title: Fluorescence studies of the secondary structure and orientation of a model ion channel peptide in phospholipid vesicles.
abstract: A 21-residue peptide of the sequence (LSSLLSL)3 forms ion channels when incorporated into planar lipid bilayer membranes of diphytanoylphosphatidylcholine (diPhy-PC). The frequency of channel openings increases with the applied voltage gradient. We investigated the molecular and structural mechanisms underlying this voltage dependence. A series of seven peptides, each containing a tryptophan substituted for a single residue in the middle heptad, was synthesized, purified, and incorporated into small, unilamellar, diPhy-PC vesicles. We measured circular dichroism, maximum fluorescence emission wave-lengths, and fluorescence quenching by both aqueous and lipid hydrocarbon-associated quenchers. Circular dichroism spectra and the observed sequence periodicity of all fluorescence and fluorescence quenching data are consistent with an alpha-helical peptide secondary structure. Energy transfer quenching measurements using N-terminally labeled (LSSLLSL)3 co-incorporated at lipid/peptide ratios greater than 100 into vesicles with one of the Trp-substituted peptides showed that the vesicle-associated peptide, in the absence of a voltage gradient across the bilayer, exists as an equilibrium mixture of monomers and dimers. Static fluorescence quenching measurements using different lipid-bound quenchers indicate that the helical axis of a representative lipid-associated peptide is, on average, oriented parallel to the surface of the membrane and located a few angstroms below the polar head group/hydrocarbon boundary. This surface orientation for the peptide is confirmed by the complementary sequence periodicity observed for Trp fluorescence emission wavelength shifts and collisional quenching by aqueous CsCl.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Du Pont Central Research & Development Department, Wilmington, Delaware 19880-0328.
authors.forename: L A
authors.initials: LA
authors.lastname: Chung
authors.forename: J D
authors.initials: JD
authors.lastname: Lear
authors.forename: W F
authors.initials: WF
authors.lastname: DeGrado
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D007473:Ion Channels / Q000737:chemistry* / Q000648:ultrastructure; D008051:Lipid Bilayers / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry
chemical_list: D007473:Ion Channels; D008051:Lipid Bilayers; D010455:Peptides; D014364:Tryptophan