Well defined helical polypeptide segments play a critical role in the assembly and activity of membrane proteins. This is particularly evident in channel proteins where transmembrane amphipathic helices associate with, insert into, and oligomerize within, the lipid bilayer to form bioactive structures. We hypothesize that these channel-forming activities arise, largely, from the interaction of the unique structural elements within amphipathic helices: a hydrophobic-- lipid interacting face, a hydrophilic--pore forming face and two asymmetric, small amino acid containing -- helix-helix interacting faces. The proposed research will describe, for the first time, the contributions each of these amphipathic helix faces play in the assembly and activity of idealized channel pores. Particular emphasis will be placed on defining the helix- helix contacts that give rise to dimers as well as higher forms of oligomerization. We will also examine the properties of hetero- oligomeric assemblies of helices, such as those proposed in the acetylcholine receptor channel and the human cystic fibrosis CFTR Cl- channel, with respect to ion selectivity and the opening and closing of channel pores. This work will be accomplished by combining two powerful techniques: solid phase peptide synthesis and biophysical analysis employing electrophysiological measurements, 2D-NMR (solution and solid phase) and Electron Spin Resonance (ESR). Synthetic peptides and template assemble helical bundles will be designed, synthesized, chemically characterized and then studied in model membranes. The biophysical methods will map the environment around each residue, lipid exposed, lining the channel or involved in the helix-helix interface as well as determine the oligomerization number and channel activity of the assembled pores. The results obtained from these studies will aid in the understanding of membrane protein assembly and channel activity. The generation of stable model channel-pore structures with high degrees of ion selectivity could herald the uses of such structures as pharmacological agents or in biomedical devices.
|Broughman, J R; Shank, L P; Prakash, O et al. (2002) Structural implications of placing cationic residues at either the NH2- or COOH-terminus in a pore-forming synthetic peptide. J Membr Biol 190:93-103|