Structural Characterization of Proteins and Assemblies by Solid State NMR

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伴侣相互作用，还是从细胞成分中纯化出来。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，以纯化形式，活细胞甚至整个生物体中表征生物分子。所使用的技术，固态核磁共振（NMR）提供了有关结构和动力学的信息。这些实验面临的一个重要挑战是，它们通常需要大量的材料，因此，这些研究往往局限于以纯化形式研究的简单大分子，而不是在原生生物环境中研究。动态核极化（DNP）是提高核磁共振灵敏度的有力技术。该项目将开发DNP实验，从活细胞中通常发现的大量物质中捕获信号。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地了解基本的生物过程。拟议的活动还将通过培训新科学家和外联活动使社会受益。该项目将利用动态核极化研究噬菌体Pf1的全病毒颗粒，这既是一种重要的生物制剂，也是纳米技术的模板。获得的有关病毒结构、分子识别、溶剂化和动力学的知识将有助于更广泛地了解自组装。该项目还将改进DNP技术。DNP的作用是将稳定自由基的未配对电子的信号强度转移到核中，然后在核磁共振实验中检测到。这些含有两个紧密间隔的稳定自由基的化合物与样品混合。为了更好地传递自由基化合物，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，标记蛋白质将有助于详细研究，以更好地了解极化转移的机制。

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.