High-Field, Wide-Bore Solid-State NMR Spectrometer for Membrane Protein Structure

描述（由申请人提供）：我们建议获得一台最先进的高场，宽口径，固态核磁共振光谱仪，用于研究膜蛋白，原纤维和其他非晶体或微晶体固体。该仪器将配备多维魔角旋转实验，在分子质量从5到至少150 kDa的肽和蛋白质中解析和分配1H， 13C和15N信号。该仪器将允许分析19F， 31P和2H核的远程距离和动力学，并可定制用于实施先前演示的总结构测定实验的目的。该仪器的新功能包括提高灵敏度和分辨率，提高3D和4D实验的操作稳定性，延长采集时间，延长低温工作时间，四重共振和快速（>40 kHz）魔角旋转。该仪器将支持伊利诺伊大学厄巴纳-香槟分校研究人员之间正在进行的合作项目，以解决涉及神经退行性疾病、心血管疾病和传染病的蛋白质结构。这些研究中的样品在天然膜、合成膜、纤维聚集体或微晶沉淀物中制备为非结晶固体；样品缺乏长程有序且不溶，因此不适合x射线晶体学或溶液核磁共振波谱学。PI开发了新的固态核磁共振方法，包括用于偶极张量精化的高分辨率结构测定的3D技术，用于固体蛋白质的第一个4D化学位移分配方法，以及用于质子检测的灵敏度增强技术。利用所提出的仪器，这些技术将用于解决膜和纤维蛋白的结构，样品制备方法和初步光谱是可用的。公共卫生相关性：拟议的仪器将能够确定与帕金森病、糖尿病、血液凝固、药物代谢和艾滋病毒/艾滋病有关的蛋白质的结构。这些蛋白质中的许多是天然存在于膜或纤维形式中，不易用其他方法研究。ssmr确定的结构将有助于对疾病病因的基本理解，并为设计更有效的药物治疗提供靶点。

DESCRIPTION (provided by applicant): We propose to acquire a state-of-the-art high field, wide-bore, solid-state NMR spectrometer for studies of membrane proteins, fibrils and other non-crystalline or microcrystalline solids. The instrument will be equipped for multidimensional magic-angle spinning experiments, to resolve and assign 1H, 13C, and 15N signals throughout peptides and proteins with molecular masses from 5 to at least 150 kDa. The instrument will permit analysis of long-range distances and dynamics with 19F, 31P and 2H nuclei, and be customized for the purpose of implementing experiments that have been previously demonstrated for total structure determination. Novel capabilities of the proposed instrument include enhanced sensitivity and resolution, improved operational stability for 3D and 4D experiments with long acquisition times, extended low temperature operation, quadruple resonance and fast (>40 kHz) magic-angle spinning. The instrument will support ongoing collaborative projects among investigators at the University of Illinois at Urbana-Champaign to solve structures of proteins involved in neurodegenerative, cardiovascular, and infectious diseases. Samples in these studies are prepared as non-crystalline solids in native membranes, in synthetic membranes, as fibrous aggregates, or as microcrystalline precipitates; the samples lack long-range order and are insoluble, and therefore are not amenable to X-ray crystallography or solution NMR spectroscopy. The PI has developed new solid- state NMR methods including 3D techniques for high-resolution structure determination with dipole tensor refinement, the first 4D chemical shift assignment methods for solid proteins, and techniques for sensitivity enhancement with proton detection. Using the proposed instrument, these techniques will be applied to solve structures of membrane and fibrous proteins for which sample preparation methods and preliminary spectra are available. PUBLIC HEALTH RELEVANCE: The proposed instrument will enable structure determination of proteins implicated in Parkinson's disease, diabetes, blood clotting, drug metabolism and HIV/AIDS. Many of these proteins are natively found in membrane or fibrous forms that are not readily studied using other methods. The SSNMR-determined structures will benefit fundamental understanding of disease etiology and provide targets for the design of more effective drug therapies.