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

 描述(由申请人提供)一种用于显著改进生物分子结构测定的Quad-Fast-MAS探针摘要在过去的15年中，魔角旋转(MAS)核磁共振在结构生物学中的应用越来越广泛，取得了稳步的进展。然而，这些方法都远远不是常规的，往往需要10-80 mg的浓缩样品，这是极其困难的分离和适当的制备。“圣杯”I固体核磁共振将是成功利用为溶液核磁共振开发的强大的核磁共振采集和自动结构确定协议的能力，这些协议主要依赖于1H检测的三重和四重共振方案(因此，对于13C和15N，通常允许比直接检测高8或30倍的S/N)，固体样品为1-10毫克。具有梯度的四通道多核探针几十年来一直是溶液核磁共振中的主力，但四共振固体探针一直没有出现-它们被认为难以设计和制造。该提案寻求资金用于开发、制造和测试H/X/Y/Z HR-FAST-MAS探头原型，该探头基于一种针对1H检测进行了优化的新型“单线圈”射频电路，适用于7-30T和0.5-5 mm的转子直径。第二阶段探测器将兼容自动样品交换、脉冲场梯度(PFG)和从90K到400K的样品温度。此外，它将基本上没有所有初级核素(1H、31P、13C、2H、15N和17O)的背景信号。固体中的1H检测方法的光谱分辨率得到了数量级的提高，这些方法结合了改进的样品过氢化、可交换位置的最佳质子化、更快的旋转和更高的极化场，参见典型的1H 刚性蛋白质的线宽从~0.4ppm下降到~0.04ppm。分析表明，剩余展宽的很大一部分是从J耦合到氘核(这不是由MAS平均的)和与自旋相关的效应--温度梯度、轴向振动和磁性。计算表明，2H J耦合对1H谱线展宽贡献了5-10赫兹，而现有数据表明，商用FAST-MAS探测器的探测器有限分辨率又贡献了6-25赫兹。一种4通道HR-MAS探头，允许13C、2H和15N的同时去耦合，实现&gt；40 kHz MAS旋转和数量级较低的温度梯度，并能够在液体1H分辨率上具有2赫兹分辨率，预计在900 MHz及以上的刚性蛋白质中的大多数残基上的1H线宽低于0.01ppm。这种新型电路还可以调谐到几乎所有感兴趣的组合，例如1H/13C/2H/15N、1H/31P/13C/2H、1H/31P/7Li/13C、1H/27Al/29Si/17O和1H/13C/29Si/103Rh，从而使其在新陈代谢、神经学、材料科学、催化和可持续能源等领域也具有不可估量的价值。

 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.