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
• 负责人: Silvia Cavagnero
• 金额: $ 33.38万
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10242819
• 关键词: 3-Dimensional; Achievement; Address; Amino Acids; Basic Science; Buffers; Carbon; Cell Nucleus; Cell-Free System; Cells; Chemicals; Client; Complex; Coupled; Crowding; Data; Data Collection; Development; Development Plans; Disease; Dyes; Environment; Exhibits; Fiber Optics; Fluorescein; Frequencies; Goals; Investigation; Laboratories; Lasers; Liquid substance; Maps; Measurement; Medical; Molecular; Molecular Chaperones; Molecular Structure; Monitor; Multidimensional NMR Techniques; NMR Spectroscopy; National Institute of General Medical Sciences; Nuclear; Nuclear Magnetic Resonance; Optics; Oxides; Oxygen; Peptides; Performance; Photosensitizing Agents; Physiologic pulse; Physiological; Procedures; Process; Proteins; Reporting; Research; Resolution; Rotation; Sampling; Scheme; Side; Solvents; Spectrum Analysis; Structure; Technology; Temperature; Testing; Time; Triplet Multiple Birth; Variant; Vertebral column; Viscosity; Work; absorption; cryogenics; experimental study; functional group; instrumentation; irradiation; macromolecule; method development; molecular dynamics; nanomolar; new technology; novel; oxidation; polypeptide; preservation; technology development; tool; triplet state; tryptophyltyrosine

Project Summary The goal of this application is to develop a novel laser-enhanced NMR technology for the highly sensitive atomic-resolution analysis of amino acids, polypeptides and proteins in solution, down to sub-micromolar concentration. The instrumentation involved in this work comprises high-power lasers (both continuous-wave and pulsed) coupled, via fiber optic, to a commercial 600 MHz NMR spectrometer equipped with a quadruple- resonance (HFCN) cryogenic probe. Transient electronic absorption measurements will also be performed to monitor the photoexcited triplet lifetime of photo-CIDNP dyes. The advances gained via this research will enable the high-resolution NMR analysis of polypeptides and proteins at unprecedentedly low concentration. The proposal further extends key method-development originally carried out in the PI’s laboratory on single amino acids to the realm of polypeptides and proteins. We will accomplish the above goals within three steps. First (Specific Aim #1), photo-CIDNP will be explored in the presence of a newly acquired cryogenic probe at 600 MHz, to further extend NMR sensitivity in solution to the nanomolar range. Our studies will employ the recently developed photosensitizer fluorescein, tailored to low-concentration photo-CIDNP, and will take advantage of thorough oxygen depletion in NMR samples, and will employ ultrafast femtosecond laser irradiation for laser-driven NMR sensitivity enhancement. Second (Specific Aim #2), we will systematically extend photo-CIDNP in solution, by extending its applicability to polypeptides, and proteins in simple buffered solution and in more complex highly crowded physiologically relevant environments. We will accomplish the above goal by testing new photosensitizer dyes tailored to optimal performance in the presence of proteins, by developing appropriate multidimensional photo-CIDNP NMR pulse sequences, and by collecting data in buffered solution, cell-free systems, and possibly live bacterial cells. Third (Specific Aim #3), we will extend photo-CIDNP to non-aromatic amino acids by implement NOE modules within photo-CIDNP pulse sequences to spread the photo-CIDNP-enhanced magnetization of polypeptides and proteins from aromatic residues to other amino acids. Finally, we will pioneer new double-laser-irradiation experiments for the transient oxidation of the carbonyl functional group of proteins, which is present in all amino acids, and is known to be particularly oxidizing in its photo-excited state. Once the proposed new technology has been developed, it will immediately become possible to collect hyperpolarized NMR data in solution on amino acids, polypeptides and proteins of biomedical relevance at sub-micromolar concentration in solution and cell-like environments.

项目概要 该应用的目标是开发一种新型激光增强核磁共振技术，用于高灵敏度 对溶液中的氨基酸、多肽和蛋白质进行原子分辨率分析，低至亚微摩尔 这项工作涉及的仪器包括高功率激光器（均为连续波）。 和脉冲）通过光纤耦合到配备有四重传感器的商用 600 MHz NMR 波谱仪 共振（HFCN）低温探针也将进行瞬态电子吸收测量。 监测光 CIDNP 染料的光激发三重态寿命将通过这项研究取得的进展。 能够以前所未有的低浓度对多肽和蛋白质进行高分辨率 NMR 分析。 该提案进一步扩展了最初在 PI 实验室针对单一项目进行的关键方法开发。 我们将通过三个步骤实现上述目标。 首先（具体目标#1），将在新获得的低温探针存在的情况下探索光-CIDNP 600 MHz，为了进一步将溶液中的 NMR 灵敏度扩展到纳摩尔范围，我们的研究将采用 最近开发的光敏剂荧光素，专为低浓度光CIDNP而设计，并将采取 核磁共振样品中彻底耗氧的优势，并将采用超快飞秒激光 第二（具体目标#2），我们将系统地进行激光驱动核磁共振的辐射。 通过将其适用性扩展到简单缓冲中的多肽和蛋白质，将光 CIDNP 扩展到溶液中 解决方案以及在更复杂、高度拥挤的生理相关环境中我们将完成该任务。 通过测试新的光敏剂染料来实现上述目标，以在蛋白质存在的情况下实现最佳性能， 开发适当的多维照片 CIDNP NMR 脉冲序列，并通过收集数据 第三（具体目标#3），我们将扩展缓冲溶液、无细胞系统和可能的活细菌细胞。 通过在 photo-CIDNP 脉冲序列中实施 NOE 模块，将 photo-CIDNP 转化为非芳香族氨基酸 将多肽和蛋白质的光 CIDNP 增强磁化从芳香残基传播到 最后，我们将开创新的双激光照射瞬时氧化实验。 蛋白质的羰基官能团存在于所有氨基酸中，并且已知特别重要 一旦所提出的新技术被开发出来，它将立即在光激发状态下氧化。 收集氨基酸、多肽和蛋白质溶液中的超极化 NMR 数据成为可能 溶液和类细胞环境中亚微摩尔浓度的生物医学相关性。