title: Structure of micelle-associated alamethicin from 1H NMR. Evidence for conformational heterogeneity in a voltage-gated peptide.
abstract: Alamethicin is a 20 amino acid peptide that produces a voltage-dependent 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 1H nuclear magnetic resonance (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 1H NMR spectrum of the peptide in micelles was assigned. The intensities of the HN-HN(i,i+1), H alpha-HN(i,i+1), H alpha-NH(i,i+3), H alpha-H beta (i,i+3), and H alpha-NH(i,i+4) cross peaks in the NOESY spectrum suggest that the N-terminal half of the peptide is predominantly alpha-helical, while the C-terminal half has a less regular or more flexible structure. The exposure of micelle bound alamethicin to the aqueous solution was 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 consisting of 175 distances (derived from NOESY spectra), five dihedral angles, 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 largely helical from residues 4-9 and 12-16. A limited number of structural forms were obtained. The main difference among forms involved the backbone conformations of MeA10, Gly11, and Leu12 and resulted in structures that were straight or had different amounts of bend. The structural forms could be easily interconverted by rotation of the psi and phi 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 position for the peptide carbonyl of MeA10. These results suggest that conformation rearrangements at or near MeA10 may play a role in the voltage-gating of alamethicin.
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville 22901.
authors.forename: J C
authors.initials: JC
authors.lastname: Franklin
authors.forename: J F
authors.initials: JF
authors.lastname: Ellena
authors.forename: S
authors.initials: S
authors.lastname: Jayasinghe
authors.forename: L P
authors.initials: LP
authors.lastname: Kelsh
authors.forename: D S
authors.initials: DS
authors.lastname: Cafiso
mesh_terms: D000408:Alamethicin / Q000737:chemistry*; D000595:Amino Acid Sequence; D007473:Ion Channels / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry; D008823:Micelles; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary
chemical_list: D007473:Ion Channels; D008565:Membrane Proteins; D008823:Micelles; D000408:Alamethicin