Structural and Functional Analysis of Microbial Phytochromes as Models for the Phytochrome Superfamily

这个项目由生物分子系统和细胞系统集群共同资助。一个复杂的光感受器阵列协调原核生物和真核生物对环境光环境的反应。其中最有影响力的是光敏色素超家族，这是一个庞大而多样的光可逆光感受器群体，使用比林(或线性四吡咯)发色团进行光检测。光敏色素通过两种相对稳定的构象来感知红光(R)和远红光(FR)，即吸收R的Pr形式和吸收FR的PFR形式。通过Pr和Pfr之间的光相互转换，光敏色素在各种信号级联中扮演光调节开关的角色。尽管光敏色素在植物和微生物中具有重要的农业意义和进化保守性，但目前还不完全清楚光敏色素是如何在分子水平上在Pr和Pfr之间进行光转化的，或者这种转换是如何启动光敏色素调控下的反应的。先前由美国国家科学基金会资助的研究在确定光敏色素如何在原子水平上发挥作用方面取得了重大突破，通过产生植物染色体组的胆碱结合结构域的第一个三维结构作为Pr。这一结构最终决定了BLIN的化学和构象，揭示了发色团口袋独特地折叠成一个罕见的八字结，确定了迄今未知的二聚接触，并为植物物理如何从其微生物前体产生提供了重要线索。这个项目继续进行结构分析，以确定光敏色素是如何从PR转化为PFR的，光敏色素是如何进化的，以及生物体一般是如何组装打结蛋白的。特别是，该项目将确定较大片段光敏色素的三维结构，最终目标是推断整个光敏色素结构，(2)通过开发更适合结构研究的新型自然产生的光敏色素来确定神秘的PFR形式的第一个三维结构，以及(3)识别参与PR到PFR光化学、二聚化、结组装以及最终通过各种生化和生物物理分析或直接响应光敏色素信号的体内系统进行信号传递的关键氨基酸。该项目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意义。更广泛的影响：该项目涉及博士后和研究生的研究培训。它还将通过学院/大学合作安排加强科学基础设施，以培训少数族裔本科生掌握现代分子技术。这个学生培训项目将为西阿拉巴马大学的本科生提供暑期研究体验。

This project is funded jointly by the Biomolecular Systems and Cellular Systems clusters.A complex array of photoreceptors coordinates the response of both prokaryotes and eukaryotes to their ambient light environment. One of the most influential is the phytochrome superfamily, a large and diverse group of photoreversible photoreceptors that use a bilin (or linear tetrapyrrole) chromophore for light detection. Phytochromes sense red (R) and far-red light (FR) through two relatively stable conformations, a R-absorbing Pr form and a FR-absorbing Pfr form. By photointerconverting between Pr and Pfr, phytochromes act as light-regulated switches in various signaling cascades. Despite their agricultural importance and evolutionary conservation among plants and microorganisms, it is not fully understood how phytochromes photoconvert between Pr and Pfr at the molecular level or how this switch initiates the responses under phytochrome control. The prior NSF-funded studies provided a major breakthrough toward determining how phytochromes work at the atomic level by generating the first 3-D structure of the bilin-binding domain of a phytochrtome as Pr. This structure conclusively determined the chemistry and conformation of the bilin, revealed that the chromophore pocket is uniquely folded into a rare figure-of-eight knot, identified a heretofore unknown dimerization contact, and provided important clues for how plant Phys arose from their microbial progenitors. This project continues the structural analysis to determine how phytochromes photoconvert from Pr to Pfr, how phytochromes evolved, and how organisms assemble knotted proteins in general. In particular, the project will determine the 3-D structures of larger fragments of phytochromes, toward the eventual goal of deducing an entire phytochrome structure, (2) determine the first 3-D structure of the enigmatic Pfr form by exploiting novel naturally-occurring phytochromes that are more amenable to structural studies, and (3) identify key amino acids that participate in Pr to Pfr photochemistry, dimerization, knot assembly, and ultimately signal transmission by various biochemical and biophysical assays or in vivo systems that directly respond to phytochrome signals. This project will 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 both food and biofuel crops. Broader Impacts:The project involves research training of postdoctoral and graduate students. It will also enhance scientific infrastructure via a cooperative college/university arrangement for the training of minority undergraduate students in modern molecular techniques. This student training program will provide summer research experience to undergraduate students from University of West Alabama.