Development and Application of In-Cell NMR Techniques

描述(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，核磁共振是唯一能够在接近生理条件下的溶液中提供这一信息的方法。然而，即使是核磁共振结构仍然是在体外确定的，而且通常选择缓冲条件并不是因为它们与蛋白质的自然环境最匹配，而是为了优化实验参数，如溶解度和灵敏度，或者将可能干扰的核磁共振缓冲信号降到最低。根据自然宿主细胞和确切的细胞室，这些核磁共振缓冲条件可能与蛋白质的自然环境有很大不同，并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，核磁共振波谱作为一种非侵入性的光谱技术，应该能够提供关于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，在活的大肠杆菌中，确实可以通过核磁共振观察到蛋白质的构象和动力学。显然，发展这些真核细胞的“细胞内”核磁共振实验将为研究蛋白质的行为以及它们在自然环境中与其他细胞成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变化。此外，这些技术还可以用来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在制药行业中已经是一种常见的工具，但“细胞内”药物筛选不仅可以识别潜在的感兴趣的分子，还可以显示这些分子是否可以穿透细胞膜并与其细胞内的目标相互作用。基于理论考虑，在真核细胞中进行细胞内核磁共振实验应该是可行的。在这项拨款申请中，我们建议将我们为细菌开发的细胞内核磁共振技术扩展到真核模型系统。特别是，我们将利用在酵母和SF9昆虫细胞中获得的高过表达水平。此外，我们建议探索将纯化的蛋白质注入非洲爪哇卵母细胞并使用这些细胞进行细胞内核磁共振的可能性。

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.