Development of LED-Assisted NMR Technologies for the Atomic-Resolution Analysis of Medically Relevant Biomolecules in Solution at Submicromolar Concentration

开发 LED 辅助 NMR 技术，对亚微摩尔浓度溶液中的医学相关生物分子进行原子分辨率分析

• 批准号: 10659378
• 负责人: Silvia Cavagnero
• 金额: $ 56.9万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-08 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10659378
• 关键词: 3-Dimensional; Address; Amino Acids; Basic Science; Buffers; Carbon; Cells; Client; Communities; Data Collection; Dependence; Development; Development Plans; Devices; Disease; Dyes; Exhibits; Funding; Goals; Investigation; Isotope Labeling; Liquid substance; Medical; Methodology; Methods; Molecular Chaperones; Molecular Structure; NMR Spectroscopy; National Institute of General Medical Sciences; Nuclear; Nuclear Magnetic Resonance; Peptides; Photosensitizing Agents; Physiologic pulse; Protein Analysis; Proteins; Reporting; Research; Resolution; Sampling; Scheme; Solvents; Spectrum Analysis; Structure; Technology; Temperature; Testing; Time; Tryptophan; Variant; Vertebral column; Viscosity; Work; detection sensitivity; experimental study; high dimensionality; improved; light emission; magnetic field; molecular dynamics; nanomolar; new technology; novel; rapid detection; success; technology development; tool

Project Summary The goal of this R01 renewal application is to further develop the LED-enhanced NMR technology (LC-photo- CIDNP) that was established during the prior cycle of funding, and extend the method to the facile and ultra- rapid 1D-to-3D NMR spectroscopy of proteins at sub-micromolar concentration. We will focus on NMR studies in solution and will target folded, unfolded and intrinsically disordered proteins in either buffered solution or cell- like media. We will accomplish the above goals within three steps. First (Specific Aim #1), we will incorporate a tryptophan (Trp) isotopolog bearing a quasi-isolated 1H-13C spin pair (QISP) within soluble proteins to achieve unprecedented NMR sensitivity for the detection of solvent-exposed Trp in proteins at nanomolar and sub-nanomolar levels. We will then employ the above technology in combination with field-cycling to achieve further NMR sensitivity enhancements. Second (Specific Aim #2) we will extend LC-photo-CIDNP to amino acids other than Trp and Tyr within proteins. This goal will be accomplished via through-space and through- bond polarization transfer methodologies. Third (Specific Aim #3), we will extend LC-photo-CIDNP to higher- dimensionality (>2D) NMR spectroscopy by developing novel 3D (and possibly 4D) 1H,13C heteronuclear spectroscopy pulse sequences tailored to the analysis of side-chain and backbone 1H-13C resonance pairs. This effort will include non-uniform-sampling (NUS) data collection schemes. We will then combine theoretical calculations and experiments to develop better LC-photo-CIDNP dyes with optimized g-factor values and long photoexcited-state lifetimes, for optimal LC-photo-CIDNP data collection. We will also exploit the peculiar field dependence of LED-enhanced NMR and implement 2D LC-photo-CIDNP on benchtop NMR spectrometers. Finally, we will test the success of the improved LC-photo-CIDNP technologies developed in this work by studying the interaction of an aggregation-prone client protein (SH3 variant) with the Hsp70 molecular chaperone at sub-micromolar concentration.

项目摘要 此R01更新应用的目的是进一步开发具有LED增强的NMR技术（LC-Photo- cidnp）是在上一家资金周期中建立的，并将方法扩展到了易于和超级 在亚微摩尔浓度下蛋白质的快速1d到3D NMR光谱。我们将专注于NMR研究 在溶液中，将在缓冲溶液或细胞中靶向折叠，展开和本质上无序的蛋白 喜欢媒体。我们将在三个步骤内完成上述目标。首先（特定目标＃1），我们将合并 色氨酸（TRP）同位素学轴承固定蛋白内的准溶解1HA-13C自旋对（QISP） 在纳摩尔和 亚纳摩尔水平。然后，我们将使用上述技术与现场循环结合以实现 进一步的NMR灵敏度增强。第二（特定目标＃2）我们将将LC-Photo-Cidnp扩展到氨基 蛋白质内TRP和TYR以外的酸。这个目标将通过空间和通行来实现 键化转移方法。第三（特定目标＃3），我们将将LC-Photo-Cidnp扩展到更高 维度（> 2D）NMR光谱通过开发新型3D（和可能的4D）1H，13C异核。 光谱脉冲序列是针对侧链和骨干1H-13C共振对分析的分析的。 这项工作将包括非均匀抽样（NUS）数据收集方案。然后，我们将结合理论 计算和实验，以开发具有优化G因子值和长时间的更好的LC-Photo-CidNP染料 光激发状态的寿命，用于最佳的LC-Photo-CIDNP数据收集。我们还将利用特殊的领域 LED增强NMR和实现2D LC-Photo-Cidnp对台式NMR光谱仪的依赖性。 最后，我们将测试在这项工作中开发的改进的LC-Photo-CIDNP技术的成功 研究易于聚集的客户蛋白（SH3变体）与HSP70分子的相互作用 亚微摩尔浓度下的伴侣。