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

行政补充：基因表达必需的 RNA 复合物的生物物理学研究

• 批准号: 10174007
• 负责人: Samuel E Butcher
• 金额: $ 7.92万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-15 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10174007
• 关键词: Administrative Supplement; Biomolecular Nuclear Magnetic Resonance; Biophysics; Cell Nucleus; Chad; Complex; Crystallization; Custom; Detection; Drug Interactions; Equipment; Essential Genes; Frequencies; Gene Expression; Generations; Goals; Illinois; Investigation; Laboratories; Leadership; Magic; Magnetic Resonance; Membrane Proteins; Methods; Molecular Structure; Molecular Weight; NMR Spectroscopy; Noise; Nuclear Magnetic Resonance; RNA; RNA Sequences; Relaxation; Research Project Grants; Research Support; Resolution; Resources; Signal Transduction; Site; Structure; United States National Institutes of Health; base; design; experimental study; instrument; multi drug transporter; novel; protein TDP-43; protein complex; radio frequency; recruit; solid state nuclear magnetic resonance

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.

项目摘要/摘要 这项行政补充建议是针对定制的800 MHz固态核磁共振(SS核磁共振)探测器，它将 允许检测1H、19F、31P、13C和15N核。到目前为止，这种探头只适用于大口径。 磁体的最大频率为600 MHz。现在，菲尼克斯核磁共振公司已经开发出一种标准的钻孔魔角 带有1.6 mm转子的旋转探头，可旋转高达40 khz，具有各种调谐配置 以实现800 MHz的分子结构测定。射频螺线管线圈可以调谐 同时到三个或四个频率，包括标准所需的所有三重共振组合 应用(HPC、HCN、HPN)以及涉及19F的新型四重谐振配置，例如 HFPC、HFCN和HFPN。菲尼克斯探头是唯一可用的支持1H的标准内孔设计 在观测19F和另外两个原子核时发生去耦合。我们期待在分辨率方面的综合改进 而800 MHz的灵敏度将使生物分子和分子络合物的完全指认成为可能 20~50 kDa，以及利用偶极序参数和松弛方法进行动力学研究。探测仪 将用于Butcher和Henzler-Wildman实验室的项目，并将被安置在国家 麦迪逊大学麦迪逊分校的磁共振设施(NMRFAM)，最近招募了SS核磁共振 专家查德·里恩斯特拉教授(前伊利诺伊州大学)。因此，探测器将支持UW内的至少3个组- 麦迪逊，还将向广泛的外部用户基础访问。屠夫实验室将使用该探测器 研究Poly-UG RNA及其与TDP-43蛋白的关系。由于高度重复的RNA 序列，这些研究将利用RNA上特定部位的2‘氟基团。一个1.0？的晶体结构 对游离的RNA进行了解析，并对其进行了核磁共振指认，这将有助于结构的优化 固体核磁共振法测定该络合物的含量。Henzler-Wildman实验室将使用该探测器来研究细菌如何 EmRE多药物转运体及其与氟化底物的相互作用。HN-分辨率1H-19F 并进行了REDOR实验，验证了该方案的可行性。