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

基本信息

批准号：
8077995
负责人：
Silvia Cavagnero
金额：
$ 10.64万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-06-03 至 2013-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8077995
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 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

项目摘要

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 光谱法有一个极其严重的缺点：灵敏度非常低。该项目的目标是开发新方法来提高液态核磁共振（NMR）的灵敏度并优化含有芳香氨基酸的生物分子的检测限。这项研究将利用异核关联光化学诱导动态核极化（也称为光 CIDNP）的方法开发，并将采用 NMR 装置与能够光激发含黄素 NMR 样品的氩离子激光器。 488 nm。将追求以下三个具体研究目标。具体目标#1：开发涉及 13C-1H 异核关联的新型基于 13C 的光 CIDNP 脉冲序列。将开发几种新颖的 2D 脉冲序列，并针对游离芳香氨基酸（包括具有强 1H photo-CIDNP 的色氨酸）和属于模型蛋白 apoHmpH 的芳香氨基酸进行测试。还将系统地探讨激光功率（高达 25W）、激光持续时间、温度和粘度的影响。鉴于我们最近关于 15N1H 异核关联和初步从头计算的结果，我们认为这一目标非常令人兴奋，这有力地支持了这种方法的可行性。具体目标#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侧链的芳香族化合物进行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。计算将使用威斯康辛大学麦迪逊分校化学系提供的高斯软件包进行。该目的的目的是评估光-CIDNP 中涉及的相关感兴趣核的自由基阳离子的超精细耦合。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：应用具体目标 #1 中开发的方法来研究富含芳香族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 TF 之外，源自 Hmp 蛋白（包含其残基 116-134）的富含芳香族的多肽也将用于这些研究。拟议的研究旨在在适合稀溶液中生物应用的温和、生理相关条件下突破液态核磁共振的灵敏度界限。公共健康相关性：该项目的目标是采用新方法来增强液态核磁共振 (NMR) 的灵敏度，并通过光化学诱导动态核极化 (photo-CIDNP) 优化含有芳香族氨基酸的生物分子的检测限。鉴于这种方法对蛋白质内的芳香族氨基酸和含有芳香环的抗生素的适用性，这项研究对于灵敏地检测抗生素与其生物分子靶标的结合具有直接影响，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相关，细菌翻译机制是抗生素开发的常见目标。