CONSTRUCTION OF A 500 MHZ REDOR NMR SPECTROMETER

500 MHZ REDOR NMR 波谱仪的构建

• 批准号: 2285346
• 负责人: JACOB SCHAEFER
• 金额: $ 12.77万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-15 至 1998-07-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2285346
• 关键词: biomedical equipment development; nuclear magnetic resonance spectroscopy

We propose to build an 11.75-T, 500-MHz rotational-echo double-resonance (REDOR) spectrometer for solid-state 15N NMR experiments on proteins and protein complexes. REDOR NMR is well suited to the characterization of binding sites of uniformly 15N-labeled, freeze-quenched, lyophilized, non-crystallizable protein complexes. In a single experiment, REDOR measures accurate, long-range distances for 31P, 19F, or 13C labels in substrates and inhibitors to both sidechain nitrogens (Arg, His, and Lys signals are always well separated) and backbone peptide nitrogens (REDOR difference signals from the peptide backbone are sometimes resolved). Using these distances as constraints to molecular dynamics simulations, plausible structures for protein binding sites can be determined. To enhance the resolution, sensitivity, and selectivity of the 15N experiment, we propose to build a solid-state NMR spectrometer specifically designed for 15N REDOR. The spectrometer will feature four-channel, transmission-line probes tuned for 1H-19F-31P-15N and 1H-19F-13C-15N. All rare-spin channels will permit observation with more than 100-kHz proton decoupling, including the 19F channel. Small-diameter coils will provide good filling factors for 100-kD protein samples as small as 20 mg. Quantitative 50-MHz 15N NMR can be performed with standard magic-angle spinning (5-8 kHz spinning speeds). High-sensitivity, high-efficiency, high-power transmission lines require big pipes. We plan to use a 2 1/2- inch outer conductor and a 3/4-inch inner conductor for the transmission line. This means that we need to build the spectrometer around a wide-bore magnet. Robert McKay, the inventor of the transmission-line, multi-frequency, high-power, high-sensitivity probe for solids NMR, will design and build the probes and the spectrometer. This 500Hz REDOR NMR spectrometer will be the only one of its kind in the world. It will be used to provide answers to a variety of important scientific (and practical) questions in the biomedical sciences: the structures of big, non-crystallizeable proteins and protein complexes; the architecture of cell walls, the Mechanism of cell-surface, cell-cell, and DNA-protein recognition; and the structure and dynamics of membrane proteins.

我们建议建造一个11.75-T，500-MHz旋转回波双回声 （重新）固态15N NMR实验的光谱仪和蛋白质和 蛋白质复合物。 REDOR NMR非常适合表征 统一的15N标签，冷冻，冻干的结合位点， 非结晶蛋白复合物。 在一次实验中，重新进行 衡量31p，19f或13c标签的准确，远程距离 两种侧chain氮的底物和抑制剂（Arg，His和Lys 信号总是很好分开）和骨干肽硝化物（重复 有时可以解决来自肽主链的差异信号）。 使用这些距离作为分子动力学模拟的约束， 可以确定蛋白质结合位点的合理结构。 到 提高15N的分辨率，灵敏度和选择性 实验，我们建议建立一个固态NMR光谱仪 专为15N重复设计。 光谱仪将具有 四通道的传输线探针调整为1H-19F-31P-15N和 1H-11F-13C-15N。所有稀有自旋通道都将允许观察更多 超过100 kHz质子去耦，包括19F通道。 小直径线圈将为100 kd蛋白提供良好的填充因子 样品小至20 mg。 可以执行定量50-MHz 15N NMR 具有标准的魔术角旋转（5-8 kHz旋转速度）。 高敏性，高效率，高功率传输线需要 大管。 我们计划使用2 1/2英寸的外导体和3/4英寸 传输线的内导体。 这意味着我们需要 在宽孔磁铁周围构建光谱仪。 罗伯特·麦凯（Robert McKay）， 传输线，多频，高功率的发明者 固体NMR的高敏性探针将设计和构建探针 和光谱仪。 这个500Hz重新验证NMR光谱仪将是唯一的 世界上同类人之一。 它将用于提供一个答案 各种重要的科学（和实用）问题 生物医学科学：大型非结晶蛋白的结构 和蛋白质复合物；细胞壁的结构，机制 细胞表面，细胞细胞和DNA蛋白识别；和结构 和膜蛋白的动力学。