NSF/MCB-BSF The virtues of lanthanides and fluorine for tracking in-cell protein conformation: a marriage of NMR and EPR

NSF/MCB-BSF 镧系元素和氟在追踪细胞内蛋白质构象方面的优点：NMR 和 EPR 的结合

• 批准号: 2116534
• 负责人: Angela Gronenborn
• 金额: $ 105.01万
• 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: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2116534&HistoricalAwards=false
• 关键词: NSF; MCB; BSF; virtues; lanthanides

The goal of this project is to develop experimental tools for understanding the structures of proteins inside living cells while the proteins carry out their functions. The tools to be developed will measure distances within and between proteins using a combination of in-cell nuclear magnetic resonance (NMR) spectroscopy and electron paramagnetic resonance (EPR) spectroscopy, two powerful approaches for tracking the structure and movement of a protein. Development of these approaches for making measurements inside live cells will have broad application as we expect to obtain 1) benchmark data for in-cell NMR and EPR that can be used by other practitioners of these methodologies (biochemists, biophysicist) and 2) biological insight that may open new strategies for understanding protein function in cells and when bound by drugs. As such, the proposed research will impact several areas of science and engineering, including physical chemistry, structural biochemistry, magnetic resonance spectroscopy, biotechnology, and pharmacology. The proposed innovative research program will provide unique training for students at all levels in state-of-the-art experimental methods. Importantly, a program within this project will empower emerging female scientists in magnetic resonance and provide a framework for populating the next generation of females in leadership roles in the global STEM workforce.Biophysical analyses of biomolecular structure and properties is typically performed on the isolated biomolecule, removed from its native environment, such as the cell. By necessity, such analyses ignore or grossly simplify cellular influences. Although this strategy has provided and continues to provide indispensable information on molecular structure, it leaves open many questions about how that information relates to interactions and function within the cell, where innumerable organelles and molecular machines engage in an intricate dance, spatially and temporally, interacting and changing shape as they perform their tasks. The main objective of this project is to begin addressing these open questions by developing an integrated 19F NMR/EPR approach that informs on the structures of proteins in their native cellular environments. Specifically, the program will establish a combined 19F-PRE (paramagnetic relaxation enhancement) and 19F-PCS (pseudo contact shift) NMR approach to measure distances in the range of 5-50 Å and a combined Gd(III)-Gd(III) DEER (double electron-electron resonance) and Gd(III)-19F ENDOR/ED-NMR (electron nuclear double resonance/electron-electron double resonance (ELDOR)-detected NMR) approach to measure distances in the 5-80 Å range. Together, the united methodology will provide complementary information that is inaccessible when using either approach alone and will permit a more complete characterization of structure and intra- or intermolecular interactions in the context of function. This project is funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences.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.

该项目的目标是开发实验工具，以便在蛋白质执行其功能时了解活细胞内蛋白质的结构。即将开发的工具将使用细胞内核磁共振(核磁共振)光谱和电子顺磁共振(EPR)光谱的组合来测量蛋白质内部和之间的距离，这两种方法是跟踪蛋白质结构和运动的两种强大方法。这些在活细胞内进行测量的方法的开发将有广泛的应用，因为我们希望获得1)细胞内核磁共振和EPR的基准数据，这些数据可以被这些方法的其他从业者(生物化学家、生物物理学家)使用，以及2)生物学洞察力，可能为理解细胞中的蛋白质功能和当被药物结合时打开新的策略。因此，拟议的研究将影响科学和工程的几个领域，包括物理化学、结构生物化学、磁共振光谱学、生物技术和药理学。拟议的创新研究计划将为各级学生提供最先进的实验方法方面的独特培训。重要的是，该项目中的一个项目将赋予新兴女性科学家磁共振方面的能力，并提供一个框架，让下一代女性在全球STEM工作队伍中担任领导角色。生物分子结构和性质的生物物理分析通常是在分离的生物分子上进行的，这些生物分子从其自然环境中移除，如细胞。不可避免地，这样的分析忽略或粗略地简化了细胞影响。尽管这一策略已经并将继续提供关于分子结构的必不可少的信息，但它留下了许多问题，即这些信息如何与细胞内的相互作用和功能有关，在细胞内，无数的细胞器和分子机器在空间和时间上进行复杂的舞蹈，在执行任务时相互作用和改变形状。该项目的主要目标是通过开发一种集成的~(19)F核磁共振/电子顺磁共振方法来开始解决这些开放的问题，该方法可以了解蛋白质在其天然细胞环境中的结构。具体地说，该计划将建立19F-PRE(顺磁松弛增强)和19F-PCS(伪接触位移)核磁共振相结合的方法来测量5-50°范围内的距离，并建立Gd(III)-Gd(III)鹿(双电子-电子共振)和Gd(III)-19F Endor/ED-核磁共振(电子核双共振/电子-电子双共振(Eldor)检测的核磁共振)相结合的方法来测量5-80°范围内的距离。总而言之，联合方法学将提供单独使用任何一种方法时无法获得的补充信息，并将允许在功能范围内更完整地描述结构和分子内或分子间相互作用。该项目由分子和细胞生物科学部的分子生物物理组资助。这一奖项反映了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

Gd III ‐ 19 F Distance Measurements for Proteins in Cells by Electron‐Nuclear Double Resonance

通过电子-核双共振测量细胞中蛋白质的 Gd III – 19 F 距离

• DOI: 10.1002/anie.202218780
• 发表时间: 2023
• 期刊: Angewandte Chemie International Edition
• 作者: Seal, Manas;Zhu, Wenkai;Dalaloyan, Arina;Feintuch, Akiva;Bogdanov, Alexey;Frydman, Veronica;Su, Xun‐Cheng;Gronenborn, Angela M.;Goldfarb, Daniella
• 通讯作者: Goldfarb, Daniella