title: Membrane binding, structure, and localization of cecropin-mellitin hybrid peptides: a site-directed spin-labeling study.
abstract: The interaction of antimicrobial peptides with membranes is a key factor in determining their biological activity. In this study we have synthesized a series of minimized cecropin-mellitin hybrid peptides each containing a single cysteine residue, modified the cysteine with the sulfhydryl-specific methanethiosulfonate spin-label, and used electron paramagnetic resonance spectroscopy to measure membrane-binding affinities and determine the orientation and localization of peptides bound to membranes that mimic the bacterial cytoplasmic membrane. All of the peptides were unstructured in aqueous solution but underwent a significant conformational change upon membrane binding that diminished the rotational mobility of the attached spin-label. Apparent partition coefficients were similar for five of the six constructs examined, indicating that location of the spin-label had little effect on peptide binding as long as the attachment site was in the relatively hydrophobic C-terminal domain. Depth measurements based on accessibility of the spin-labeled sites to oxygen and nickel ethylenediaminediacetate indicated that at high lipid/peptide ratios these peptides form a single alpha-helix, with the helical axis aligned parallel to the bilayer surface and immersed approximately 5 A below the membrane-aqueous interface. Such a localization would provide exposure of charged/polar residues on the hydrophilic face of the amphipathic helix to the aqueous phase, and allow the nonpolar residues along the opposite face of the helix to remain immersed in the hydrophobic phase of the bilayer. These results are discussed with respect to the mechanism of membrane disruption by antimicrobial peptides.
authors.affiliation: Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin 53326, USA.
authors.forename: Kalpana
authors.initials: K
authors.lastname: Bhargava
authors.forename: Jimmy B
authors.initials: JB
authors.lastname: Feix
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000737:chemistry*; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D019476:Insect Proteins / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D008555:Melitten / Q000737:chemistry*; D008563:Membrane Lipids / Q000737:chemistry*; D008567:Membranes, Artificial; D011485:Protein Binding; D011487:Protein Conformation; D013113:Spin Labels / Q000138:chemical synthesis
chemical_list: D023181:Antimicrobial Cationic Peptides; D019476:Insect Proteins; D008081:Liposomes; D008563:Membrane Lipids; D008567:Membranes, Artificial; D013113:Spin Labels; C406844:cecropin protein, Anopheles; D008555:Melitten