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

该项目的目标是开发实验工具，以了解活细胞内蛋白质的结构，同时蛋白质执行其功能。待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。这些方法的发展，使活细胞内的测量将有广泛的应用，因为我们希望获得1）细胞内NMR和EPR的基准数据，可以使用这些方法的其他从业者（生物化学家，生物制药师）和2）生物学的见解，可能会打开新的策略，了解蛋白质功能的细胞和药物结合时。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的独特培训。重要的是，该项目中的一个项目将赋予新兴的女性科学家在磁共振领域的能力，并为全球STEM劳动力中的下一代女性领导角色提供一个框架。生物分子结构和特性的生物物理分析通常是在分离的生物分子上进行的，从其天然环境中取出，如细胞。这种分析必然会忽略或严重简化细胞的影响。尽管这种策略已经并将继续提供关于分子结构的不可或缺的信息，但它留下了许多关于这些信息如何与细胞内的相互作用和功能相关的问题，在细胞内，无数的细胞器和分子机器在空间和时间上进行着复杂的舞蹈，它们在执行任务时相互作用并改变形状。该项目的主要目标是开始通过开发一种集成的19 F NMR/EPR方法来解决这些悬而未决的问题，该方法可以告知蛋白质在其天然细胞环境中的结构。具体地说，该计划将建立一个19 F-PRE组合（顺磁弛豫增强）和19 F-PCS（伪接触位移）NMR方法来测量5-50 nm范围内的距离，以及组合的Gd（III）-Gd（III）DEER（双电子-电子共振）和Gd（III）-19F ENDOR/ED-NMR在一些实施方案中，使用电子核双共振/电子-电子双共振（ELDOR）检测的NMR）方法来测量5-80 nm范围内的距离。总之，统一的方法将提供互补的信息，这是无法单独使用任何一种方法时，将允许更完整的表征结构和内部或分子间的相互作用的功能。该项目由分子和细胞生物科学部的分子生物物理学小组资助。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.