1 and 2D IR structural analyses of Influenza M2 channel

描述（由申请人提供）：流行性感冒是目前西方世界因传染病导致死亡的主要原因。金刚烷胺是目前仅有的两种抗流感药物之一，因此，金刚烷胺耐药性的出现显然对生物医学构成了威胁。金刚烷胺靶向流感A M2， pH激活的H+通道，在那里它作为通道阻断抗烟道剂。为了了解金刚烷胺的作用机制及其耐药性的出现，需要对蛋白质和蛋白质-药物复合物的分子结构有一个了解。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红外方法的结构精度，该方法与位点特异性同位素标记相结合，非常适合于快速阐明跨膜螺旋束（如M2）的骨干结构。在本提案中，同位素标记的M2肽（例如1-13C=18O和1-13C=16O）将用ATR-FTIR和2D IR光谱进行研究，以获得主链羰基之间的角度和距离的约束。ATR-FTIR将用于测量羰基与膜的相对角度，二维红外将给出羰基之间的相对角度和振动耦合产生的距离。约束分子动力学模拟将用于构建M2的结构模型。同时，二维红外光子回波测量将通过其振动动力学来定义孔隙衬里残留物，这将为结构模型提供独立的测试。由于流感A M2 H+是为数不多的跨膜螺旋束之一，其结构已被广泛表征，这为评估这种新方法的准确性提供了一个独特的机会。一旦建立，舞台将为一系列严格的

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.