A Quad-Fast-MAS probe for Dramatically Improved Biomolecular Structure Determinations

 描述（由申请人提供）用于显著改进的生物分子结构测定的四快速MAS探针摘要过去15年中，在将魔角旋转（MAS）NMR应用于结构生物学中越来越广泛的应用方面取得了稳步进展。然而，这些方法都远非常规方法，并且通常需要10-80 mg的浓缩样品，这非常难以分离和适当制备。“圣杯”i固体NMR将是成功利用为溶液NMR开发的强大的NMR采集和自动结构测定方案套件的能力，其主要依赖于1H检测的三重和四重共振方案（因此通常允许比直接检测高8或30倍的S/N，分别用于13 C和15 N），固体样品为1-10 mg。 具有梯度的四通道多核探针几十年来一直是溶液NMR中的主力，但四共振固体探针还没有出现-它们被认为难以设计和构建。该提案寻求资金来开发、建造和测试原型H/X/Y/Z HR-fast-MAS探头，该探头基于针对1H检测优化的新型“单线圈”射频电路，适合在7-30 T的磁场和0.5-5 mm的转子直径下使用。第二阶段探头将与自动样本交换、脉冲场梯度（PFG）、样品温度为90 ~ 400 K。此外，它将基本上没有所有主要核素（1H、31 P、13 C、2 H、15 N和17 O）的背景信号。 对于固体中的1H检测方法，已经证明了光谱分辨率的数量级改进，这些改进来自改进的样品全氘化、可交换位点的最佳质子化、更快的旋转和更高的极化场的组合，参见典型的1H 刚性蛋白质中的线宽从约0.4 ppm降低至约0.04 ppm。分析表明，大部分剩余的加宽是来自于氘核的J-耦合（这不是MAS的平均值）和自旋相关效应-热梯度，轴向振动和磁性。计算表明2 H J-耦合对1H谱线展宽贡献5- 10 Hz，并且可用数据表明市售快速MAS探针中的探针有限分辨率贡献了另外6-25 Hz。4通道HR-MAS探针允许13 C、2 H和15 N的同时解耦，实现具有数量级较低热梯度的>40 kHz MAS旋转，并且能够在液体1H分辨率上具有2 Hz分辨率，预期能够在900 MHz及以上在刚性蛋白质中的大多数残基上实现低于0.01 ppm的1H线宽。该新型电路还可以调谐到几乎所有感兴趣的组合，例如1H/13 C/2 H/15 N，1H/31 P/13 C/2 H，1H/31 P/7 Li/13 C，1H/27 Al/29 Si/17 O和1H/13 C/29 Si/103 Rh，从而使其在代谢，神经学，材料科学，催化和可持续能源等领域也具有价值。

 DESCRIPTION (provided by applicant) A Quad-Fast-MAS probe for Dramatically Improved Biomolecular Structure Determinations Abstract The last 15 years have seen steady progress in applying magic angle spinning (MAS) NMR to an increasingly wide range of applications in structural biology. However, the methods are all far from routine and often require 10-80 mg of a concentrated sample that is extremely difficult to isolate and prepare suitably. The "Holy Grail" i solids NMR would be the ability to successfully utilize the powerful suite of NMR acquisition and automated structure determination protocols developed for solution NMR, which rely mostly on 1H-detected triple- and quad-resonance schemes (as such generally permit 8 or 30 times higher S/N than direct detection, for 13C and 15N respectively) with solid samples of 1-10 mg. Four-channel multinuclear probes with gradients have been the workhorse in solution NMR for decades, but quad-resonance solids probes have not been available - they have been perceived to be impractically difficult to design and build. This proposal seeks funding to develop, build, and test a prototype H/X/Y/Z HR-fast-MAS probe based on a novel "single-coil" rf circuit optimized for 1H detection and suitable for use at fields from 7-30 T and rotor diameters from 0.5-5 mm. The Phase-II probe will be compatible with automated sample exchange, pulsed-field gradients (PFG), and sample temperatures from 90 K to 400 K. Moreover, it will be essentially devoid of background signals for all the primary nuclides (1H, 31P, 13C, 2H, 15N, and 17O). Order-of-magnitude improvements in spectral resolution have been demonstrated for 1H-detected methods in solids from the combination of improved sample perdeuteration, optimal protonation of exchangeable sites, faster spinning, and higher polarizing fields, seeing typical 1H linewidths in rigid proteins decrease from ~0.4 ppm to ~0.04 ppm. Analysis suggests that a substantial portion of the remaining broadening is from J-couplings to the deuterons (which is not averaged by MAS) and spinner-dependent effects - thermal gradients, axial vibration, and magnetism. Calculations suggest 2H J-couplings contribute 5- 10 Hz to 1H line broadening, and available data suggest the probe-limited resolution in commercially available fast-MAS probes has contributed another 6-25 Hz. A 4-channel HR-MAS probe that permits simultaneous decoupling of 13C, 2H, and 15N, achieves >40 kHz MAS rotation with order-of-magnitude lower thermal gradients, and is capable of 2 Hz resolution on liquids 1H resolution, is expected to enable 1H linewidths below 0.01 ppm on most of the residues in rigid proteins at 900 MHz and above. The novel circuit will also be tunable to virtually all combinations of interest, such as 1H/13C/2H/15N, 1H/31P/13C/2H, 1H/31P/7Li/13C, 1H/27Al/29Si/17O, and 1H/13C/29Si/103Rh, thereby making it also invaluable in such areas as metabolism, neurology, materials science, catalysis, and sustainable energy.