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

用于显着改进生物分子结构测定的 Quad-Fast-MAS 探针

• 批准号: 9045315
• 负责人: Francis DAVID Doty
• 金额: $ 22.43万
• 依托单位: DOTY SCIENTIFIC, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9045315
• 关键词: Area; Biological; Biomedical Research; Budgets; Caliber; Catalysis; Cellular Membrane; Data; Databases; Deposition; Detection; Evaluation; Funding; Laboratories; Liquid substance; Magic; Magnetism; Membrane Proteins; Metabolism; Methods; Modeling; NBL1 gene; Neurology; Nuclear Magnetic Resonance; Performance; Phase; Physiologic pulse; Proteins; Protocols documentation; Publishing; Research Personnel; Resolution; Rotation; Sampling; Scheme; Signal Transduction; Site; Solid; Speed; Structure; Surface; System; Techniques; Temperature; Testing; Time; Validation; abstracting; base; design; deuteron; improved; interest; macromolecule; materials science; novel; protein structure; protonation; prototype; public health relevance; simulation; solid state nuclear magnetic resonance; structural biology; tool; vibration

 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.

 描述（由申请人提供） 用于显着改进生物分子结构测定的四极快速 MAS 探针 摘要 在过去 15 年中，魔角旋转 (MAS) NMR 在结构生物学中日益广泛的应用中取得了稳步进展。然而，这些方法都与常规方法相去甚远，通常需要 10-80 毫克的浓缩样品，而这种样品很难适当分离和制备。 “圣杯”i 固体 NMR 将能够成功利用为溶液 NMR 开发的强大的 NMR 采集和自动结构测定协议套件，该协议主要依赖于 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，分别针对 13C 和 15N），固体样品为 1-10 mg。 几十年来，具有梯度的四通道多核探针一直是溶液核磁共振的主力，但四共振固体探针还没有出现——它们被认为设计和制造起来非常困难。该提案寻求资金来开发、构建和测试原型 H/X/Y/Z HR-fast-MAS 探头，该探头基于新型“单线圈”射频电路，针对 1H 检测进行了优化，适用于 7-30 T 的磁场和 0.5-5 mm 的转子直径。 II 期探针将兼容自动样品交换、脉冲场梯度 (PFG) 和 90 K 至 400 K 的样品温度。此外，它基本上没有所有主要核素（1H、31P、13C、2H、15N 和 17O）的背景信号。 通过改进样品重氘化、可交换位点的最佳质子化、更快的旋转和更高的偏振场的组合，1H 检测固体方法的光谱分辨率得到了数量级的改进，可以看到典型的 1H 刚性蛋白质的线宽从 ~0.4 ppm 减少到 ~0.04 ppm。分析表明，剩余展宽的很大一部分来自于 J 耦合到氘核（未通过 MAS 进行平均）以及与旋转器相关的效应 - 热梯度、轴向振动和磁性。计算表明，2H J 耦合对 1H 线展宽贡献了 5-10 Hz，现有数据表明，市售快速 MAS 探头中探头有限的分辨率又贡献了 6-25 Hz。 4 通道 HR-MAS 探头允许同时解耦 13C、2H 和 15N，实现 >40 kHz MAS 旋转，并具有较低数量级的热梯度，并且能够在液体上实现 2 Hz 分辨率 1H 分辨率，预计能够在 900 MHz 及以上频率下对刚性蛋白质中的大多数残留物实现低于 0.01 ppm 的 1H 线宽。该新型电路还可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、神经病学、材料科学、催化作用，以及 可持续能源。