Low Temperature Solid-State NMR Technologies for Protein Structure Determination

用于蛋白质结构测定的低温固态核磁共振技术

• 批准号: 8710289
• 负责人: Chad M Rienstra
• 金额: $ 18.95万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8710289
• 关键词: Address; Alcohols; Amplifiers; Amyloid Fibrils; Benchmarking; Caliber; Case Study; Chemicals; Communities; Consumption; Data; Data Collection; Data Set; Detection; Drug Formulations; Drug Targeting; Engineering; Freezing; Glycols; Hour; Label; Lipid Binding; Lipids; Magic; Measurement; Membrane; Membrane Proteins; Methods; NMR Spectroscopy; Neurodegenerative Disorders; Nuclear Magnetic Resonance; Parkinson Disease; Pattern; Peptides; Performance; Preparation; Procedures; Proteins; Protocols documentation; Protons; Reagent; Recycling; Relative (related person); Relaxation; Research; Research Project Grants; Resolution; Salts; Sampling; Site; Spectrum Analysis; Structure; Technology; Temperature; Tertiary Protein Structure; Testing; Time; alpha synuclein; aqueous; base; cold temperature; computerized data processing; experience; improved; methyl methanethiosulfonate; nanocrystal; novel strategies; protein complex; protein structure; prototype; public health relevance; solid state; solid state nuclear magnetic resonance; structural biology; synuclein; vector

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.

描述（由申请人提供）：在该项目中，将开发新型固态核磁共振（SSNMR）低温（~100 K）探针技术，以及最有效利用该技术的样品制备、数据收集和数据处理能力近年来，膜蛋白和原纤维的结构研究将有助于提高对膜蛋白（目前的大多数药物靶点）以及与帕金森病和其他神经退行性疾病有关的蛋白质的结构的理解。 SSNMR 结构研究取得了实质性进展，多个研究小组已成功确定蛋白质结构，不仅包括纳米晶体，还包括寡聚膜肽、淀粉样原纤维和大膜蛋白复合物，这些工作主要依赖于 SSNMR 数据的质量，包括两者。灵敏度和分辨率，共同决定结构测定的可行性和最终的结构质量。 通过追求三个目标，将评估和改进低温下的 SSNMR 谱，特别是膜蛋白和原纤维，目标#1 是完成基于 600 MHz 的 750 MHz 冷魔角旋转探针的制造和安装。原型，包括灵敏度的关键特性（3.2 mm 转子直径；冷 RF 电路；具有低 E 场 1H 谐振器和 13C/15N 螺线管的交叉线圈谐振器）、分辨率（20 kHz MAS、<0.05 ppm B0 均匀性、1H 谐振器上的高功率处理能力）、吞吐量（气动样品插入和弹出；样品更换时的稳定时间 <15 分钟）和长期可操作性（~2 L/小时 N2（ l) 消耗，稳定的射频调谐性能，以延长数据收集周期。目标#2是开发改进的样品制备协议，以提高低温下的光谱分辨率和灵敏度。利用有机醇、二醇和盐的稀疏同位素标记模式和冷冻保护程序可通过使用顺磁弛豫增强试剂（通过共价标签以及可溶性水性和脂质结合掺杂剂定位到蛋白质）来缩短 T1 弛豫时间，从而提高灵敏度。 #3 是使用原型四跨膜螺旋膜蛋白 (DsbB) 和大的、主要是进行分配和结构确定的案例研究¿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。