Membrane proteins are of critical importance to nearly every aspect of cell physiology, comprising one quarter to one third of all proteins encoded by eubacterial, archaean, and eukaryotic organisms. Nevertheless, our understanding of their folding and structures is at a very primitive state when compared to that of water-soluble proteins. The M2 proton channel from influenza A virus provides an attractive system for understanding the folding and function of membrane proteins. This proton channel is essential to the survival of the virus, and is the target of the anti-influenza drugs, amantadine and rimantadine. The M2 proton channel is formed by the intermolecular association of four identical transmembrane helices, and a peptide (tmM2) spanning only the transmembrane helix of M2 forms amantadine- dependent channels in bilayers. In the past period, we have studied the thermodynamics of assembly of tmM2 and the full- length protein. Diffraction-quality crystals of tmM2 have also been obtained at low pH, where the channel is maximally active, as well as at pH 8.0, near the pH optimum for inhibition by the drug amantadine.
Our specific aims for the current period are to: 1. Determine the structure of functional fragments of M2 using NMR, IR, and X-ray crystallography. 2. Determine what features in the sequence of M2 contribute to its thermodynamic stability. 3. Determine the mechanism of proton conductance by M2, and its inhibition by amantadine. 4. Design and structurally characterize a water-soluble version of M2, which retains the tertiary structure of M2 as well as its ability to bind amantadine.
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