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.

项目概要 此次 R01 更新应用的目标是进一步开发 LED 增强 NMR 技术（LC-photo- CIDNP）是在上一个资助周期中建立的，并将该方法扩展到简单和超 亚微摩尔浓度蛋白质的快速 1D 至 3D NMR 光谱。我们将重点关注核磁共振研究 在溶液中，将靶向缓冲溶液或细胞中的折叠、未折叠和本质上无序的蛋白质 像媒体一样。我们将通过三步走来实现上述目标。首先（具体目标#1），我们将纳入 色氨酸 (Trp) 同位素同位素在可溶性蛋白质中带有准隔离的 1Hα-13Cα 自旋对 (QISP) 实现前所未有的 NMR 灵敏度，可检测纳摩尔和蛋白质中溶剂暴露的色氨酸 亚纳摩尔水平。然后我们将采用上述技术结合现场循环来实现 进一步增强 NMR 灵敏度。第二（具体目标#2）我们将 LC-photo-CIDNP 扩展到氨基 蛋白质中除色氨酸和酪氨酸外的酸。这一目标将通过穿越空间和穿越来实现 键极化转移方法。第三（具体目标#3），我们将把 LC-photo-CIDNP 扩展到更高的- 通过开发新型 3D（可能还有 4D）1H,13C 异核来实现维度 (>2D) NMR 光谱 专为分析侧链和主链 1H-13C 共振对而定制的光谱脉冲序列。 这项工作将包括非均匀采样（NUS）数据收集方案。然后我们将结合理论 计算和实验开发更好的 LC-photo-CIDNP 染料，具有优化的 g 因子值和长 光激发态寿命，用于最佳的 LC-photo-CIDNP 数据收集。我们还将开拓这个特殊的领域 LED 增强 NMR 的依赖性，并在台式 NMR 波谱仪上实施 2D LC-photo-CIDNP。 最后，我们将测试本工作中开发的改进的 LC-photo-CIDNP 技术是否成功。 研究易于聚集的客户蛋白（SH3 变体）与 Hsp70 分子的相互作用 亚微摩尔浓度的伴侣。