Structural Characterization of Protein and Protein Assemblies by Solid State NMR

通过固态 NMR 进行蛋白质和蛋白质组装体的结构表征

• 批准号: 1913885
• 负责人: Ann McDermott
• 金额: $ 115万
• 依托单位: Columbia University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1913885&HistoricalAwards=false
• 关键词: Structural; Characterization; Protein; Assemblies; Solid

Structures inside living cells are often built from numerous copies of one protein. Like a brick wall made from many individual bricks, an oligomer can have size and strength unattainable by individual proteins. For example, during bacterial growth and division, a cell is pinched into two daughter cells by the action of a protein called FtsZ, many copies of which assemble into a ring that can constrict. In another example, when cells are stressed they respond by halting cellular activity. To do this they create a "mesh" of protein from many copies of TIA-1, which stalls protein synthesis by trapping mRNA in the "mesh". Oligomeric structures can be exquisitely sensitive to events in the cell, much more than an individual protein could be, as illustrated by the classic example of oligomeric hemoglobin which is much more sensitive to oxygen than a single hemoglobin protein. Although oligomeric structures are crucially important, little is known about how the protein molecules pack together to form the larger structures. It is expected that knowledge of the oligomeric structures gained through this project will enhance our understanding of the biological role of these proteins in the cell. This project will have broad impact through mentorship, teaching and outreach. The investigator has a strong record of mentoring women in science and these efforts will continue. Research activities will be showcased through outreach activities for middle and high school students.Although our knowledge of protein structure has advanced considerably over the past 50 years, oligomeric proteins specifically have lagged behind, because they are difficult to study using traditional structural methods. This project pursues oligomeric structures through technical developments in solid state NMR, a promising new method that is well suited for determining structures of oligomeric proteins. In these studies, the signal strength is enhanced through the use of low temperatures and by transfer of signal from electron spin to nuclear spin, in a method called Dynamic Nuclear Polarization. Optimization of sample freezing protocols, and multidimensional detection schemes pursued in this project are expected to improve the low temperature linewidths and enable the process of determining structures. Although most studies will be carried out with in vitro samples, additional methods are developed for elucidating structures within living cells. The strategy for detection within the cell is to selectively enhance signals for the oligomeric protein of interest using a combination of selective isotopic enrichment and selective transfer of polarization from radicals tagged onto the protein of interest.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.

活细胞内的结构通常是由一种蛋白质的许多拷贝组成的。就像由许多单独的砖块组成的砖墙一样，寡聚体可以具有单个蛋白质无法达到的尺寸和强度。例如，在细菌生长和分裂过程中，一个细胞在一种名为FtsZ的蛋白质的作用下被捏成两个子细胞，其中许多拷贝组装成一个可以收缩的环。在另一个例子中，当细胞受到压力时，它们通过停止细胞活动来做出反应。为了做到这一点，他们从TIA-1的许多拷贝中创建了一个蛋白质“网格”，通过将mRNA捕获在“网格”中来阻止蛋白质合成。寡聚体结构可以对细胞中的事件非常敏感，远远超过单个蛋白质，如寡聚血红蛋白的经典例子所示，它对氧气的敏感性远远超过单个血红蛋白。虽然寡聚体结构至关重要，但人们对蛋白质分子如何聚集在一起形成更大的结构知之甚少。预计通过该项目获得的寡聚体结构的知识将增强我们对这些蛋白质在细胞中的生物学作用的理解。 该项目将通过指导、教学和外联产生广泛影响。 该研究员在指导科学领域的妇女方面有着良好的记录，这些努力将继续下去。 通过面向初中生和高中生的宣传活动展示研究活动。虽然我们对蛋白质结构的认识在过去50年中有了很大的进步，但寡聚蛋白质特别落后，因为它们很难用传统的结构方法进行研究。该项目通过固态NMR技术的发展来研究寡聚体结构，这是一种非常适合确定寡聚体蛋白质结构的有前途的新方法。 在这些研究中，通过使用低温和通过将信号从电子自旋转移到核自旋来增强信号强度，这种方法称为动态核极化。在这个项目中，样品冷冻方案的优化和多维检测方案有望改善低温线宽，并使确定结构的过程成为可能。虽然大多数研究将在体外样品中进行，但还开发了其他方法来阐明活细胞内的结构。 细胞内检测的策略是选择性地增强目标寡聚蛋白的信号，使用选择性同位素富集和选择性转移标记在目标蛋白上的自由基的极化。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。

项目成果

Zinc Alters the Supramolecular Organization of Nucleic Acid Complexes with Full-Length TIA1

锌改变全长 TIA1 核酸复合物的超分子组织

• 发表时间: 2023
• 期刊: bioRxiv
• 作者: Yizhuo Yang, Keith J.

Contribution of protein conformational heterogeneity to NMR lineshapes at cryogenic temperatures.

低温下蛋白质构象异质性对 NMR 线形的贡献。

• DOI: 10.1073/pnas.2301053120
• 发表时间: 2024
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Yi,Xu;Fritzsching,KeithJ;Rogawski,Rivkah;Xu,Yunyao;McDermott,AnnE
• 通讯作者: McDermott,AnnE

Stability of nitroxide biradical TOTAPOL in biological samples

• DOI: 10.1016/j.jmr.2019.04.013
• 发表时间: 2019-06-01
• 期刊: JOURNAL OF MAGNETIC RESONANCE
• 影响因子: 2.2
• 作者: McCoy, Kelsey M.;Rogawski, Rivkah;McDermott, Ann E.
• 通讯作者: McDermott, Ann E.

Micellar TIA1 with folded RNA binding domains as a model for reversible stress granule formation

• DOI: 10.1073/pnas.2007423117
• 发表时间: 2020-12-15
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Fritzsching, Keith J.;Yang, Yizhuo;McDermott, Ann E.
• 通讯作者: McDermott, Ann E.