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

基本信息

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

项目摘要

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.

该项目将开发实验和模拟方法来阐明铜绿假单胞菌蛋白 CueR 的传感和转录机制。 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地努力，以增加代表性不足的群体参与科学的机会。其中包括向高中生进行推广、向两个主要本科院校提供电子顺磁共振波谱 (EPR) 仪器、为本科生课程创建新实验室以及对不同学生群体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态的技术。此外，点对点距离、弹性网络模型和分子动力学将用于获取转录周期中不同功能状态的铜绿假单胞菌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学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。该项目得到了生物科学理事会分子和细胞生物科学部分子生物物理学和遗传机制集群的支持。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值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

期刊论文数量（22）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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
影响因子：
0
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