Sensitivity enhancement in solution NMR through dynamic nuclear polarization

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，而核磁共振现象中固有的信息提供了更多的信息。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，但由于其异常不敏感，溶液核磁共振光谱仍然从根本上受到限制。无法在远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，这对未来的应用构成了严重的限制。然而，原则上，解决方案核磁共振提供了通过其他方式很难获得的信息的访问。例如，在一个巨大的时间范围内获得动力学，获得配体结合的细节，在不寻常的背景下获得结构等等。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量，并开辟一个较低的浓度区域，使有限溶解度的蛋白质可以获得。考虑到这一点，又出现了一种“老”现象--动态核极化(DNP)。这个想法是利用自由电子在磁场中极大的极化，使氢等原子核的极化程度远远超过由原子核本身的性质所决定的玻尔兹曼分布。这一过程背后的物理基础可能相当复杂，特别是在固体状态下，有几种极化机制在起作用。在解决方案中，通常认为这种DNP转移将主要通过奥弗豪泽效应发生。可以想象，感光度提高几百倍是可以实现的。对于溶液核磁共振，基本的策略是使稳定的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，水的氢自旋又会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。主要原因是合适的自由基的电子跃迁频率位于水被强烈吸收的亚太赫兹光谱中。因此，辐射会对样品造成灾难性的加热和破坏。这里 我们将利用胶囊化蛋白质溶解在具有合适介电性质的低粘度溶剂中的溶液的物理性质。这样的样本基本上是透明的 它可以提供所需的亚太赫兹频率，从而避免在电子跃迁饱和时产生大量热量。各种蛋白质，从小的到大的可溶性蛋白质；酸性碱性蛋白质；完整的和锚定的膜蛋白质；边缘稳定的蛋白质和核酸可以以高结构保真度被包裹。因此，反胶束技术与DNP的合并将大大提高蛋白质和核酸的溶液核磁共振光谱的灵敏度。

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.