Development and Application of In-Cell NMR Techniques

细胞内核磁共振技术的开发与应用

• 批准号: 6505439
• 负责人: JAMES L THOMAS
• 金额: $ 11.05万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6505439
• 关键词: Sf9 cell line; Xenopus oocyte; conformation; intermolecular interaction; method development; molecular cloning; molecular dynamics; nuclear magnetic resonance spectroscopy; posttranslational modifications; protein binding; protein folding; protein protein interaction; protein structure function; technology /technique development; yeasts

DESCRIPTION (provided by applicant): Of all methods currently available for obtaining high resolution structures of biological macromolecules, NMR is the only one that can provide this information in solution under near physiological conditions. However, even NMR structures are still determined in vitro, and often buffer conditions are not selected for their closest match to the natural environment of the protein but to optimize experimental parameters such as solubility and sensitivity or to minimize NMR buffer signals that could interfere. Depending on the natural host cell and the exact cellular compartment, these NMR buffer conditions can be substantially different from a protein's natural environment and may influence its structure and dynamics. Furthermore, interactions with other cellular (macro-) molecules and post-translational modifications can alter the conformation. In principle, NMR spectroscopy, as a non-invasive spectroscopic technique, should be able to provide infonnation about the structure and dynamics of biological macromolecules inside living cells. Recently, we have demonstrated that the conformation and the dynamics of proteins can indeed be observed by NMR inside living E. coli bacteria. Clearly, developing these "in-cell" NMR experiments for eukaryotic cells would open new avenues to study the behavior of proteins and their interaction with other cellular components in their natural environment. The biggest advantage of these techniques would not be to determine structures, but to observe structural changes that can; for example, be caused by posttranslational modifications or binding to other cellular components. In addition, these techniques could be used to study the interaction of proteins inside the cell with potential drugs. While NMR-based drug screens are already a common tool in the pharmaceutical industry, an "in-cell" drug screen would, not only identify potentially interesting molecules, but could also show if these molecules can penetrate the cellular membrane and interact with their target inside a cell. Based on theoretical considerations, in-cell NMR experiments should be feasible in eukaryotic cells. In this grant application, we propose to extend our in-cell NMR techniques that we have developed for bacteria to eukaryotic model systems. In particular, we will take advantage of the high overexpression levels obtainable in yeast and in SF9 insect cells. In addition, we propose to explore the possibility to inject purified proteins into xenopus oocytes and to use these cells for in-cell NMR.

描述（由申请人提供）：在目前可用于获得生物大分子的高分辨率结构的所有方法中，NMR是唯一一种可以在接近生理条件下在溶液中提供该信息的方法。然而，即使NMR结构仍然在体外确定，并且通常缓冲液条件不是为了与蛋白质的天然环境最接近匹配而选择的，而是为了优化实验参数，例如溶解度和灵敏度，或者最小化可能干扰的NMR缓冲液信号。根据天然宿主细胞和确切的细胞区室，这些NMR缓冲液条件可以与蛋白质的天然环境显著不同，并且可能影响其结构和动力学。此外，与其他细胞（大）分子的相互作用和翻译后修饰可以改变构象。原则上，NMR光谱学作为一种非侵入性光谱技术，应该能够提供有关活细胞内生物大分子的结构和动力学的信息。最近，我们已经证明，蛋白质的构象和动力学确实可以通过核磁共振在活的大肠杆菌中观察到。大肠杆菌显然，为真核细胞开发这些“细胞内”NMR实验将为研究蛋白质的行为及其与自然环境中其他细胞组分的相互作用开辟新的途径。这些技术的最大优势不是确定结构，而是观察可能的结构变化;例如，由翻译后修饰或与其他细胞成分结合引起的结构变化。此外，这些技术可用于研究细胞内蛋白质与潜在药物的相互作用。虽然基于核磁共振的药物筛选已经是制药行业的常用工具，但“细胞内”药物筛选不仅可以识别潜在的感兴趣分子，还可以显示这些分子是否可以穿透细胞膜并与细胞内的靶标相互作用。基于理论上的考虑，细胞内NMR实验在真核细胞中应该是可行的。在这项资助申请中，我们建议将我们为细菌开发的细胞内NMR技术扩展到真核模型系统。特别地，我们将利用在酵母和SF 9昆虫细胞中可获得的高过表达水平。此外，我们建议探索的可能性，注射纯化的蛋白质到非洲爪蟾卵母细胞，并使用这些细胞的细胞内NMR。