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 这项应用的目标是开发一种新的激光增强核磁共振技术，用于高灵敏 溶液中氨基酸、多肽和蛋白质的原子分辨分析，精度可达亚微摩尔 集中精神。这项工作中涉及的仪器包括高功率激光器(两种连续波 和脉冲)通过光纤耦合到商用600 MHz核磁共振光谱仪，该光谱仪配备有四倍- 共振(HFCN)低温探头。还将进行瞬时电子吸收测量，以 监测光敏CIDNP染料的光激发三重态寿命。通过这项研究取得的进展将 以前所未有的低浓度实现多肽和蛋白质的高分辨率核磁共振分析。 该提案进一步扩展了关键方法--最初在PI的实验室中进行的单机开发 氨基酸属于多肽和蛋白质的范畴。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测器存在的情况下探索PHOTO-CIDNP 600 MHz，进一步将溶液中的核磁共振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，专为低浓度的光敏剂CIDNP，并将采取 核磁共振样品彻底耗氧的优势，并将使用超快飞秒激光 激光驱动的核磁共振灵敏度增强的辐照。第二(具体目标2)，我们将系统地 通过将其适用范围扩展到简单缓冲中的多肽和蛋白质，将Photo-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 通过测试新的光敏剂染料在蛋白质存在下的最佳性能来实现上述目标，通过 开发合适的多维Photo-CIDNP核磁共振脉冲序列，并通过收集 缓冲溶液，无细胞系统，可能还有活的细菌细胞。第三(具体目标3)，我们将延长 在光敏CIDNP脉冲序列中实现NOE模块将光敏CIDNP转化为非芳香族氨基酸 将多肽和蛋白质的光CIDNP增强磁化作用从芳香族残基扩展到 其他氨基酸。最后，我们将开创用于瞬变氧化的新的双激光辐照实验 蛋白质的羰基官能团，它存在于所有氨基酸中，已知尤其是 在其光激发状态下氧化。一旦提议的新技术被开发出来，它将立即 在溶液中收集氨基酸、多肽和蛋白质的超极化核磁共振数据成为可能 溶液和类细胞环境中亚微摩尔浓度的生物医学相关性。

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.