Construction of a 500-MHz REDOR NMR Spectrometer

500 MHz REDOR NMR 波谱仪的构建

• 批准号: 9400072
• 负责人: Jacob Schaefer
• 金额: $ 104.22万
• 依托单位: Washington University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-15 至 1998-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9400072&HistoricalAwards=false
• 关键词: Construction; 500; MHz; REDOR; NMR

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- crystallizeable protein complexes. In a single experiment, REDOR measures accurate, long-range distances from 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-31C-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 filing 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 500-MHz 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) quest ions in the biological sciences: the structures of big, non-crystallizeable 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.

我们建议建造一辆11.75-T的。500 mhz旋转回波双共振（REDOR）光谱仪，用于蛋白质和蛋白质复合物的固态15N核磁共振实验。REDOR NMR非常适合于表征均匀的15n标记，冷冻淬灭，冻干，不可结晶的蛋白质复合物的结合位点。在单个实验中，REDOR测量底物和抑制剂中31P， 19F或13C标记到侧链氮（Arg， His和Lys信号总是很好地分离）和主肽氮（有时可以分辨肽主链的REDOR差异信号）的准确，远距离距离。利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。为了提高15N实验的分辨率、灵敏度和选择性，我们建议建立一个专门为15N REDOR设计的固态核磁共振光谱仪。该光谱仪将配备针对1H-19F-31C-15N进行调谐的四通道在线传输探头。所有稀有自旋通道，包括19F通道，将允许观察超过100千赫的质子去耦。小直径线圈将为小至20毫克的100-kD蛋白质样品提供良好的归档因子。定量50-MHz 15N NMR可以用标准魔角纺丝（5-8 kHz纺丝速度）进行。高灵敏度、高效率、大功率的输电线路需要大管道。我们计划使用2 1/2英寸的外导体和3/4英寸的内导体作为传输线。这意味着我们需要在一个大口径磁铁周围建造光谱仪。罗伯特·麦凯（Robert McKay）是固体核磁共振在线传输、多频率、高功率、高灵敏度探针的发明者，他将设计和制造探针和光谱仪。这台500兆赫的REDOR核磁共振光谱仪将是世界上唯一的同类光谱仪。它将被用来为生物科学中各种重要的科学（和实际）问题提供答案：大的、不可结晶的蛋白质复合物的结构；细胞壁结构、细胞表面细胞-细胞机制、dna -蛋白质识别；以及膜蛋白的结构和动力学。