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

HXYZ-g HR-FAST-MAS探针在生物分子结构测定中的应用 抽象的。 液态核磁共振波谱可以说是确定可溶性化合物结构的最好工具之一。 蛋白质。该方法为中等分子量蛋白质和/或其复合体提供了原子分辨率。 当体系的相对分子质量引起慢分子运动时，该方法开始困难， 这又增加了线宽，超出了有用的解析点。固体核磁共振(ss核磁共振)方法 在过去的15年里取得了显著的进步，可以改善这些情况的解决方案，但他们 仍然远远没有达到液状拆分生物大分子的目标，例如膜 蛋白质和纤维是阿尔茨海默氏症的核心。核磁共振中的“圣杯”将是 要成功地利用强大的核磁共振获取和自动结构确定协议套件 专为溶液核磁共振而开发，它依赖于检测到的三氢和四氢去耦合方案(AS 这样通常允许用固体直接检测的S/N比直接检测分别高8倍或30倍(分别为C和⁵5N 样品1-10毫克。 此二期应用程序的主要目标是完成四通道的开发 多核单核磁共振探头(HXYZ)，能够在？H下提供？C/？⁵N/？H关联 利用中等(15 KHz)到快速(&gt；35 kHz)魔角旋转(MAS)解耦，同时检测？H。 产生的分辨率，特别是利用所提出的新的脉冲序列，将接近于典型液体的分辨率 蛋白质的国家实验。 具有梯度的四通道多核探针一直是溶液核磁共振中的主力 几十年来，但它们还没有用于ss核磁共振-它们被认为是不切实际的困难 设计和建造。第一阶段展示了H/X/Y/Z窄口径(NB)MAS探测器的可行性，该探测器基于 一种新型的单线圈射频电路，优化了？H检测，适用于7-31T的磁场。 探头将与自动样品交换、脉冲场梯度(PFG)、Nb磁体和 用于动态核极化(DNP)的新型Nb微波辐射方法。 计算表明，2 H J耦合对刚性中剩余的H谱线展宽贡献了5-10赫兹 蛋白质和现有数据表明，探测器的分辨率有限(根据温度梯度和磁性)。 商业上可用的FAST-MAS探测器又贡献了6-25赫兹。一种4通道MAS探头，带订单- 更低的温度梯度，能够在液体上实现2赫兹？H的分辨率，有望实现？H 在900 MHz及以上频率下，刚性蛋白质中的大部分残基的线宽都在0.01ppm以下。一种新颖的电路 还可针对几乎所有感兴趣的组合进行调整，例如：⁵N、P/⁷、LI/C、 ⁷Al/⁹Si/⁷O和H/⁹Si/⁰Rh，从而使其在新陈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。

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.