An HXYZ-g HR-Fast-MAS probe for Dramatically Improved Biomolecular Structure Determinations

用于显着改进生物分子结构测定的 HXYZ-g HR-Fast-MAS 探针

• 批准号: 9988618
• 负责人: Francis DAVID Doty
• 金额: $ 64.31万
• 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 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9988618
• 关键词: Alzheimer' s Disease; Amyloid beta-Protein; Area; Biological; Biomedical Research; Budgets; COSY; Caliber; Catalysis; Cellular Membrane; Complex; Data; Deposition; Detection; Development; Diffusion; Equipment; Funding; Goals; Health; Inferior; Laboratories; Liquid substance; Magic; Magnetism; Measurement; Membrane Proteins; Metabolism; Methods; Modeling; Molecular; Molecular Weight; Motion; NMR Spectroscopy; Neurology; Nuclear; Nuclear Magnetic Resonance; Performance; Phase; Physiologic pulse; Platelet Factor 4; Price; Proteins; Protocols documentation; Publishing; RF coil; Research Personnel; Resolution; Sampling; Scheme; Side; Solid; Solvents; Sonication; Speed; Structural Protein; Structure; Surface; System; Techniques; Technology; Time; Width; base; data warehouse; design; experimental study; improved; innovation; insight; interest; irradiation; macromolecule; materials science; microwave electromagnetic radiation; next generation; novel; operation; protein structure; solid state nuclear magnetic resonance; structural biology; tool; vibration; virtual

An HXYZ-g HR-Fast-MAS probe for Dramatically Improved Biomolecular Structure Determinations Abstract. Liquid state NMR spectroscopy is arguably one of the best tools for structure determination for soluble proteins. The method provides atomic resolution for modest molecular weight proteins and/or their complexes. The method begins to have difficulty when the molecular weight of the system causes slow molecular motion, which in turn increases the linewidth beyond the point of useful resolution. Solid state NMR (ssNMR) methods have progressed remarkably over the past 15 years to permit improved resolution for these conditions, but they still come well short of the goal of liquid-like resolution on biological macromolecules, such as membrane proteins and the fibrils that are central to Alzheimer’s Disease. The “Holy Grail” in ssNMR would be the ability to successfully utilize the powerful suite of NMR acquisition and automated structure determination protocols developed for solution NMR, which rely on ¹H-detected triple- and quad-resonance ²H-decoupled schemes (as such generally permit 8 or 30 times higher S/N than direct detection, for ¹³C and¹⁵5N respectively) with solid samples of 1-10 mg. The main objective of this Phase II application is to complete the development a four-channel multinuclear ssNMR probe (HXYZ) that has the capability of providing ¹³C/¹⁵N/¹H correlations under ²H decoupling utilizing modest (15 kHz) to fast (> 35 kHz) Magic Angle Spinning (MAS) while detecting ¹H. The resulting resolution, particularly with proposed novel pulse sequences, will be close to that of a typical liquid state experiment on proteins. Four-channel multinuclear probes with gradients have been the workhorse in solution NMR for decades, but they have not been available for ssNMR – they have been perceived to be impractically difficult to design and build. The Phase-I demonstrated feasibility of an H/X/Y/Z narrow-bore (NB) MAS probe based on a novel “single-coil” rf circuit optimized for ¹H detection and suitable for use at fields from 7-31 T. The Phase-II probe will be compatible with automated sample exchange, pulsed-field gradients (PFG), NB magnets, and novel NB microwave irradiation methods for Dynamic Nuclear Polarization (DNP). Calculations suggest ²H J-couplings contribute 5-10 Hz to the remaining ¹H line broadening in rigid proteins, and available data suggest the probe-limited resolution (from thermal gradients and magnetism) in commercially available fast-MAS probes has contributed another 6-25 Hz. A 4-channel MAS probe with order- of-magnitude lower thermal gradients that is capable of 2 Hz ¹H resolution on liquids 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.

HXYZ-g HR-Fast-MAS探针用于显著改进的生物分子结构测定 抽象。 液态NMR光谱可以说是用于可溶性化合物的结构测定的最佳工具之一。 proteins.该方法为中等分子量蛋白质和/或其复合物提供原子分辨率。 当体系的分子量引起缓慢的分子运动时，该方法开始有困难， 这又将线宽增加到超过有用分辨率的点。固态NMR（ssNMR）方法 在过去的15年中取得了显着的进步，以提高对这些条件的分辨率，但它们 但仍远未达到对生物大分子（如膜）进行类液体拆分的目标， 蛋白质和纤维是阿尔茨海默病的核心。ssNMR中的“圣杯”将是 成功利用强大的NMR采集和自动结构测定方案套件 为溶液NMR开发，其依赖于<$H检测的三重和四重共振<$H解耦方案（如 这通常允许比直接检测S/N高8或30倍（分别对于<$3C和<$45N）， 样品为1-10 mg。 第二阶段申请的主要目标是完成四通道的开发 多核ssNMR探针（HXYZ），能够在2 H下提供<$3C/<$3N/<$3H相关性 利用适度（15 kHz）到快速（> 35 kHz）魔角旋转（MAS）去耦，同时检测<$H。的 所得到的分辨率，特别是使用所提出的新颖脉冲序列，将接近于典型液体的分辨率 关于蛋白质的实验 具有梯度的四通道多核探针已经成为溶液NMR的主力， 几十年来，但它们还没有用于ssNMR -它们被认为是不切实际的困难， 设计和建造。第一阶段证明了H/X/Y/Z窄孔（NB）MAS探头的可行性， 一种新型的“单线圈”射频电路，针对1/H检测进行了优化，适用于7-31 T的磁场。第二阶段 探头将与自动样本交换、脉冲场梯度（PFG）、NB磁体兼容， 新的NB微波辐照方法用于动态核极化（DNP）。 计算表明，²H J耦合对刚性中剩余的<$H谱线展宽贡献5-10 Hz。 蛋白质，和现有的数据表明，探针有限的分辨率（从热梯度和磁性）， 市售的快速MAS探针贡献了另外6-25 Hz。4通道MAS探头， 能够在液体上实现2 Hz <$H分辨率的量级较低的热梯度预计能够实现<$H 在900 MHz及以上频率下，刚性蛋白质中大多数残基的线宽低于0.01 ppm。该新颖电路 也可调谐到几乎所有感兴趣的组合，例如C/P/C/P/LiLi/C， <$H/<$Al/<$Si/<$O和<$H/<$C/<$Si/<$Rh，从而使其在代谢等领域也是无价的， 神经学、材料科学、催化和可持续能源。