The Tangled Signaling Pathway of a Bacterial Phytochrome

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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.