Construction of a 500-MHz REDOR NMR Spectrometer

我们计划建造一艘11.75吨的。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15 N NMR实验。 REDOR NMR非常适合于表征均匀15 N标记的、冷冻淬灭的、冻干的、不可结晶的蛋白质复合物的结合位点。 在单个实验中，REDOR测量从底物和抑制剂中的31 P、19 F或13 C标记到侧链氮（Arg、His和Lys信号总是很好地分离）和主链肽氮（来自肽主链的REDOR差异信号有时被解析）的精确的长距离。 使用这些距离作为分子动力学模拟的约束条件，可以确定蛋白质结合位点的合理结构。 为了提高15 N实验的分辨率、灵敏度和选择性，我们建议建立一个专门为15 N REDOR设计的固态NMR光谱仪。 该光谱仪将采用四通道传输线探头，调谐为1H-19 F-31 C-15 N。 所有罕见的自旋通道将允许观察超过100 kHz的质子去耦，包括19 F通道。 小直径弹簧圈将为小至20 mg的100 kD蛋白质样品提供良好的填充因子。 定量50-MHz 15 N NMR可以用标准魔角旋转（5-8 kHz旋转速度）进行。 高灵敏度、高效率、大功率的输电线路需要大管道。 我们计划在传输线上使用2 1/2英寸的外导体和3/4英寸的内导体。 这意味着我们需要围绕宽口径磁体构建光谱仪。 罗伯特·麦凯是固体核磁共振传输线、多频、高功率、高灵敏度探头的发明者，他将设计和建造探头和光谱仪。 这台500 MHz的REDOR NMR光谱仪将是世界上唯一的一台。 它将被用来回答生物科学中各种重要的科学（和实践）问题：大的，不可结晶的蛋白质复合物的结构;细胞壁的结构，细胞表面细胞-细胞和DNA-蛋白质识别的机制;以及膜蛋白的结构和动力学。

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.