Development and Application of In-Cell NMR Techniques

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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.