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

该项目由生物分子系统和细胞系统集群联合资助。一系列复杂的光感受器协调原核生物和真核生物对其周围光环境的反应。 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，使用胆色素（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，R-吸收Pr形式和FR-吸收Pfr形式感测红光（R）和远红光（FR）。 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充当光调节开关。 尽管它们在植物和微生物中的农业重要性和进化保守性，但尚未完全理解光敏色素如何在分子水平上在Pr和Pfr之间进行光转换，或者这种转换如何在光敏色素控制下启动响应。 先前NSF资助的研究提供了一个重大突破，通过生成植物色素组的胆色素结合结构域的第一个3-D结构作为Pr，确定了光敏色素在原子水平上如何工作。该结构最终确定了胆色素的化学和构象，揭示了发色团口袋独特地折叠成罕见的8字形结，确定了迄今未知的二聚化接触，并为植物Phys如何从其微生物祖先中产生提供了重要线索。 该项目继续进行结构分析，以确定光敏色素如何从Pr光转换为Pfr，光敏色素如何进化，以及生物体如何组装打结蛋白。 特别是，该项目将确定光敏色素较大片段的三维结构，最终目标是推导出整个光敏色素结构，（2）通过利用更适合结构研究的新型天然光敏色素确定神秘的Pfr形式的第一个三维结构，（3）确定参与Pr到Pfr光化学，二聚化，结组装，并最终通过各种生物化学和生物物理测定或直接响应光敏色素信号的体内系统进行信号传递。 该项目将有助于阐明微生物和植物如何感知其光环境，这可能对理解微生物生态系统，控制重要的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战略产生重要影响。 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/大学的合作安排加强科学基础设施，培训少数民族大学生掌握现代分子技术。这个学生培训项目将为来自西亚拉巴马大学的本科生提供暑期研究经验。

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.