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

基本信息

  • 批准号:
    10659378
  • 负责人:
  • 金额:
    $ 56.9万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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。

项目成果

期刊论文数量(0)
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Silvia Cavagnero其他文献

Silvia Cavagnero的其他文献

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{{ truncateString('Silvia Cavagnero', 18)}}的其他基金

Development of a Laser-Assisted NMR Technology for the Atomic-Resolution Analysis of Medically Relevant Biomolecules in Solution at Submicromolar Concentration
开发激光辅助核磁共振技术,对亚微摩尔浓度溶液中医学相关生物分子进行原子分辨率分析
  • 批准号:
    10020189
  • 财政年份:
    2018
  • 资助金额:
    $ 56.9万
  • 项目类别:
Development of a Laser-Assisted NMR Technology for the Atomic-Resolution Analysis of Medically Relevant Biomolecules in Solution at Submicromolar Concentration
开发激光辅助核磁共振技术,对亚微摩尔浓度溶液中医学相关生物分子进行原子分辨率分析
  • 批准号:
    10242819
  • 财政年份:
    2018
  • 资助金额:
    $ 56.9万
  • 项目类别:
Development of Laser-Mediated Hyper-Sensitive NMR in Liquids
激光介导液体超灵敏核磁共振的发展
  • 批准号:
    8757756
  • 财政年份:
    2014
  • 资助金额:
    $ 56.9万
  • 项目类别:
Development of Laser-Mediated Hyper-Sensitive NMR in Liquids
激光介导液体超灵敏核磁共振的发展
  • 批准号:
    8898152
  • 财政年份:
    2014
  • 资助金额:
    $ 56.9万
  • 项目类别:
Analysis of De Novo Protein Folding by Fluorescence Resonance Energy Transfer
通过荧光共振能量转移分析从头蛋白质折叠
  • 批准号:
    8373308
  • 财政年份:
    2012
  • 资助金额:
    $ 56.9万
  • 项目类别:
Analysis of De Novo Protein Folding by Fluorescence Resonance Energy Transfer
通过荧光共振能量转移分析从头蛋白质折叠
  • 批准号:
    8550099
  • 财政年份:
    2012
  • 资助金额:
    $ 56.9万
  • 项目类别:
Analysis of De Novo Protein Folding by Fluorescence Resonance Energy Transfer
通过荧光共振能量转移分析从头蛋白质折叠
  • 批准号:
    8852633
  • 财政年份:
    2012
  • 资助金额:
    $ 56.9万
  • 项目类别:
Analysis of De Novo Protein Folding by Fluorescence Resonance Energy Transfer
通过荧光共振能量转移分析从头蛋白质折叠
  • 批准号:
    8668100
  • 财政年份:
    2012
  • 资助金额:
    $ 56.9万
  • 项目类别:
CONFORMATION OF HSP70-BOUND PEPTIDE SUBSTRATES PROBED USING NMR SPECTROSCOPY
使用核磁共振波谱探测 HSP70 结合肽底物的构象
  • 批准号:
    8361245
  • 财政年份:
    2011
  • 资助金额:
    $ 56.9万
  • 项目类别:
Ultra-sensitive NMR via Photochemically induced dynamic nuclear polarization
通过光化学诱导动态核极化的超灵敏核磁共振
  • 批准号:
    7991252
  • 财政年份:
    2010
  • 资助金额:
    $ 56.9万
  • 项目类别:

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