Development of structural biology methods for solid-state NMR utilizing sensitivity enhancement via dynamic nuclear polarization (DNP) and their application to drug-binding of membrane proteins

利用动态核极化 (DNP) 增强灵敏度开发固态 NMR 的结构生物学方法及其在膜蛋白药物结合中的应用

• 批准号: 211335566
• 负责人: Dr. Marcel Reese
• 金额: --
• 依托单位: Francis Bitter Magnet Laboratory
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2012-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/211335566?language=en
• 关键词: Development; structural; biology; methods; solid

Solid state NMR spectroscopy (ssNMR) is a powerful tool of structural biology. In contrast to crystallography it does not require molecular long-range order. This facilitates sample preparation and allows for more physiological sample conditions. SsNMR finds an application to structural studies of membrane proteins and their interaction with drugs.One membrane protein of major interest is M2 of influenza A. It has been the target of the antiviral drugs rimantadine and amantadine. Due to mutations of M2 these drugs lost their efficacy completely. To facilitate the development of alternative drugs major efforts have been invested to unravel the structural details of the M2-drug interaction. Unfortunately the contradictory data does not lead to a conclusive picture. One of the reasons seem to be structural artefacts introduced by the used membrane mimetics.SsNMR allows for structure determination of membrane proteins in native and physiological lipid bilayers; while the intrinsically low sensitivity of NMR requires large sample amounts, expensive isotope labelling and long measurement times.Dynamic nuclear polarization (DNP) can be used to transfer the large electronic polarization of stable radicals to nuclear spins to amplify the NMR signal by two orders of magnitude. Depending on the sample under investigation the resonance line width under cryogenic DNP conditions can be increased, possibly complicating resonance assignment.Novel methods for sensitivity and resolution enhancement shall be developed and implemented in DNP spectrometers of up to 800 MHz proton frequency.These techniques shall allow for the development of novel high-dimensional NMR spectra to facilitate resonance assignment and internuclear distance determination and will find application in the structure determination of M2 in complex with its drugs.

固体核磁共振波谱（ssNMR）是结构生物学研究的有力工具。与晶体学相反，它不需要分子长程有序。这有助于样品制备，并允许更多的生理样品条件。ssNMR可应用于膜蛋白的结构研究及其与药物的相互作用，其中一种主要的膜蛋白是甲型流感病毒的M2。它一直是抗病毒药物金刚烷胺和金刚烷胺的靶点。由于M2的突变，这些药物完全失去了效力。为了促进替代药物的开发，已投入大量精力来解开M2-药物相互作用的结构细节。不幸的是，这些相互矛盾的数据并不能得出结论性的结论。其原因之一似乎是所使用的膜模拟物引入的结构假象。ssNMR允许天然和生理脂质双层中膜蛋白的结构测定;虽然NMR固有的低灵敏度需要大量的样品，昂贵的同位素标记和长的测量时间。动态核极化（DNP）可用于将稳定自由基的大电子极化转移到核自旋，以将NMR信号放大两个数量级。根据所研究的样品，可以增加低温DNP条件下的谐振线宽，应开发提高灵敏度和分辨率的新方法，并在高达800 MHz质子频率的DNP谱仪中实施。这些技术应允许开发新的高分辨率、高灵敏度和高分辨率的DNP谱仪。三维核磁共振谱，以促进共振分配和核间距的测定，并将发现在M2的结构确定与其药物的复杂应用。