CONSTRUCTION OF A 500 MHZ REDOR NMR SPECTROMETER

点击翻译按钮获取中文摘要

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.