Structural Characterization of Proteins and Assemblies by Solid State NMR

通过固态 NMR 进行蛋白质和组装体的结构表征

• 批准号: 1412253
• 负责人: Ann McDermott
• 金额: $ 116.99万
• 依托单位: Columbia University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1412253&HistoricalAwards=false
• 关键词: Structural; Characterization; Proteins; Assemblies; Solid

Purification is a normal requirement for determining the structure of biological macromolecules. However, these structures may differ depending on whether the macromolecule is in the cell interacting with its partner, or purified from cellular components. Thus, the physiological relevance of many structures remains unknown. This project will develop new experiments to characterize biological molecules in purified form, in living cells and even in whole organisms. The technique to be used, solid-state nuclear magnetic resonance (NMR) provides information about both the structure and dynamics. An important challenge for these experiments is that they often require large amounts of material, and, therefore, the studies are often limited to simple macromolecules studied in their purified forms, rather than in a native biological context. Dynamic nuclear polarization (DNP) is a powerful technique for enhancing NMR sensitivity. This project will develop DNP experiments that capture signals from amounts of material typically found in living cells. Knowledge of structures of biological macromolecules benefits society because it enables detailed understanding of basic biological processes. The proposed activities will also benefit society through training of new scientists and outreach activities.The project will use dynamic nuclear polarization to study full viral particles of the bacteriophage Pf1, which is both an important biological agent and a template for nanotechnology. The knowledge gained regarding the virus structure, molecular recognition, solvation, and dynamics, will contribute to a broader understanding of self-assembly. The project will also develop improvements in DNP technology. DNP operates by transferring signal strength from the unpaired electrons of stable free radicals to nuclei that are then detected in the NMR experiments. These compounds, containing two closely spaced stable free radicals, are mixed with the sample. To better deliver the radical compounds, the project will develop ways to attach them to proteins in ways that facilitate specific enhancements. Moreover, tagging the proteins will facilitate detailed studies to better understand the mechanism of polarization transfer.

纯化是确定生物大分子结构的正常要求。 然而，这些结构可能会有所不同，这取决于大分子是在细胞中与其配偶体相互作用，还是从细胞组分中纯化。因此，许多结构的生理相关性仍然未知。该项目将开发新的实验来表征纯化形式、活细胞甚至整个生物体中的生物分子。所使用的技术，固态核磁共振（NMR）提供有关结构和动力学的信息。这些实验的一个重要挑战是，它们通常需要大量的材料，因此，研究往往限于以其纯化形式研究的简单大分子，而不是在天然生物学背景下。动态核极化（DNP）是一种提高核磁共振灵敏度的有效技术。该项目将开发DNP实验，从活细胞中通常发现的大量材料中捕获信号。生物大分子结构的知识有益于社会，因为它使人们能够详细了解基本的生物过程。拟议的活动还将通过培训新的科学家和外联活动造福社会，该项目将利用动态核极化研究噬菌体Pf1的完整病毒颗粒，噬菌体Pf1既是一种重要的生物制剂，也是纳米技术的模板。所获得的关于病毒结构，分子识别，溶剂化和动力学的知识，将有助于更广泛地理解自组装。该项目还将改进DNP技术。DNP通过将信号强度从稳定自由基的未成对电子转移到核来工作，然后在NMR实验中检测到核。这些化合物含有两个紧密间隔的稳定自由基，与样品混合。为了更好地提供自由基化合物，该项目将开发将它们附着在蛋白质上的方法，以促进特定的增强。此外，标记的蛋白质将有助于详细的研究，以更好地了解极化转移的机制。

项目成果

Pf1 bacteriophage hydration by magic angle spinning solid-state NMR

• DOI: 10.1063/1.4903230
• 发表时间: 2014-12-14
• 期刊: JOURNAL OF CHEMICAL PHYSICS
• 影响因子: 4.4
• 作者: Sergeyev, Ivan V.;Bahri, Salima;McDermott, Ann E.
• 通讯作者: McDermott, Ann E.

Efficient assignment and NMR analysis of an intact virus using sequential side-chain correlations and DNP sensitization

• DOI: 10.1073/pnas.1701484114
• 发表时间: 2017-05-16
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Sergeyev, Ivan V.;Itin, Boris;McDermott, Ann E.
• 通讯作者: McDermott, Ann E.