Low Temperature Solid-State NMR Technologies for Protein Structure Determination

描述（由申请人提供）：在该项目中，将开发新的固态核磁共振（SSNMR）低温（~100 K）探针技术，沿着最有效地利用这种能力进行膜蛋白和原纤维结构研究的样品制备，数据收集和数据处理能力。这些研究将有助于改善膜蛋白的结构理解，这些膜蛋白是目前大多数药物靶点，以及参与帕金森病和其他神经退行性疾病的蛋白质原纤维。近年来，SSNMR结构研究取得了很大进展，几个研究小组已经成功地确定蛋白质结构，不仅包括纳米晶体，但寡聚膜肽，淀粉样蛋白原纤维和大的膜蛋白复合物。这些工作主要依赖于SSNMR数据的质量，包括灵敏度和分辨率，它们共同决定了结构测定的可行性和最终结构质量。在这里，灵敏度和分辨率 SSNMR谱在低温下将进行评估和改进，特别是膜蛋白和纤维，追求三个目标。目标#1是在600 MHz原型的基础上完成750 MHz冷魔角旋转探头的制造和安装，包括灵敏度的关键特性（3.2 mm转子直径;冷射频电路;交叉线圈谐振器，带低E场1H谐振器和13 C/15 N螺线管），分辨率（20 kHz MAS，<0.05 ppm B 0均匀性，1H谐振器上的高功率处理），吞吐量（气动样品插入和弹出;样品更换后稳定时间<15分钟），以及长期可操作性（~2 L/小时N2（l）消耗量，稳定的RF调谐性能，延长数据收集周期）。目标#2是开发改进的样品制备方案，以提高低温下的光谱分辨率和灵敏度。将通过稀疏同位素标记模式和利用有机醇、乙二醇和盐的冷冻保护程序来提高分辨率。通过使用顺磁弛豫增强试剂（通过共价标签定位于蛋白质）以及可溶性水性和脂质结合掺杂剂来减少T1弛豫时间，可以提高灵敏度。目的#3是进行分配和结构确定的案例研究与典型的四跨膜螺旋膜蛋白（DsbB）和大，主要是<$-折叠纤维（<$-突触核蛋白）。这些样品在室温附近得到了很好的表征，因此基于新的SSNMR探针技术，为评估光谱分辨率和灵敏度以及所得结构质量提供了极好的基准。

DESCRIPTION (provided by applicant): In this project, new solid-state nuclear magnetic resonance (SSNMR) low temperature (~100 K) probe technology will be developed, along with sample preparation, data collection and data processing capabilities that most effectively leverage this capability for structural studies of membrane proteins and fibrils. These studies wil facilitate improved structural understanding of membrane proteins, which are the majority of current drug targets, and fibrils of proteins involved in Parkinson's disease and other neurodegenerative disorders. In recent years, SSNMR structural studies have advanced substantially and several research groups have been successful in determining protein structures, including not only nanocrystals but oligomeric membrane peptides, amyloid fibrils, and large membrane protein complexes. Such efforts rely critically upon the quality of the SSNMR data, including both sensitivity and resolution, which together determine the feasibility of structure determination and the final structural quality. Here the sensitivity and resolution of SSNMR spectra at low temperature will be evaluated and improved, specifically for membrane proteins and fibrils, by pursuit of three aims. Aim #1 is to complete the fabrication and installation of a 750 MHz cold magic-angle spinning probe, based on a 600 MHz prototype, including key features for sensitivity (3.2 mm rotor diameter; cold RF circuit; crossed coil resonator with low E field 1H resonator and 13C/15N solenoid), resolution (20 kHz MAS, <0.05 ppm B0 homogeneity, high power handling on 1H resonator), throughput (pneumatic sample insert and eject; <15 minute stabilization time upon sample change), and long-term operability (~2 L/hour N2(l) consumption, stable RF tuning performance for extended data collection periods). Aim #2 is to develop improved sample preparation protocols for enhancing spectral resolution and sensitivity at low temperature. Resolution will be enhanced by sparse isotopic labeling patterns and cryoprotection procedures utilizing organic alcohols, glycols and salts. Sensitivity will be improved by reducing T1 relaxation times with paramagnetic relaxation enhancement reagents, localized to the protein by covalent tags, as well as soluble aqueous and lipid-bound dopants. Aim #3 is to perform case studies of assignment and structure determination with a prototypical fourtransmembrane helix membrane protein (DsbB) and large, predominantly ¿-sheet fibril (¿-synuclein). These samples are well characterized near room temperature and thus present excellent benchmarks for evaluating the spectral resolution and sensitivity, as well as resultant structure quality, based on the new SSNMR probe technology.