Roles of Heme and Protein Conformation in Ligand Binding Cooperativity and Selectivity in Bacterial Globin Coupled Sensors

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With the support of the Chemistry of Life Processes program in the Division of Chemistry, Emily Weinert and her research group at Pennsylvania State University will investigate the signaling mechanisms of proteins that allow bacteria to respond to oxygen and nitric oxide. Bacteria are essential for the health of plants, animals, humans, and ecosystems, but some species also can cause detrimental effects, such as infections. To respond to changes in their environment, bacteria use proteins to sense oxygen/nitric oxide and then alter metabolism and phenotypes to enable survival. By understanding how bacteria respond to changing oxygen/nitric oxide levels, methods can be developed to alter bacterial behavior or engineer bacteria to elicit a specific function. The proposed research will characterize how sensing proteins recognize and respond to oxygen and/or nitric oxide and will use this knowledge to investigate the possibility of rewiring bacterial regulation. This project will provide specialized training for students in protein chemistry, spectroscopy, and structural mass spectrometry. In addition, the outreach program will integrate research on bacterial sensing and signaling into lesson plans, experiments using readily available materials, and a board game for high school and undergraduate students. Dissemination of the outreach materials online and through presentations at high schools in rural communities surrounding Penn State University is expected to allow the results of the research program to reach a wide audience.This research project seeks to characterize the mechanisms of ligand binding cooperativity and selectivity within a family of bacterial heme proteins, termed globin coupled sensors. Globin coupled sensor proteins are widely distributed within bacteria and often contain output domains involved in cyclic di-GMP metabolism, which controls phenotypes such as biofilm formation, motility, and virulence. A range of techniques, including equilibrium ligand binding, enzyme kinetics, site-directed mutagenesis, resonance Raman spectroscopy, and hydrogen-deuterium exchange mass spectrometry will be used to interrogate the interactions that allow for communication between heme domains and ligand-selective effects on the linked c-di-GMP metabolic domains. Studies in bacteria will interrogate the effects of ligand binding cooperativity and selectivity on c-di-GMP signaling and phenotypes within bacteria. Results from this study are expected to provide new information about the mechanisms controlling cooperative ligand binding, bacterial gas-sensing proteins, c-di-GMP metabolic domain regulation, and modulation of bacterial biofilm formation and virulence.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.