Protein ion channels are essential for the generation and propagation of electrical activity in a variety of cells. Two features important for their activities are ion selectivity and rectification (asymmetry in current/voltage relationships). De novo design is an attractive approach to probe the minimal features required for these functions. The design process often reflects the hierarchy of forces required for folding and function, beginning with relatively non-specific hydrophobic and polar interactions. Specific electrostatic and H-bonded interactions are subsequently added to achieve more complex functions. Recently, this group designed two simple alpha-helical models for ion channel proteins using only Leu and Ser residues. One forms proton-selective channels while the second forms channels selective for cations smaller than 8 angstroms in diameter. These peptides are believed to form helical bundles with a central core that conducts ions across membranes. Derivatives of these peptides will be chemically synthesized, functionally characterized by single channel studies, and dynamically modeled to explore the structural determinants of ion selectivity and rectification. To aid in the determination of their structures, they will be covalently attached to rigid macrocyclic templates. Next, these methods will be applied to an ion channel protein (M2) from the influenza A virus. M2 has 97-residues and a single transmembrane helix domain, and forms alpha-helical tetramers in membranes. It is essential for cell infection and is the target of the only approved anti-influenza drugs. Derivatives of M2 will be chemically synthesized and attached to macromolecular templates. These and the natural protein will be incorporated into planar bilayers and their ion conduction properties evaluated at the single channel level. Features promoting assembly into ion-conducting tetramers will be evaluated and modeled using molecular dynamics.
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