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.

项目摘要 该应用程序的目的是为高度敏感的新型激光增强NMR技术开发 溶液中氨基酸，多肽和蛋白质的原子分辨率分析，至微摩尔 专注。这项工作所涉及的仪器包括高功率激光器（均连续波动 并通过光纤与配备四倍体 - 共振（HFCN）低温探针。瞬态电子滥用测量也将进行 监视Photo-CIDNP染料的光激发三胞胎寿命。通过这项研究获得的进步将 在前所未有的低浓度下，启用对多肽和蛋白质的高分辨率NMR分析。 该提案进一步扩展了最初在PI实验室进行的关键方法开发 氨基酸到多肽和蛋白质领域。我们将在三个步骤内完成上述目标。 首先（特定目标＃1），将在有新获得的低温探针的情况下探索照片cidnp 600 MHz，以进一步扩展溶液中的NMR灵敏度到纳摩尔范围。我们的研究将采用 最近开发的光敏剂荧光素，适合低浓度摄影光周期，并将采用 NMR样品中彻底的氧气耗竭的优势，并将使用超飞秒激光 激光驱动的NMR灵敏度增强的辐照。第二个（特定目标＃2），我们将系统地 通过将其适用性扩展到多肽和简单缓冲的蛋白 解决方案以及更复杂的人体相关环境。我们将完成 通过测试在蛋白质存在下定制的新的光敏剂染料，通过测试新的光敏剂染料， 开发适当的多维照片cidnp nmr脉冲序列，并通过收集数据 缓冲溶液，无细胞系统和可能的活细胞细胞。第三（特定目标＃3），我们将扩展 通过在Photo-Cidnp脉冲序列中实现NOE模块的照片盘对非芳香氨基酸 将多肽和蛋白质的光周期增强的磁化分布从芳香族保留到 其他氨基酸。最后，我们将开拓新的双激光 - 辐照实验，以进行瞬态氧化 所有氨基酸中存在的羰基官能蛋白功能群，尤其是 以其光激发状态氧化。一旦提出了新技术，它将立即 可以在氨基酸，多肽和蛋白质的溶液中收集超极化的NMR数据 溶液和细胞样环境中亚微摩尔浓度的生物医学相关性。