1 and 2D IR structural analyses of Influenza M2 channel

描述（由申请人提供）：流感目前是西方世界传染病导致死亡的主要原因。因此，对金刚烷胺（仅有的两（2）种可用于对抗流感的药物类别之一）的耐药性的出现是明显的生物医学威胁。金刚烷胺靶向甲型流感病毒M2，pH激活H+通道，在那里它作为一种通道阻断剂。为了了解金刚烷胺的机制和对它的耐药性的出现，需要对蛋白质和蛋白质-药物复合物的结构进行分子理解。当试图解决膜蛋白的结构时，标准的结构方法遇到了严重的困难。因此，我们建议开发和评估集成1/2D红外方法的结构准确性，该方法与位点特异性同位素标记相结合，非常适合快速阐明跨膜螺旋束（例如M2）的骨架结构。在这个提议中，同位素标记的M2肽（例如1- 13 C = 18 O和1- 13 C = 16 O）将使用ATR-FTIR和2D IR光谱进行研究，以产生对骨架羰基之间的角度和距离的限制。ATR-FTIR将用于测量羰基相对于膜的角度，2D IR将给出羰基之间的相对角度和源自振动耦合的距离。约束分子动力学模拟将用于构建M2的结构模型。同时，2D IR光子回波测量将通过其振动动力学来定义孔衬里残留物，这将提供结构模型的独立测试。由于甲型流感病毒M2 H+是其结构已被广泛表征的极少数跨膜螺旋束之一，因此它为评估这种新方法的准确性提供了独特的机会。一旦建立，将为一系列严格的 在金刚烷胺存在下和在低pH条件下对M2的结构进行实验，这是目前其他方法无法实现的。

DESCRIPTION (provided by applicant): Influenza is currently the leading cause of death due to an infectious disease in the western world. As such, the emeregence of resistance towards amantadine, one of only two (2) drug classes available against Influenza, is a clear biomedical threat. Amantadine targets the Influenza A M2, pH activated H+ channel, where it acts as a channel blocking anti-flue agent. In order to understand the mechanism of amantadine and the emergence of resistance towards it, a molecular understanding of the structure of the protein and the protein-drug complex is required. Standard structural approaches encounter severe difficulties when attempting to solve the structures of membrane proteins. As such, we propose to develop and assess the structural accuracy of an integrated 1/2D infrared approach, that in conjunction with site-specific isotope labeling, is well-suited for elucidating the backbone structure of transmembrane helical bundles, such as M2, in a rapid manner. In this proposal, isotopically labeled M2 peptides (e.g. 1-13C=18O and 1-13C=16O) will be studied with ATR-FTIR and 2D IR spectroscopies to yield constraints on the angles and distances between backbone carbonyl groups. ATR-FTIR will be used to measure the carbonyl group angles relative to the membrane and 2D IR will give relative angles between carbonyl groups and distances derived from vibrational couplings. Constrained molecular dynamics simulations will be used to construct a structural model of M2. In parallel, 2D IR photon echo measurements will define the pore lining residues by their vibrational dynamics that will provide an independent test of the structural model. Since, Influenza A M2 H+ is one (1) of very few transmembrane helical bundles whose structure has been extensively characterized, it provides a unique opportunity to assess the accuracy of this novel approach. Once established, the stage will be set for a rigorous series of experiments on the structure of M2 in the presence of amantadine and under low pH conditions that are currently not possible with other methods.