800 MHz Solid-State NMR Spectrometer for Biomacromolecular Structure and Dynamics

描述（由申请人提供）：我们建议获得最先进的800 MHz宽口径固态NMR光谱仪，用于研究复杂的生物大分子组装体，包括朊病毒蛋白淀粉样纤维和大核小体阵列。这些系统在蛋白质构象疾病的发病机制、基因调控和DNA修复等多种生物现象中发挥着重要作用，对人类健康至关重要。在这些研究中使用的大多数样品天然地作为高分子量非结晶或纤维聚集体存在。由于这种制剂是不溶性的，缺乏长程有序，它们不容易通过常规的X射线晶体学或溶液态NMR方法进行询问。另一方面，它们是通过固态NMR进行分析的理想目标。拟议的仪器将配备先进的多维魔角旋转NMR实验，这将使整个13 C，15 N标记的蛋白质的分子量高达~400 kDa的复合物中的1H，13 C和15 N信号的分辨率和分配。该仪器还将允许测定远距离原子间1H-1H，13 C-13 C和13 C-15 N距离限制高达~5-8，以及在特定位点用顺磁性Cu 2+螯合标签修饰的蛋白质分子中15 N-Cu 2+和13 C-Cu 2+距离高达~20-25。这种类型的距离限制是固态NMR结构测定方案的关键成分。此外，该仪器将提供容易地检测肽、蛋白质和核酸中的31 P、2 H和17 O核的能力，以进行结构和动态测量，并对标准探针调谐元件进行简单调整。该仪器的新功能对实验的成功至关重要，包括相对于中低场光谱仪显着增强的灵敏度和分辨率，2D，3D和4D NMR实验的出色长期操作稳定性，低温下的扩展操作，以及在频率<$40 kHz下的快速魔角旋转。拟议的仪器将支持在俄亥俄州州立大学和凯斯西储大学在克利夫兰，俄亥俄州与NIH R 01资金的核心组的研究人员正在进行的项目，也使超高场魔角旋转固态NMR的应用研究项目在俄亥俄州国家可以显着受益于这种方法，但尚未利用它由于缺乏设备。PI在应用于生物大分子的高级魔角旋转固态NMR方面接受了广泛的培训，并开发了用于蛋白质分配、三维蛋白质结构细化和快速获取具有增强灵敏度的蛋白质NMR数据的多维NMR方法。这些和其他新方法将在新的800 MHz NMR仪器上实施，以使在较低磁场下难以或不可能研究的生物大分子系统的研究成为可能。

DESCRIPTION (provided by applicant): We propose to acquire a state-of-the-art 800 MHz wide-bore solid-state NMR spectrometer for studies of complex biomacromolecular assemblies including prion protein amyloid fibrils and large nucleosome arrays. These systems play a major role in diverse biological phenomena such as the pathogenesis of protein conformational diseases, gene regulation and DNA repair, and are of fundamental importance to human health. Most samples employed in these studies exist natively as high-molecular weight non-crystalline or fibrous aggregates. Since such preparations are insoluble and lack long-range order they are not readily interrogated by conventional X-ray crystallography or solution-state NMR methods. On the other hand, they are ideal targets for analysis by solid-state NMR. The proposed instrument will be equipped for advanced multidimensional magic-angle spinning NMR experiments, that will enable the resolution and assignment of 1H, 13C and 15N signals throughout 13C,15N-labeled proteins within complexes having molecular masses up to ~400 kDa. The instrument will also permit determination of long-range interatomic 1H-1H, 13C-13C and 13C-15N distance restraints up to ~5-8 ¿, as well as 15N-Cu2+ and 13C-Cu2+ distances up to ~20-25 ¿ in protein molecules modified at specific sites with paramagnetic Cu2+-chelating tags. Distance restraints of this type are a key ingredient of solid-state NMR structure determination protocols. Moreover, the instrument will provide the capability to readily detect 31P, 2H and 17O nuclei in peptides, proteins and nucleic acids for structural and dynamic measurements, with simple adjustments of the standard probe tuning elements. The novel capabilities of the proposed instrument, which are critical to the success of the proposed experiments, include significantly enhanced sensitivity and resolution relative to low- to moderate-field spectrometers, excellent long-term operational stability for 2D, 3D and 4D NMR experiments, extended operation at low temperatures, and rapid magic-angle spinning at frequencies ¿ 40 kHz. The proposed instrument will support ongoing projects among a core group of investigators at The Ohio State University and Case Western Reserve University in Cleveland, OH with NIH R01 funding, and also enable the application of ultrahigh-field magic-angle-spinning solid-state NMR to research projects at Ohio State which can significantly benefit from this methodology but have not exploited it as of yet due to lack of equipment. The PI has extensive training in advanced magic-angle-spinning solid-state NMR applied to biological macromolecules and has developed multidimensional NMR methods for protein assignments, three-dimensional protein structure refinement and rapid acquisition of protein NMR data with enhanced sensitivity. These and other new methodologies will be implemented on the new 800 MHz NMR instrument to enable studies of biomacromolecular systems that are difficult or impossible to investigate at lower magnetic fields.