Sensitivity enhancement in solution NMR through dynamic nuclear polarization

通过动态核极化提高溶液 NMR 的灵敏度

• 批准号: 8729503
• 负责人: A. JOSHUA WAND
• 金额: $ 20万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-02 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8729503
• 关键词: Access to Information; Address; Alkanes; Biological; Biological Process; Biopolymers; Cell Nucleus; Crystallography; Development; Electromagnetic Energy; Electronics; Electrons; Encapsulated; Exercise; Experimental Designs; Free Radicals; Frequencies; Future; Heat Losses; Heating; Human Resources; Hydrogen; Knowledge; Ligand Binding; Liquid substance; Magnetic Resonance; Measurement; Membrane Proteins; Micelles; Mind; NMR Spectroscopy; Nature; Nuclear; Nuclear Magnetic Resonance; Nucleic Acids; Performance; Physics; Population; Preparation; Process; Property; Proteins; Relative (related person); Relaxation; Request for Applications; Research; Resolution; Sampling; Scheme; Signal Transduction; Solubility; Solutions; Solvents; Source; Spin Labels; Staging; Structure; System; Technology; Time; Viscosity; Water; aqueous; base; dielectric property; improved; instrumentation; interest; irradiation; macromolecule; magnetic field; nucleic acid stability; physical property; protein structure; public health relevance; research study; response; solid state; solid state nuclear magnetic resonance; surfactant; tool

DESCRIPTION (provided by applicant): The structural and dynamic aspects of proteins have been at center stage of our understanding of the basis of their function. Nuclear magnetic resonance in solution has contributed significantly to this advancement and the information inherent in the NMR phenomena offers much more. Yet, despite tremendous advances in technology, experimental design and analytical strategies, solution NMR spectroscopy remains fundamentally restricted due to its extraordinary insensitivity. The inability to investigate protens and other biopolymers at well below sub-millimolar concentrations and using sub-micromole amounts presents severe limitations on future applications. Nevertheless, solution NMR offers, in principle, access to information that is very difficult to obtain by other means. Examples include access to dynamics over an enormous range of time scales, to details of ligand binding, to structures in unusual contexts and so on. Thus, it seems important to improve the sensitivity of the solution NMR experiment in order to reduce experiment time, lower the absolute quantities of sample required and open a lower concentration regime where proteins of limited solubility can be accessed. With this in mind there has been a revival of an "old" phenomenon - dynamic nuclear polarization (DNP). The idea is to use the enormously greater polarization of a radical electron in a magnetic field to polarize nuclei such as hydrogen to a much greater degree than the Boltzmann distribution dictated by the properties of the nuclei themselves. The physics underlying this process can be quite complicated, particularly in the solid state where several mechanisms for polarization are operative. In solution, it is generally thought that such DNP transfer will occur primarily through the Overhauser effect. One can imagine that increases in sensitivity of several hundred folds are accessible. For solution NMR, the basic strategy is to saturate the electronic transition of a stable free radical and transfer this non-equilibrium polarization to the hydrogen spins of water, which will in turn transfer this polarization to the hydrogens of the dissolved macromolecule. Unfortunately, technical aspects of this approach seem to prove fatal to the idea in its current form. The primary reason is that the frequency of the electron transition of suitable radicals lies in the subTHz spectrum where water absorbs strongly. Thus, irradiation results in catastrophic heating of the sample and its destruction. Here we will take advantage of the physical properties of solutions of encapsulated proteins dissolved in low viscosity solvents of suitable dielectric character. Such samples are largely transparent to the subTHz frequencies required and thereby avoid significant heating during saturation of the electronic transition. A variety of proteins ranging from small to large soluble proteins; acidic t basic proteins; integral and anchored membrane proteins; proteins of marginal stability and nucleic acids can be encapsulated with high structural fidelity. Thus the merging of the reverse micelle technology with DNP will provide a significant increase in the sensitivity of the solution NMR spectroscopy of proteins and nucleic acids.

描述（由申请人提供）：蛋白质的结构和动态方面一直是我们对其功能基础理解的中心阶段。溶液中的核磁共振对这一进步做出了重大贡献，核磁共振现象中固有的信息提供了更多。然而，尽管在技术、实验设计和分析策略方面取得了巨大的进步，但溶液核磁共振波谱由于其异常的不灵敏度，仍然从根本上受到限制。无法在低于亚毫摩尔浓度和使用亚微摩尔量的情况下研究蛋白质和其他生物聚合物，这严重限制了未来的应用。然而，解决方案NMR原则上提供了通过其他方式很难获得的信息。例子包括在巨大的时间尺度范围内获得动力学，了解配体结合的细节，了解不寻常背景下的结构等等。因此，提高溶液核磁共振实验的灵敏度似乎很重要，以减少实验时间，降低所需样品的绝对数量，并打开一个低浓度的区域，在那里可以获得有限溶解度的蛋白质。考虑到这一点，一种“老”现象——动态核极化（DNP）又复活了。这个想法是利用自由基电子在磁场中极大的极化，使原子核（如氢）极化到比由原子核本身的性质决定的玻尔兹曼分布大得多的程度。这个过程背后的物理原理可能相当复杂，特别是在固态中，有几种极化机制在起作用。在解决方案中，一般认为这种DNP转移将主要通过奥弗豪泽效应发生。可以想象，将灵敏度提高几百倍是可以实现的。对于溶液核磁共振，基本策略是饱和稳定自由基的电子跃迁，并将这种非平衡极化转移到水的氢自旋上，而水的氢自旋又将这种极化转移到溶解的大分子的氢上。不幸的是，这种方法的技术方面似乎对当前形式的想法是致命的。主要原因是合适自由基的电子跃迁频率位于水吸收较强的次太赫兹谱。因此，辐照会导致样品的灾难性加热和破坏。在这里