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.

项目概要/摘要 本行政补充提案适用于定制的 800 MHz 固态 NMR (SSNMR) 探头，该探头将 允许检测 1H、19F、31P、13C 和 15N 原子核 到目前为止，此类探头仅适用于大口径。 最大频率为 600 MHz 的磁铁 现在，Phoenix NMR 开发了一种标准孔径魔角。 带有 1.6 mm 转子的旋转探头，旋转频率高达 40 kHz，具有全方位的调谐配置 能够在 800 MHz 下确定分子结构 可以调谐射频螺线管线圈。 同时三个或四个频率，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 的新颖四重共振配置，例如 HFPC、HFCN 和 HFPN 探头是唯一支持 1H 的标准孔设计。 我们预计分辨率会得到综合提高。 800 MHz 的灵敏度将能够完成生物分子和分子量复合物的分配 20 至 50 kDa，以及利用偶极序参数和弛豫方法进行动力学研究。 将用于 Butcher 和 Henzler-Wildman 实验室的项目，并将放置在国家实验室 威斯康星大学麦迪逊分校麦迪逊磁共振设施 (NMRFAM) 最近招募了 SSNMR 专家查德·里恩斯特拉（Chad Rienstra）教授（前伊利诺伊大学）因此，该调查将支持威斯康星大学内部的至少 3 个小组。 麦迪逊和屠夫实验室也将使用该探测器来访问广泛的外部用户群。 由于高度重复的 RNA，研究多聚 UG RNA 及其与蛋白质 TDP-43 的关联。 序列，这些研究将利用 RNA 的 1.0 Å 晶体结构上的位点特异性 2' 氟基团。 游离RNA已被解析并已进行NMR分配，这将有利于结构 Henzler-Wildman 实验室将使用该探针来研究细菌如何通过 SSNMR 测定复合物。 EmrE 多药物转运蛋白及其与氟化底物 HN 解析的 1H-19F 的相互作用。 REDOR 实验已经进行，证明了该项目的可行性。