CONSTRUCTION OF A 500 MHZ REDOR NMR SPECTROMETER

我们建议建造一个11.75-T，500-MHz旋转回波双共振 用于蛋白质和蛋白质固体15N核磁共振实验的(REDOR)光谱仪 蛋白质复合体。Redor核磁共振仪非常适合表征 均一标记的15N，冷冻淬火，冻干， 不可结晶的蛋白质复合体。在一次实验中，雷多尔 测量31P、19F或13C标签的精确远距离 两边链氮(Arg、His和Lys)的底物和抑制剂 信号总是被很好地分离)和主链多肽氮(红色 来自多肽主干的不同信号有时会被解析)。 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部位的合理结构。至 提高15N的分辨率、灵敏度和选择性 实验中，我们建议搭建一台固态核磁共振波谱仪 专为15N红色设计。分光计将以 四通道传输线探头，针对1H-19F-31P-15N和 1H-19F-13C-15N。所有罕见的自旋通道将允许观测到更多 超过100 kHz的质子去耦合，包括19F通道。 小直径线圈将为100-kD蛋白质提供良好的填充因子 小到20毫克的样品。可以进行定量的50-MHz 15N核磁共振 具有标准魔角纺纱(5-8 kHz纺丝速度)。 高灵敏度、高效率、高功率的输电线路要求 大管子。我们计划使用2.5英寸的外导体和3/4英寸的外导体 传输线的内导体。这意味着我们需要 围绕着一块大口径磁铁建造光谱仪。罗伯特·麦凯， 发明者传输线，多频，大功率， 高灵敏度固体核磁共振探头，将设计和制造探头 还有分光计。这台500赫兹的红外线核磁共振波谱仪将是唯一的 是世界上同类产品中的一家。它将用于为 各种重要的科学(和实际)问题在 生物医学：大而不可结晶的蛋白质的结构 和蛋白质复合体；细胞壁的结构， 细胞-表面、细胞-细胞和DNA-蛋白质识别；以及结构 以及膜蛋白的动态变化。

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.