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.
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