Administrative Supplement: Biophysical investigations of RNA complexes essential for gene expression

项目总结/摘要 本行政补充提案是针对定制的800 MHz固态NMR（SSNMR）探头， 允许检测1H、19 F、31 P、13 C和15 N核。到目前为止，这种探头仅适用于宽口径 最高频率为600 MHz的磁铁。现在凤凰核磁共振仪已经开发出一种标准孔径魔角 带有1.6 mm转子的旋转探头，旋转频率高达40 kHz，具有全范围的调谐配置 以实现在800 MHz下的分子结构测定。射频电磁线圈可以调谐 同时达到三个或四个频率，包括标准所需的所有三重共振组合。 应用（HPC，HCN，HPN）以及涉及19 F的新型四重共振配置，例如 HFPC、HFCN和HFPN。Phoenix探针是唯一支持1H的标准孔设计 在观察19 F和另外两个原子核的同时进行去耦。我们预计， 在800 MHz的灵敏度将使生物分子和分子量的复合物的完整分配 20至50 kDa，以及利用偶极序参数和弛豫方法的动力学研究。探针 将用于Butcher和Henzler-Wildman实验室的项目，并将被放置在国家实验室。 威斯康星大学麦迪逊分校的麦迪逊磁共振设施（NMRFAM）最近招募了SSNMR 专家Chad Rienstra教授（原美国大学教授）。Illinois）。因此，探针将支持来自UW内的至少3个组。 麦迪逊，也将访问一个广泛的外部用户群。布彻实验室会用这个探测器 研究poly-UG RNA及其与TDP-43蛋白的相关性。由于高度重复的RNA 序列，这些研究将利用RNA上的位点特异性2′氟基团。一种1.0 μ m的晶体结构， 游离RNA已被解析，NMR归属也已完成，这将有助于结构的构建 通过SSNMR测定络合物。Henzler-Wildman实验室将使用该探针研究细菌是如何 EmrE多药物转运蛋白及其与氟化底物的相互作用。HN分辨1H-19 F REDOR实验证明了该项目的可行性。

PROJECT SUMMARY/ABSTRACT This administrative supplement proposal is for a custom-built 800 MHz solid state NMR (SSNMR) probe that will allow detection of 1H, 19F, 31P, 13C and 15N nuclei. Until now, such probes have only been available for wide bore magnets at a maximum frequency of 600 MHz. Now, Phoenix NMR has developed a standard bore magic-angle spinning probe with a 1.6 mm rotor capable of spinning up to 40 kHz, with the full range of tuning configurations to enable molecular structure determination at 800 MHz. The radio frequency solenoid coil can be tuned simultaneously to three or four frequencies, including all the triple resonance combinations required for standard applications (HPC, HCN, HPN) as well as novel, quadruple resonance configurations involving 19F, such as HFPC, HFCN, and HFPN. The Phoenix probe is the only standard bore design available that supports 1H decoupling while observing 19F and two other nuclei. We anticipate the combined improvements in resolution and sensitivity at 800 MHz will enable complete assignments of biomolecules and complexes of molecular weight 20 to 50 kDa, as well as studies of dynamics utilizing dipolar order parameter and relaxation methods. The probe will be used for projects in the Butcher and Henzler-Wildman laboratories and will be placed in the National Magnetic Resonance Facility at Madison (NMRFAM) at UW-Madison, which has recently recruited SSNMR expert Prof. Chad Rienstra (formerly U. Illinois). Thus, the probe will support at least 3 groups from within UW- Madison and will also be accessible to an extensive outside user base. The Butcher lab will use the probe to investigate poly-UG RNA and its association with the protein TDP-43. Due to the highly repetitive RNA sequence, these studies will make use of site-specific 2′ fluoro groups on the RNA. A 1.0 Å crystal structure of the free RNA has been solved and NMR assignments have been made, which will facilitate structure determination of the complex by SSNMR. The Henzler-Wildman lab will use the probe to study how the bacterial EmrE multi-drug transporter and its interaction with fluorinated substrates. HN-resolved 1H-19F REDOR experiments have been performed that demonstrate the feasibility of the project.