Development of a Laser-Assisted NMR Technology for the Atomic-Resolution Analysis of Medically Relevant Biomolecules in Solution at Submicromolar Concentration

项目摘要 本申请的目标是开发一种新型的激光增强核磁共振技术，用于高灵敏度的 对溶液中的氨基酸、多肽和蛋白质进行原子分辨率分析，分析精度可达亚微摩尔 浓度.在这项工作中涉及的仪器包括高功率激光器（连续波 和脉冲的）通过光纤耦合到配备有四重NMR谱仪的商用600 MHz NMR光谱仪。 谐振（HFCN）低温探头。还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。通过这项研究取得的进展将 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实验室中进行的单一方法开发， 从氨基酸到多肽和蛋白质。我们将分三步走实现上述目标。 首先（具体目标#1），将在新获得的低温探针的存在下探索光CIDNP， 600 MHz，以进一步将溶液中的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，专门针对低浓度的光CIDNP，并将采取 利用NMR样品中的彻底氧耗尽，并将采用超快飞秒激光 用于激光驱动NMR灵敏度增强的辐照。第二（具体目标#2），我们将系统地 通过将其适用性扩展到简单缓冲溶液中的多肽和蛋白质， 解决方案和更复杂的高度拥挤的生理相关环境中。我们将实现 通过测试在蛋白质存在下定制为最佳性能的新光敏剂染料， 开发适当的多维光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菌细胞。第三（具体目标#3），我们将扩大 通过在光CIDNP脉冲序列内实施NOE模块将光CIDNP转化为非芳香族氨基酸 将多肽和蛋白质的光CIDNP增强的磁化从芳香残基扩散到 其他氨基酸最后，我们将开创新的双激光辐照瞬态氧化实验 蛋白质的羰基官能团，它存在于所有氨基酸中，并且已知是特别重要的。 在其光激发态氧化。一旦拟议中的新技术开发出来，它将立即 成为可能收集超极化NMR数据的氨基酸，多肽和蛋白质的解决方案， 在溶液和细胞样环境中亚微摩尔浓度的生物医学相关性。

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.