STRUCTURE OF MICELLE-ASSOCIATED ALAMETHICIN FROM H-1-NMR - EVIDENCE FOR CONFORMATIONAL HETEROGENEITY IN A VOLTAGE-GATED PEPTIDE

Alamethicin is a 20 amino acid peptide that produces a voltage-depende nt conductance in membranes. To understand the mechanism by which this peptide becomes voltage-gated, the structure of alamethicin bound to micelles was examined using high-resolution H-1 nuclear magnetic reson ance (NMR). Two-dimensional correlation and nuclear Overhauser effect spectroscopy (NOESY) were carried out on alamethicin incorporated into perdeuterated sodium dodecyl sulfate (SDS) micelles, and the H-1 NMR spectrum of the peptide in micelles was assigned. The intensities of t he HN-HN(i,i+1), Halpha-HN(i,i+1), Halpha-NH(i,i+3), Halpha-Hbeta(i,i3), and Halpha-NH(i,i+4) cross peaks in the NOESY spectrum suggest tha t the N-terminal half of the peptide is predominantly alpha-helical, w hile the C-terminal half has a less regular or more flexible structure . The exposure of micelle bound alamethicin to the aqueous solution wa s determined by examining the effect of aqueous paramagnetic reagents on the line widths of the peptide protons. These measurements suggest that alamethicin is buried in the micelle. A set of restraints consist ing of 175 distances (derived from NOESY spectra), five dihedral angle s, and two hydrogen bond distances were used in a simulated annealing procedure that yielded structures for micelle associated alamethicin, The structures that were generated with simulated annealing were large ly helical from residues 4-9 and 12-16. A limited number of structural forms were obtained. The main difference among forms involved the bac kbone conformations of MeA10, Gly11, and Leu12 and resulted in structu res that were straight or had different amounts of bend. The structura l forms could be easily interconverted by rotation of the phi and psi angles of residues 10-12. The rotational freedom at or near MeA10 may be a result of Pro14, which would be the normal hydrogen-bonding posit ion for the peptide carbonyl of MeA10. These results suggest that conf ormation rearrangements at or near MeA10 may play a role in the voltag e-gating of alamethicin.