Low Temperature Solid-State NMR Technologies for Protein Structure Determination

点击翻译按钮获取中文摘要

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.