The Tangled Signaling Pathway of a Bacterial Phytochrome

光敏色素是一种对光敏感的感觉分子，但细菌究竟是如何感知光的，以及这些信息是如何被用来适应光水平的变化的，目前还不清楚。本项目将探讨蛋白质折叠如何影响可溶性光敏色素的信号通路。此外，该项目将研究其他蛋白质（称为伴侣蛋白）如何促进适当光敏色素活性所需的蛋白质折叠。这些实验有望为光敏色素提供普遍适用的见解，此外，光敏色素将被修改以创建新的显微成像工具，这将允许生物学家窥视生物体以了解更多基本的生物过程。该项目包括推广活动，促进科学家和艺术家之间的互动，以加强科学交流和学习。该项目的目的是了解细菌光敏色素的不寻常的打结拓扑结构如何决定这种光调节组氨酸激酶的信号特性和折叠途径。光敏色素是少数已知的深结蛋白之一，其最终的8字形拓扑结构的路径是完全未知的。为了确定光敏色素的打结蛋白结构及其同源反应调节剂的链状二聚体界面是否有助于分子内和分子间信号传导，该项目将利用激酶、磷酸转移酶和磷酸酶对天然和未打结的（工程的）细菌光敏色素及其同源反应调节剂进行分析。为了确定光敏色素的折叠途径如何依赖于富含脯氨酸的套索环的特征，该项目将研究在含有伴侣的脯氨酸异构酶存在和不存在的情况下，该环的序列和长度变化对光敏色素折叠的影响。为了创建具有理想的近红外激发最大值的小遗传编码荧光团，该项目将通过去除仅用于信号转导的结构域，同时保留稳定折叠和发色团相互作用所需的元素，对光敏色素进行逆向工程。

Phytochromes are light-responsive sensory molecules, but exactly how light is perceived by bacteria and how that information is used to bring about adaptation to changes in light levels is not well understood. This project will explore how protein folding influences the signaling pathways of a soluble phytochrome. Additionally, this project will investigate how other proteins (called chaperones) facilitate the protein folding required for proper phytochrome activity. These experiments promise to provide generally applicable insights into phytochromes, and in addition phytochromes will be modified to create new microscopic imaging tools which will permit biologists to peer into living organisms to learn more about basic biological processes. The project includes outreach activities that facilitate interaction between scientists and artists to enhance science communication and learning. The objective of this project is to understand how the unusual knotted topology of bacterial phytochrome determines the signaling properties and folding pathway of this light-regulated histidine kinase. Phytochrome is one of few deeply knotted proteins known, and the route to its figure-of-eight final topology is completely unknown. To determine whether the knotted protein structure of phytochrome and the catenated dimeric interface of its cognate response regulator contribute to intra- and intermolecular signaling, the project will utilize kinase, phosphotransferase, and phosphatase assays on native and untangled (engineered) bacterial phytochromes and their cognate response regulators. To determine how the folding pathway of knotted phytochrome is dependent on characteristics of the proline-rich lasso loop the project will investigate the effects of sequence and length variation in this loop on folding of phytochromes in the presence and absence of a prolyl-isomerase containing chaperone. To create small genetically encoded fluorophores with desirable near infrared excitation maxima, the project will reverse engineer phytochrome by removing domains required only for signal transduction while maintaining elements required for stable folding and chromophore interaction.