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（由适用提供）用于大大改进的生物分子结构确定的四及时探针摘要摘要在将魔法角度旋转（MAS）NMR应用于结构生物学中越来越广泛的应用中，在应用魔法角度旋转（MAS）NMR方面稳步进步。但是，这些方法远非常规，通常需要10-80毫克的集中样品，这些样品极为难以隔离和适当。 “圣杯” I固体NMR将是成功地使用针对解决方案NMR开发的强大的NMR获取和自动化结构确定协议的能力，该协议主要依赖于1H检测的三重和四倍共振方案（因此，通常允许S/N比直接检测高于13C和15n的S/N比直接检测高8或30倍）。数十年来，四通道多核问题一直是解决方案NMR的主力，但是四谐固体固体问题尚未可用 - 人们认为它们在设计和构建上是不切实际的。该提案寻求资金来开发，构建和测试原型H/X/Y/Z HR-FAST-MAS探针基于优化可用于1H检测的小说“单线圈” RF电路，适用于从0.5-5 mm处使用的7-30 T和转子直径的田间使用。 II期探针将与自动样品交换，脉冲场梯度（PFG）和从90 K到400 K的样品温度兼容。此外，它将基本上没有所有原发性核所的背景信号（1H，31P，13C，13C，2H，15N和17O）。通过改进的样品perdeuteration，最佳可交换位点的最佳质子化，更快的旋转和较高的极化场的结合，已经证明了光谱分辨率的稳定顺序改善光谱分辨率的改善，从 刚性蛋白的线宽从〜0.4 ppm降至〜0.04 ppm。分析表明，剩余的很大一部分是从J耦合到杜特隆（不是由MAS平均）和旋转器依赖性效应 - 热梯度，轴向振动和磁性的。计算表明2H J耦合对1H线的扩展贡献了5-10 Hz，并且可用的数据表明，在市售的快速MAS探针中，探测限制的分辨率又贡献了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 residuals in rigid proteins at 900 MHz and above.新颖的电路还可以调节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/29SI/17O，以及1H/13C/13C/29SI/103RH，也可以进行材料，并在此材料中进行构成，并在此类材料中进行策略。可持续能源。