Ultra-sensitive NMR via Photochemically induced dynamic nuclear polarization

说明（由申请人提供）：NMR光谱学具有极其严重的缺点：灵敏度非常低。该项目的目标是开发新的方法来提高液态核磁共振（NMR）的灵敏度，并优化含有芳香族氨基酸的生物分子的检测限。这项研究将利用异质结相关光化学诱导的动态核极化（也称为photo-CIDNP）的方法发展，并将采用NMR装置与氩离子激光器结合，能够在488 nm处光激发含黄素的NMR样品。将追求以下三个具体研究目标。具体目标#1：开发涉及13 C-1H异质结相关的新型13 C基光CIDNP脉冲序列。将开发几种新的2D脉冲序列，并在游离芳香族氨基酸（包括Trp，其表现出强1H光CIDNP）和属于模型蛋白apoHmpH的芳香族氨基酸上进行测试。激光功率（高达25 W），激光持续时间，温度和粘度的影响也将系统地探讨。我们认为这一目标是非常令人兴奋的，鉴于我们最近的结果15 N1 H的异质结相关性和初步的从头计算，这有力地支持了这种方法的可行性。具体目标#2：模拟取代吲哚和其它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化学系提供的Gaussian软件包进行。这个目标的目的是评估参与光CIDNP的自由基阳离子的超精细耦合，为相关的核的利益。这些研究的结果将为具体目标#1中的方法开发提供信息。具体目标#3：应用特定目标#1中开发的方法研究富含芳香族氨基酸的多肽与低浓度细菌触发因子（TF）分子伴侣的结合。除了E.大肠杆菌中，一种来源于Hmp蛋白（包含其残基116-134）的富含芳香族的多肽将用于这些研究。拟议的研究旨在推动液态NMR的灵敏度界限下温和的，生理相关的条件下，适合生物应用在稀溶液。 公共卫生相关性：该项目的目标是通过光化学诱导动态核极化（photo-CIDNP）来提高液态核磁共振（NMR）的灵敏度并优化含芳香族氨基酸的生物分子的检测限的新方法。鉴于这种方法适用于蛋白质内的芳香族氨基酸和含芳香环的抗生素，这项研究对灵敏检测抗生素与其生物分子靶标的结合具有直接影响，这是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，这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.