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: 8575416
• 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/8575416
• 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理解其功能基础的中心阶段。溶液中的核磁共振对这一进步做出了重大贡献，并且 NMR 现象中固有的信息提供了更多信息。然而，尽管技术、实验设计和分析策略取得了巨大进步，溶液核磁共振波谱由于其非凡的不敏感性，仍然受到根本上的限制。无法在远低于亚毫摩尔浓度和使用亚微摩尔量下研究蛋白质和其他生物聚合物，这对未来的应用造成了严重限制。尽管如此，溶液核磁共振原则上可以提供通过其他方式很难获得的信息。例子包括了解大范围时间尺度的动力学、配体结合的细节、不寻常背景下的结构等等。因此，提高溶液 NMR 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量并打开较低的浓度范围，以获取溶解度有限的蛋白质。考虑到这一点，一种“古老”的现象——动态核极化（DNP）又复活了。这个想法是利用磁场中自由基电子的极大极化来使氢等原子核极化，其程度比由原子核本身特性决定的玻尔兹曼分布要大得多。这一过程背后的物理原理可能非常复杂，特别是在多种极化机制起作用的固态下。在解决方案中，人们普遍认为这种 DNP 转移主要通过奥弗豪瑟效应发生。可以想象，灵敏度可以提高数百倍。对于溶液核磁共振，基本策略是使稳定自由基的电子跃迁饱和，并将这种非平衡极化转移到水的氢自旋上，水又将这种极化转移到溶解大分子的氢上。不幸的是，这种方法的技术方面似乎对当前形式的想法来说是致命的。主要原因是合适自由基的电子跃迁频率位于水强烈吸收的亚太赫兹光谱中。因此，辐射会导致样品灾难性的加热及其破坏。这里 我们将利用溶解在具有适当介电特性的低粘度溶剂中的封装蛋白质溶液的物理特性。此类样本在很大程度上是透明的 所需的亚太赫兹频率，从而避免电子跃迁饱和期间的显着加热。各种蛋白质，从小到大的可溶性蛋白质；酸性和碱性蛋白质；整合膜蛋白和锚定膜蛋白；边缘稳定性的蛋白质和核酸可以以高结构保真度封装。因此，反胶束技术与 DNP 的结合将显着提高蛋白质和核酸溶液 NMR 光谱的灵敏度。