DNP approach for structural determination of large protein-RNA complexes by solid-state NMR

在本研究项目中，我们旨在探索动态核极化(DNP)辅助实验用于用固体核磁共振研究大的蛋白质-RNA复合体结构的可行性。固体核磁共振已成功地应用于淀粉样纤维、膜蛋白和大的蛋白质复合体的研究；最近首次报道了蛋白质-RNA复合体的单核磁共振研究。虽然可以在合理的时间内获得小型蛋白质-RNA复合体的结构信息，但由于缺乏敏感性和严重的信号重叠，对大型复合体的研究要耗费更多的时间，而且往往是不可行的。DNP通过将顺磁极化剂的电子自旋极化转移到核自旋，大大提高了核磁共振实验的灵敏度。核磁共振信号增强因子通常达到两个数量级。这节省了数万个数万的时间，并为在几天内进行3D和4D实验打开了可能性。我们的研究小组通过MAS核磁共振和DNP增强的MAS核磁共振活跃在RNA结构生物学方面的独特合作，将使我们能够开发出在接近自然的环境中以原子分辨率研究大型RNP络合物的结构的方法。在这个项目中开发的方法论将在结构生物学的几个领域找到广泛的适用性。

In this research project we aim to investigate the feasibility of dynamic nuclear polarization (DNP) aided experiments for structural studies of large protein-RNA complexes by solid-state NMR. Solid-state NMR has been successfully applied for studies of amyloid fibrils, membrane proteins and large protein complexes; first ssNMR studies on protein-RNA complexes have been reported recently. While structural information on small protein-RNA complexes can be obtained in a reasonable time, studies of large complexes are much more time consuming - and often unfeasible - due to lack of sensitivity and severe signal overlap. DNP provides a significant boost in sensitivity for NMR experiments by transferring electron spin polarization of a paramagnetic polarizing agent to nuclear spins. NMR signal enhancement factors typically reach two orders of magnitude. This leads to time savings of the order of tens of thousands and opens the possibility to perform 3D and 4D experiments within few days. The unique collaboration of our research groups, being active in RNA structural biology by MAS NMR as well as DNP-enhanced MAS NMR, will allow us to develop methods for structural studies of large RNP complexes in close-to-native environment at atomic resolution. The methodology developed within this project will find broad applicability in several areas of structural biology.