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

固体核磁共振是结构生物学的有力工具。与结晶学不同，它不需要分子的长程有序。这便于样品制备，并允许更多的生理样品条件。Ss核磁共振在膜蛋白的结构及其与药物相互作用的研究中得到了应用。甲型流感病毒M2是一种主要的膜蛋白，它一直是抗病毒药物金刚烷胺和金刚烷胺的靶标。由于M2基因突变，这些药物完全失去了疗效。为了促进替代药物的发展，已经投入了重大努力来揭示M2与药物相互作用的结构细节。不幸的是，相互矛盾的数据并不能得出决定性的结论。其中一个原因似乎是所使用的膜模拟物引入的结构伪影。Ss核磁共振可以确定天然和生理脂质双层中的膜蛋白的结构；而核磁共振固有的低灵敏度需要大量样品，昂贵的同位素标记和较长的测量时间。动态核极化(DNP)可以用来将稳定自由基的大电子极化转移到核自旋上，将核磁共振信号放大两个数量级。根据被研究样品的不同，低温DNP条件下的共振线宽度可能会增加，这可能会使共振指定复杂化。新的灵敏度和分辨率增强方法将在质子频率高达800 MHz的DNP光谱仪中开发和实施。这些技术将允许开发新的高维核磁共振谱，以促进共振指定和核间距离的确定，并将在M2及其药物的络合物结构确定中得到应用。

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.