We propose to explore the gating and pH activation of the proton-selective M2 and BM2 viral membrane channels at atomic resolution using nuclear magnetic resonance (NMR) and biochemical techniques. The M2 and BM2 channels from influenzas A and B, respectively, play central roles in viral entry and replication. The membrane proteins will be reconstituted into DHPC micelles and structural information will come primarily from sets of interatomic vector orientations calculated from residual dipolar couplings. Using such approaches, we will (1) solve the high-resolution structure of M2 channel in the closed conformation, and (2) evaluate structural and dynamic changes that occur within the channel upon activation at low pH, (3) look for structural determinants governing the interactions between M2 and the antiviral drug amantadine, and (4) conduct similar studies on the BM2 channel. Features of the two channels, including their structure, dynamics, and drug-interactions will be compared to elucidate mechanisms of ion gating and channel assembly. These studies will assist in the discovery of true channel blockers for anti-viral therapy, and lead the way toward investigation of other viral channels.
