框架。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。

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.