Ultra-sensitive NMR via Photochemically induced dynamic nuclear polarization

通过光化学诱导动态核极化的超灵敏核磁共振

• 批准号: 7991252
• 负责人: Silvia Cavagnero
• 金额: $ 23.5万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-03 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7991252
• 关键词: Amino Acids; Antibiotics; Applications Grants; Area; Argon; Aromatic Amino Acids; Aromatic Compounds; Binding; Biological; Biological Process; Biology; Cations; Cell Nucleus; Cells; Chemicals; Chemistry; Complex; Computer software; Detection; Development; Diffusion; Disadvantaged; Electrons; Engineering; Environment; Escherichia coli; Exhibits; Flavins; Gases; Goals; Hand; Indoles; Investigation; Ions; Lasers; Light; Liquid substance; Medicine; Methods; Modeling; Molecular; Molecular Chaperones; NMR Spectroscopy; National Institute of Allergy and Infectious Disease; Nuclear; Nuclear Magnetic Resonance; Physiologic pulse; Principal Investigator; Proteins; Research; Research Proposals; Resolution; Role; Sampling; Science; Shapes; Side; Simulate; Solid; Solutions; Solvents; Techniques; Temperature; Testing; Translations; Uncertainty; Viscosity; analog; base; contextual factors; density; electron density; experience; improved; interest; method development; novel; novel strategies; polypeptide; protein folding; public health relevance; research study; tool

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.

描述（由申请人提供）：NMR光谱法患有极为严重的缺点：灵敏度非常低。该项目的目的是开发新的方法，以增强液态核磁共振（NMR）的灵敏度，并优化含有芳香族氨基酸的生物分子的检测极限。这项研究将利用异核相关性光化学诱导的动态核极化（也称为Photo-CIDNP）中的方法开发，并将使用NMR设置以及能够为488 nm的氩离子激光结合使用NMR设置。将追求以下三个具体研究目标。特定目的＃1：开发基于13C的新型光盘脉冲序列，涉及13C-1H异核相关性。将开发和测试几种新型的2D脉冲序列，并在游离芳族氨基酸（包括表现出强烈的1H光蛋白）和属于模型蛋白APOHMPH的芳族氨基酸上进行测试。激光功率（最大25W），激光持续时间，温度和粘度的影响也将系统探索。鉴于我们最近对15N1H异核相关性和初步计算的结果，我们认为这一目标非常令人兴奋，这强烈支持了这种方法的可行性。特定目的＃2：对模型取代的吲哚和其他类似于天然存在的氨基酸和氨基酸类似物的侧链的其他芳族化合物进行的电子密度计算。这些计算将基于限制的Hartree-Fock和密度功能方法。这些计算将使用UW-Madison化学系可用的高斯软件包进行。该目的的目的是评估相关的相关核，以评估参与光蛋白的自由基阳离子的超精细耦合。这些研究的结果将为特定目标1中的方法发展提供信息。特定目的＃3：在特定目标1中开发的方法的应用在低浓度下研究了富含芳香族氨基酸与细菌触发因子（TF）分子伴侣的多肽与细菌触发因子（TF）的结合。除了来自大肠杆菌的TF外，还将利用源自HMP蛋白（包括其残基116-134）的芳族富含芳香族的多肽。拟议的研究旨在在温和的生理相关条件下推动液态NMR的敏感性边界，适合于稀释溶液中的生物应用。 公共卫生相关性：该项目以新颖的方法来增强液态核磁共振（NMR）的灵敏度，并通过光化学诱导的动态核极化（Photo-CIDNP）优化含有芳香族氨基酸的生物分子的检测极限。鉴于这种方法适用于蛋白质中的芳族氨基酸和含有芳香环的抗生素的适用性，这项研究对抗生素结合的敏感检测与其生物分子靶靶标具有直接影响，这是NIAID的目标之一。所提出的对细菌伴侣触发因子（TF）的应用与细菌翻译机械有关，这是抗生素发育的常见靶标。