Well defined helical segments of proteins play a critical role in several cell biological processes including protein translocation into or across membranes and the flow of ions across the lipid bilayer. The proposed research will investigate the contribution ordered alpha-helices have as both leader sequences and ion channels. The planned studies will describe, for the first time, the roles of the leader sequence in influencing the structure and processing of a protein as well as defining conclusively the structure and stoichiometry of the membrane pores for two different ion channels. This work will be accomplished by combining two powerful techniques: 2D-NMR (solution and solid-phase) and solid-phase peptide synthesis. Solid-phase peptide synthesis is essential in order to prepare otherwise unobtainable protein/peptide species for study by physical methods as well as permitting great flexibility in the design of both the domains to be studied and the experimental techniques to be utilized for the analysis. In the area of protein translocation, solid phase synthesis will enable us to: 1) elucidate the three-dimensional structure of a precursor form of a protein; 2) assess the role of particular amino acid residues in influencing the preprotein's conformations; 3) characterize kinetically the processing enzyme - leader peptidase; and 4) design of inhibitors for leader peptidase. IN the area of ion channels, solid-phase peptide synthesis will allow us 1) to define the three-dimensional structure of the ion channels formed by amphipathic helices for both the Torpedo acetylcholine receptor and the mammalian brain sodium channel; and 2) observe the dynamics that open and close these channels. Solid-phase template synthesis will be employed to define the number of helices comprising the channels.
