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: 8575754
• 负责人: Chad M Rienstra
• 金额: $ 18.98万
• 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/8575754
• 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(申请人提供)：在这个项目中，将开发新的固态核磁共振(SS核磁共振)低温(~100K)探头技术，以及最有效地利用这种能力进行膜蛋白和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对膜蛋白和与帕金森病和其他神经退行性疾病有关的蛋白质纤维的结构理解，膜蛋白是目前主要的药物靶标。近年来，SS核磁共振的结构研究取得了长足的进步，一些研究小组已经成功地确定了蛋白质的结构，不仅包括纳米晶，而且还包括低聚膜肽、淀粉样纤维和大的膜蛋白复合体。这些努力严重依赖于SS核磁共振数据的质量，包括灵敏度和分辨率，这两者共同决定了结构确定的可行性和最终的结构质量。这里的敏感度和分辨率 通过追求三个目标，将评估和改进低温下的SS核磁共振谱，特别是膜蛋白和纤维。第一个目标是在600 MHz样机的基础上完成750 MHz冷魔角旋转探头的制造和安装，包括灵敏度(3.2 mm转子直径；冷射频电路；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；0.05ppm B0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；&lt；更换样品的稳定时间为15分钟)和长期的可操作性(约2 L/小时氮气(L)消耗，延长数据采集期的稳定射频调谐性能)。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。将通过稀疏的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。通过共价标签定位于蛋白质的顺磁性驰豫增强剂，以及可溶的水和脂结合的掺杂剂，可以通过减少T1驰豫时间来提高灵敏度。目的#3是用典型的四跨膜螺旋膜蛋白(DsbB)和大的、以片状为主的纤维蛋白(？-突触核蛋白)进行指定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SS核磁共振探针技术，为评估光谱分辨率和灵敏度以及所产生的结构质量提供了极好的基准。