Ultra-sensitive NMR via Photochemically induced dynamic nuclear polarization

描述(申请人提供)：核磁共振光谱学有一个极其严重的缺点：非常低的灵敏度。本项目的目标是开发新的方法来提高液体核磁共振的灵敏度，并优化含有芳香氨基酸的生物分子的检测下限。这项研究将利用异核相关光化学诱导的动态核极化(也称为Photo-CIDNP)方法的发展，并将使用核磁共振装置与能够在488 nm处光激发含有黄素的核磁共振样品的Argon离子激光相结合。将追求以下三个具体研究目标。具体目标#1：开发包含13C-1H异核相关的基于13C的新型光CIDNP脉冲序列。几个新的2D脉冲序列将被开发并在游离芳香氨基酸(包括显示出很强的1H光CIDNP的Trp)和属于模型蛋白质apoHmpH的芳香氨基酸上进行测试。此外，还将系统地探讨激光功率(25W以下)、激光持续时间、温度和粘度的影响。鉴于我们最近对15N1H异核关联和初步从头计算的结果，我们认为这个目标是非常令人兴奋的，这有力地支持了这一方法的可行性。具体目标#2：模型取代吲哚和其他类似天然氨基酸和氨基酸类似物侧链的芳香族化合物的从头算电子密度计算。这些计算将基于受限的Hartree-Fock和密度泛函方法。计算将使用UW-Madison化学部提供的Gauss软件包进行。这一目标的目的是评估光-CIDNP中涉及的自由基阳离子的超精细耦合，对于相关的核。这些研究的结果将为特定目标#1的方法开发提供参考。特定目标#3：应用在特定目标#1中开发的方法来研究富含芳香氨基酸的多肽在低浓度下与细菌触发因子(TF)分子伴侣的结合。除了来自大肠杆菌的转铁蛋白外，来自HMP蛋白(包括其残基116-134)的富含芳香族的多肽将被用于这些研究。拟议的研究旨在推动液态核磁共振在温和的生理相关条件下的灵敏度界限，适合于稀溶液中的生物应用。 与公众健康相关：该项目旨在通过光化学诱导动态核极化(PHOTO-CIDNP)提高液态核磁共振(NMR)的灵敏度，并优化含有芳香氨基酸的生物分子的检测下限。鉴于这种方法既适用于蛋白质中的芳香氨基酸，也适用于含有芳环的抗生素，这项研究对灵敏地检测抗生素与其生物分子靶点的结合具有直接意义，这也是NIAID的目标之一。细菌伴侣触发因子(Tf)的拟议应用与细菌翻译机制有关，细菌翻译机制是抗生素开发的常见目标。

DESCRIPTION (provided by applicant): NMR spectroscopy suffers from an extremely serious disadvantage: very low sensitivity. The goal of this project is to develop novel approaches to enhance the sensitivity of liquid state nuclear magnetic resonance (NMR) and optimize the detection limits of biomolecules containing aromatic amino acids. This research will take advantage of method developments in heteronuclear correlation photo-chemically-induced dynamic nuclear polarization, also known as photo-CIDNP, and it will employ NMR setups in conjunction with an Argon Ion laser able to photo-excite flavin-containing NMR samples at 488 nm. The following three specific research aims will be pursued. Specific Aim #1: Development of novel 13C-based photo-CIDNP pulse sequences involving 13C-1H heteronuclear correlation. Several novel 2D pulse sequences will be developed and tested on both free aromatic amino acids (including Trp, which exhibits a strong 1H photo-CIDNP) and aromatic amino acids belonging to the model protein apoHmpH. The effect of laser power (up to 25W), laser duration, temperature, and viscosity will also be systematically explored. We perceive this aim as extremely exciting, in light of our recent results on 15N1H heteronuclear correlation and preliminary ab initio calculations, which strongly support the feasibility of this approach. Specific Aim #2: Ab initio electron density calculations on model substituted indoles and other aromatic compounds resembling the side chain of naturally occurring amino acids and amino acid analogs. These calculations will be based on restricted Hartree-Fock and Density Functional approaches. The computations will be carried out with the Gaussian software package, available in the UW-Madison Department of Chemistry. The goal of this aim is to evaluate the hyperfine couplings of radical cations involved in photo-CIDNP, for the relevant nuclei of interest. The results of these studies will inform the method developments in Specific Aim #1. Specific Aim #3: Application of the methods developed in Specific Aim #1 to study the binding of polypeptides rich in aromatic amino acids to the bacterial trigger factor (TF) molecular chaperone at low concentrations. In addition to the TF from E. coli, an aromatic-rich polypeptide derived from the Hmp protein (comprising its residues 116-134) will be utilized for these studies. The proposed studies are aiming at pushing the sensitivity boundaries of liquid state NMR under mild, physiologically relevant conditions suitable for biological applications in dilute solutions. PUBLIC HEALTH RELEVANCE: This project targets novel approaches to enhance the sensitivity of liquid state nuclear magnetic resonance (NMR) and optimize the detection limits of biomolecules containing aromatic amino acids by photo-chemically- induced dynamic nuclear polarization (photo-CIDNP). Given the applicability of this approach to both aromatic amino acids within proteins and aromatic-ring-containing antibiotics, this research has direct implications for the sensitive detection of antibiotic binding to their biomolecular targets, one of the aims of NIAID. The proposed applications to the bacterial chaperone trigger factor (TF) are relevant to the bacterial translation machinery, a frequent target for antibiotic development.