Heteronuclear Multi-dimensional In-Cell NMR Spectroscopy

描述(申请人提供)：核磁共振波谱是对分子体系进行详细表征的最具选择性的方法之一。它在生物大分子研究中的重要性已得到公认，特别是在结构确定和在纯化或重组系统中探测分子动力学方面。然而，使用核磁共振波谱来表征细胞环境中的大分子受到检测灵敏度和用于获取详细分析所需的异核多维核磁共振数据的传统方法的限制。关于活细胞中拥挤而复杂的隔室中大分子成分的基本性质的问题，对于我们理解正常和致病条件下的生物功能和涉及的系统生物学是至关重要的。这一探索性项目旨在开发两种新的、有前途的核磁共振技术以及先进的生物合成表达策略，目的是克服过去的限制，并应用先进的核磁共振方法直接研究活细胞和细胞环境中的蛋白质和蛋白质相互作用。在600、800和900 MHz的高场核磁共振波谱仪中使用低温冷却探头，同时采用常规和耐盐探头设计，以提高实验的灵敏度。数据将通过降维实验获得，并通过投影重建方法进行分析，以最大限度地减少多维实验所需的时间。这种类型的快速核磁共振方法被用来收集与细胞存活率相适应的时间范围内的数据。我们设想在活细胞中直接观察异核多维核磁共振谱，并将其用于蛋白质共振的从头分配。这些数据为评估蛋白质的全球组织和监测体内蛋白质-蛋白质、蛋白质-配体和蛋白质-DNA相互作用提供了基础。 该项目的主要目标是开发强大的磁共振波谱(核磁共振)方法来表征活细胞中的蛋白质。了解单个蛋白质在复杂和拥挤的细胞环境中的行为对于系统生物学的研究至关重要。新的快速数据采集方法和新一代超灵敏核磁共振探头为这种细胞内光谱提供了使能技术。

DESCRIPTION (provided by applicant): Nuclear Magnetic Resonance Spectroscopy is one of the most selective methods for detailed characterization of molecular systems. Its importance in studies of biological macromolecules is well recognized particularly for structure determination and for probing molecular dynamics in purified or reconstituted systems. The use of NMR spectroscopy to characterize macromolecules in cellular environments, however, has been limited by sensitivity of detection and by the conventional approaches used for acquiring the heteronuclear multi- dimensional NMR data needed for detailed analysis. Fundamental questions regarding the properties of macromolecular components in the crowded and complex compartments contained in living cells are of prime importance to our understanding of biological function and the systems biology involved under normal and pathogenic conditions. This exploratory project is designed to exploit two new and promising NMR technologies along with advanced biosynthetic expression strategies for the purpose of overcoming past limitations and applying advanced NMR methods to study proteins and protein interactions directly in living cells and cellular environments. Cryogenically cooled probes in high field NMR spectrometers operating at 600, 800 and 900 MHz with both conventional and salt tolerant probe designs will be used to enhance the sensitivity of the experiment. Data will be acquired with reduced dimensionality experiments and analyzed by projection reconstruction methods to minimize the time needed for the multi-dimensional experiments. Fast NMR methods of this type are used to collect data on a timeframe compatible with cell viability. We envision direct observation of heteronuclear, multi-dimensional NMR spectra in living cells and their use in de novo assignment of protein resonances. These data provide the foundation for assessing global organization of proteins and monitoring protein-protein, protein-ligand, and protein-DNA interactions in vivo. The primary aim of this project is to develop powerful magnetic resonance spectroscopy (NMR) methods to characterize proteins in living cells. Understanding the behavior of individual proteins in the complex and crowded environment of the cell is critical to studies of systems biology. New fast data acquisition methods along with new generation ultra sensitive NMR probes provide enabling technologies for this in-cell spectroscopy.