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

NSF-BSF：顺磁金属离子标记方法测量铜绿假单胞菌转录激活机制

• 批准号: 2006154
• 负责人: Sunil Saxena
• 金额: $ 86.74万
• 依托单位: University of Pittsburgh
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2006154&HistoricalAwards=false
• 关键词: NSF; BSF; Paramagnetic; metal; ion

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.

本项目将开发实验和模拟方法来阐明铜绿假单胞菌CueR蛋白的传感和转录机制。CueR以非常高的亲和力与细胞内的铜离子结合，导致该蛋白质与DNA上的特定位置结合。这反过来又激活了蛋白质的转录，将金属离子带出细胞，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，并允许继续多管齐下的努力，以增加代表不足的群体参与科学的机会。这些措施包括扩展到高中生，向两个主要是本科生的机构提供电子顺磁共振(EPR)仪器的使用，为本科生课程创建新的实验室，以及对不同群体的学生进行研究生和本科生研究培训。该项目将对细菌中铜金属传感器的传感和转录机制产生全面的了解。该项目将开发用Cu2+离子对DNA进行定位、标记和建模的方法，并开发使用新型Cu2+标记蛋白质来测量特定部位动力学的技术。此外，还将使用点对点距离、弹性网络建模和分子动力学来获得铜绿假单胞菌CueR蛋白和DNA在转录周期中不同功能状态的结构信息。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和蛋白质中的结构约束和灵活性。该项目由生物科学中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智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Copper Based Site-directed Spin Labeling of Proteins for Use in Pulsed and Continuous Wave EPR Spectroscopy

用于脉冲和连续波 EPR 光谱的蛋白质铜基定点自旋标记

• DOI: 10.21769/bioprotoc.4258
• 发表时间: 2021
• 期刊: BIO-PROTOCOL
• 影响因子: 0.8
• 作者: Singewald, Kevin;Wilkinson, James;Saxena, Sunil
• 通讯作者: Saxena, Sunil

Double Histidine Based EPR Measurements at Physiological Temperatures Permit Site‐Specific Elucidation of Hidden Dynamics in Enzymes

生理温度下基于双组氨酸的 EPR 测量允许位点特定阐明酶中隐藏的动力学

• DOI: 10.1002/ange.202009982
• 发表时间: 2020
• 期刊: Angewandte Chemie
• 作者: Singewald, Kevin;Bogetti, Xiaowei;Sinha, Kaustubh;Rule, Gordon S.;Saxena, Sunil
• 通讯作者: Saxena, Sunil

Buffer effects on site directed Cu2+-labeling using the double histidine motif

• DOI: 10.1016/j.jmr.2020.106848
• 发表时间: 2020-11-01
• 期刊: JOURNAL OF MAGNETIC RESONANCE
• 影响因子: 2.2
• 作者: Jarvi, Austin Gamble;Casto, Joshua;Saxena, Sunil
• 通讯作者: Saxena, Sunil

Beyond structure: Deciphering site‐specific dynamics in proteins from double histidine‐based EPR measurements

• DOI: 10.1002/pro.4359
• 发表时间: 2022-06
• 期刊: Protein Science
• 影响因子: 8
• 作者: Kevin Singewald;James A. Wilkinson;Zikri Hasanbasri;S. Saxena

Nanoscale spin detection of copper ions using double electron-electron resonance at room temperature

• DOI: 10.1103/physrevb.104.224412
• 发表时间: 2021-01
• 期刊: Physical Review B
• 影响因子: 3.7
• 作者: Kai Zhang;Shreya Ghosh;S. Saxena;M. Dutt