NSF-BSF: Paramagnetic metal-ion labeling methods to measure mechanism of transcriptional activation in P. aeruginosa

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This project will develop experimental and simulation methodology to elucidate the sensing and transcription mechanism of protein CueR from P. aeruginosa. CueR binds to cellular Copper ions with very high affinity that leads to the binding of this protein to a specific site on DNA. This, in turn, activates the transcription of proteins that take the metal ion out of the cell to prevent toxicity and cell death. The results of this study will provide atomistic details of protein and DNA structure and conformational dynamics that enable the regulation of copper levels inside the cells of bacteria. The project will also firm up an international collaboration and allow the continuation of a multi-pronged effort to enhance opportunities for under-represented groups to participate in science. These include outreach to high-school students, providing access of Electron Paramagnetic Resonance Spectroscopy (EPR) instrumentation to two primarily undergraduate institutions, the creation of new labs for undergraduate curriculum, and the graduate and undergraduate research training of a diverse group of students.The project will generate a holistic understanding of the sensing and transcription mechanism of the copper metal sensor in bacteria. The project will develop methodology to site specifically label and model DNA with Cu2+-ions and develop techniques to measure site-specific dynamics using a novel Cu2+ label for proteins. In addition, point to point distances, elastic network modeling and molecular dynamics will be used to obtain structural information on the P. aeruginosa CueR protein and DNA in different functional states during the transcriptional cycle. Another centrally innovative aspect of this project is that the work will provide the wider biophysical community with new spin labeling and simulation approaches to measure structural constraints and flexibility in both DNA as well as proteins. This project is supported by the Molecular Biophysics and Genetic Mechanism Clusters of the Division of Molecular and Cellular Biosciences in Biological Sciences Directorate.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.