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 此次R 01更新申请的目标是进一步开发LED增强型NMR技术（LC-照片- CIDNP），并将该方法扩展到简易和超 蛋白质在亚微摩尔浓度下的快速1D到3D NMR光谱。我们将专注于核磁共振研究 在溶液中，并且将靶向缓冲溶液或细胞中的折叠、未折叠和内在无序的蛋白质。 比如媒体。我们将分三步走实现上述目标。首先（具体目标#1），我们将纳入一个 色氨酸（Trp）同位素，在可溶性蛋白质中具有准分离的1H-13 C自旋对（QISP）， 实现前所未有的NMR灵敏度，用于检测纳摩尔浓度下蛋白质中溶剂暴露的Trp， 亚纳摩尔水平。然后，我们将采用上述技术结合现场循环，以实现 进一步提高NMR灵敏度。其次（具体目标#2），我们将LC-photo-CIDNP扩展到氨基 蛋白质中色氨酸和酪氨酸以外的氨基酸。这一目标将通过空间和通过- 键极化转移方法。第三（具体目标#3），我们将LC-photo-CIDNP扩展到更高级别- 通过开发新的3D（和可能的4D）1H，13 C杂原子， 光谱脉冲序列定制为侧链和主链1H-13 C NMR共振对的分析。 这项工作将包括非统一抽样数据收集计划。然后我们将联合收割机 计算和实验，以开发更好的LC-光-CIDNP染料，其具有优化的g因子值和长 光激发态寿命，用于最佳LC-光-CIDNP数据收集。我们还将探索 LED增强NMR的依赖性，并在台式NMR光谱仪上实现2D LC-照片-CIDNP。 最后，我们将测试在这项工作中开发的改进的LC-照片-CIDNP技术的成功， 研究了一种易于聚集的客户蛋白（SH 3变体）与Hsp 70分子的相互作用， 亚微摩尔浓度的分子伴侣。