Paramagetic metal ion based ESR distance methods for measuring ion-channel conformations in cells and in vitro

基于顺磁金属离子的 ESR 距离方法，用于测量细胞内和体外离子通道构象

• 批准号: 1613007
• 负责人: Sunil Saxena
• 金额: $ 85万
• 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: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1613007&HistoricalAwards=false
• 关键词: Paramagetic; metal; ion; based; ESR

Proteins are large biopolymers made up of amino acids and participate in most processes within cells. Their structure and the dynamics or flexibility of that structure is essential to protein function. This project will develop Electron Spin Resonance (ESR) methodology to measure precise distance constraints that are reporters of protein backbone structure and flexibility. In this project, a Copper ion based spin probe will be developed for the ESR measurements. The copper spin probe can be assembled without modification of the protein, and is much smaller that conventional probes and thus will enable more precise distance measurements. This project will measure changes in protein structure directly in cells under conditions where functional measurements can be performed. While training a number of graduate and undergraduate students, this project will expand access to pulsed ESR instrumentation to undergraduates and graduate students at regional colleges in Pennsylvania including those serving predominantly African American populations. This project will also provide hands on science experience, career awareness and academic advising to assist pre-college historically under-represented students in STEM fields.This project will develop strategies to exploit native protein residues to site-specifically and selectively attach Copper(II) for pulsed ESR distance measurements both in-vitro and in cells; and probe initial steps in the gating mechanism of ELIC, a prokaryotic, pentameric, ligand-gated ion channel from Erwinia chrysanthemi in lipid vesicles and in cells. A Copper(II)-based spin probe that can be assembled in situ from natural amino acid residues and a metal salt, without the need for post-expression synthetic modification, will be developed and used. This probe will be used to determine distance constraints that are more narrowly defined and overcome the inherent limitation of the current technology which relies on a spin label with a highly flexible side-chain. Conformational changes in the extracellular domain of ELIC that lead to the opening of the pore upon ligand binding will be determined and such changes will then be measured directly in cells under functional conditions. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.

蛋白质是由氨基酸组成的大型生物聚合物，参与细胞内的大多数过程。 它们的结构以及该结构的动力学或灵活性对蛋白质功能至关重要。 该项目将开发电子自旋共振（ESR）方法，以测量蛋白质骨架结构和灵活性的精确距离限制。在本计画中，我们将发展一种以铜离子为基底的自旋探针，以进行ESR测量。 铜自旋探针可以在不修改蛋白质的情况下组装，并且比传统探针小得多，因此能够实现更精确的距离测量。该项目将在可以进行功能测量的条件下直接测量细胞中蛋白质结构的变化。在培训一些研究生和本科生的同时，该项目将使宾夕法尼亚州区域学院的本科生和研究生（包括主要为非洲裔美国人服务的学院）有更多机会使用脉冲ESR仪器。该项目还将提供科学经验，职业意识和学术建议，以帮助在STEM领域历史上代表性不足的大学前学生。该项目将制定策略，利用天然蛋白质残基来位点特异性和选择性地附着铜（II），用于体外和细胞中的脉冲ESR距离测量;并探索ELIC门控机制的初始步骤，ELIC是脂囊泡和细胞中来自菊欧文氏菌的原核五聚体配体门控离子通道。将开发和使用一种基于铜（II）的自旋探针，该探针可以从天然氨基酸残基和金属盐原位组装，而不需要表达后的合成修饰。该探针将用于确定更窄定义的距离约束，并克服依赖于具有高度柔性侧链的自旋标记的当前技术的固有限制。将测定ELIC的胞外结构域中导致配体结合时孔打开的构象变化，然后在功能条件下在细胞中直接测量这种变化。该项目得到了生物科学理事会分子和细胞生物科学司分子生物物理学小组的支持。

项目成果

Cu(II) EPR Reveals Two Distinct Binding Sites and Oligomerization of Innate Immune Protein Calgranulin C

• DOI: 10.1007/s00723-018-1053-7
• 发表时间: 2018-11-01
• 期刊: APPLIED MAGNETIC RESONANCE
• 影响因子: 1
• 作者: Ghosh, Shreya;Garcia, Velia;Saxena, Sunil
• 通讯作者: Saxena, Sunil

Increasing nitroxide lifetime in cells to enable in-cell protein structure and dynamics measurements by electron spin resonance spectroscopy

• DOI: 10.1016/j.jmr.2018.12.005
• 发表时间: 2019-02-01
• 期刊: JOURNAL OF MAGNETIC RESONANCE
• 影响因子: 2.2
• 作者: Singewald, Kevin;Lawless, Matthew J.;Saxena, Sunil
• 通讯作者: Saxena, Sunil

Cu2+-based distance measurements by pulsed EPR provide distance constraints for DNA backbone conformations in solution

• DOI: 10.1093/nar/gkaa133
• 发表时间: 2020-05-21
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Ghosh, Shreya;Lawless, Matthew J.;Saxena, Sunil
• 通讯作者: Saxena, Sunil

Effects of MnO2 of different structures on activation of peroxymonosulfate for bisphenol A degradation under acidic conditions

• DOI: 10.1016/j.cej.2019.03.238
• 发表时间: 2019-08-15
• 期刊: CHEMICAL ENGINEERING JOURNAL
• 影响因子: 15.1
• 作者: Huang, Jianzhi;Dai, Yifan;Zhang, Huichun
• 通讯作者: Zhang, Huichun

Rotamer Modelling of Cu(II) Spin Labels Based on the Double-Histidine Motif

• DOI: 10.1007/s00723-018-1052-8
• 发表时间: 2018-11-01
• 期刊: APPLIED MAGNETIC RESONANCE
• 影响因子: 1
• 作者: Ghosh, Shreya;Saxena, Sunil;Jeschke, Gunnar
• 通讯作者: Jeschke, Gunnar