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.

该项目的目的是开发实验工具，以了解活细胞内蛋白质的结构，而蛋白质执行其功能。要开发的工具将使用细胞内核磁共振（NMR）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，这是跟踪蛋白质结构和运动的两种强大方法。我们期望获得的这些方法的开发将具有广泛的应用，因为我们希望获得1）基于NMR和EPR的基准数据，这些方法可以由其他从业者使用这些方法（生物化学家，生物物理学家）和2）生物学见解，这些方法可能会在细胞和药物限制的情况下开放新的策略。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，生物技术和药理学。拟议的创新研究计划将为最先进的实验方法的各个级别的学生提供独特的培训。重要的是，该项目中的一个程序将使新兴的女科学家在磁共振中赋予能力，并为在全球STEM劳动力中的领导角色中填充下一代女性的框架。生物分子结构的生物物理分析和特性的生物物理分析通常在孤立的生物分子上表现为从其本地环境中取出的孤立生物分子。必要时，此类分析忽略或严重简化了细胞影响。尽管该策略已经提供并继续提供有关分子结构的必不可少的信息，但它留下了许多有关该信息如何与细胞内的相互作用和功能相关的问题，在该信息中，无数的细胞器和分子机器在空间和临时进行复杂的舞蹈，在执行任务时进行了互动和变化。该项目的主要目的是通过开发一种综合的19F NMR/EPR方法来开始解决这些开放问题，该方法在其天然细胞环境中有关蛋白质结构的信息。具体而言，该程序将建立一个组合的19F-PRE（顺磁松弛增强）和19F-PC（伪接触移动）NMR方法，以测量5-50Å和组合GD（III）-GD（III）-GD（III）-GD（III）鹿的距离（双电子 - 电子）和GD（Double Electectron-Electron Conicance）和GD（III III）-19F-NMR（III）-19F-NMR（共振/电子 - 电子双共振（ELDOR）检测的NMR）方法，用于测量5-80Å范围内的距离。联合方法共同提供了单独使用两种方法时无法访问的完整信息，并且可以在功能背景下对结构和分子间相互作用进行更完整的表征。该项目由分子和细胞生物科学划分的分子生物物理学簇资助。该奖项反映了NSF的法定任务，并使用基金会的知识分子优点和更广泛的影响审查标准，通过评估被认为是宝贵的支持。

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