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

本计画将发展实验与模拟方法，以阐明绿脓杆菌蛋白CueR的感应与转录机制。 CueR以非常高的亲和力与细胞铜离子结合，导致该蛋白质与DNA上的特定位点结合。这反过来又激活了将金属离子带出细胞的蛋白质的转录，以防止毒性和细胞死亡。这项研究的结果将提供蛋白质和DNA结构以及构象动力学的原子细节，从而能够调节细菌细胞内的铜水平。 该项目还将加强国际合作，并允许继续进行多管齐下的努力，以增加代表性不足的群体参与科学的机会。 这些措施包括推广到高中学生，提供电子顺磁共振光谱（EPR）仪器访问两个主要的本科院校，为本科课程的新实验室的创建，以及研究生和本科生的研究培训的一个多元化的学生群体。该项目将产生一个整体的理解的传感和转录机制的铜金属传感器在细菌中。该项目将开发用Cu2+离子对DNA进行位点特异性标记和建模的方法，并开发使用新型蛋白质Cu2+标记来测量位点特异性动力学的技术。此外，点对点距离，弹性网络建模和分子动力学将用于获得在转录周期中不同功能状态下铜绿假单胞菌CueR蛋白和DNA的结构信息。该项目的另一个核心创新方面是，这项工作将为更广泛的生物物理界提供新的自旋标记和模拟方法，以测量DNA和蛋白质的结构约束和灵活性。该项目由生物科学理事会分子和细胞生物科学部的分子生物物理学和遗传机制集群支持。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.