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续订应用程序的目标是进一步开发LED增强的核磁共振技术(LC-照片- CIDNP)，并将该方法扩展到方便和超 亚微摩尔浓度蛋白质的快速一维至三维核磁共振波谱。我们将专注于核磁共振研究 在溶液中，并将靶向在缓冲溶液或细胞中折叠、未折叠和内在无序的蛋白质- 就像媒体一样。我们将分三步走实现上述目标。首先(具体目标1)，我们将合并一个 在可溶性蛋白质中含有准隔离的1H-13C自旋对的色氨酸同位素 获得前所未有的核磁共振灵敏度，用于检测纳分子和蛋白质中暴露于溶剂的色氨酸 亚纳摩尔水平。然后，我们将利用上述技术与野外自行车相结合来实现 进一步增强了核磁共振的灵敏度。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以外的酸。这一目标将通过穿透空间和通过- 键极化转移方法。第三(具体目标3)，我们将LC-PHOTO-CIDNP扩展到更高的- 发展新的3D(可能是4D)1H，13C异核的维度(&gt；2D)核磁共振波谱 为分析侧链和主链1H-13C共振对量身定做的光谱脉冲序列。 这项工作将包括非均匀抽样(NUS)数据收集计划。然后我们将结合理论 优化g因子值和长时间的LC-Photo-CIDNP染料的计算和实验 最佳LC-PHOTO-CIDNP数据收集的光激发态寿命。我们还将开发这一特殊的油田 LED增强核磁共振的依赖性，并在台式核磁共振光谱仪上实现2D LC-PHOTO-CIDNP。 最后，我们将通过以下方式测试在本工作中开发的改进的LC-PHOTO-CIDNP技术的成功 易于聚集的客户蛋白(SH3变异体)与Hsp70分子相互作用的研究 亚微摩尔浓度的伴侣。