alamethicin and ampullosporin

The (2)H solid-state NMR spectra of deuterated fatty acyl chains provide direct access to the order of the hydrophobic membrane interior. From the deuterium order parameter profiles of perdeuterated fatty acyl chains the membrane hydrophobic thickness can be calculated. Here we show data obtained from POPC, POPE and mixed POPE/POPG bilayers, representative of bacterial membranes, in the presence of cholesterol or ergosterol and antimicrobial peptaibols. Whereas sterols have a strong ordering effect also on these membranes, the peptides exhibit neutral or disordering effects. By comparing with data from the literature it becomes obvious that cationic amphipathic peptides that probably reside within the interface of phospholipid membranes tend to strongly disorder the packing of the fatty acyl chains, an effect that has been correlated to antimicrobial and DNA transfection activities. In contrast transmembrane sequences or hydrophobic peptides that probably partition deeply into the membrane tend to have only modest disordering activities. The (2)H solid-state NMR approach has also been used to monitor the lateral separation of domains rich in anionic phospholipids in the presence of cationic peptides and has thereby provided important insights into their mechanisms of action.

Topics: Alamethicin; Anti-Bacterial Agents; Cholesterol; Deuterium; Ergosterol; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Magnetic Resonance Spectroscopy; Peptaibols; Peptides

Ampullosporin A and alamethicin are two members of the peptaibol family of antimicrobial peptides. These compounds are produced by fungi and are characterized by a high content of hydrophobic amino acids, and in particular the alpha-tetrasubstituted amino acid residue ?-aminoisobutyric acid. Here ampullosporin A and alamethicin were uniformly labeled with (15)N, purified and reconstituted into oriented phophatidylcholine lipid bilayers and investigated by proton-decoupled (15)N and (31)P solid-state NMR spectroscopy. Whereas alamethicin (20 amino acid residues) adopts transmembrane alignments in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes the much shorter ampullosporin A (15 residues) exhibits comparable configurations only in thin membranes. In contrast the latter compound is oriented parallel to the membrane surface in 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine and POPC bilayers indicating that hydrophobic mismatch has a decisive effect on the membrane topology of these peptides. Two-dimensional (15)N chemical shift -(1)H-(15)N dipolar coupling solid-state NMR correlation spectroscopy suggests that in their transmembrane configuration both peptides adopt mixed alpha-/3(10)-helical structures which can be explained by the restraints imposed by the membranes and the bulky alpha-aminoisobutyric acid residues. The (15)N solid-state NMR spectra also provide detailed information on the helical tilt angles. The results are discussed with regard to the antimicrobial activities of the peptides.

Topics: Alamethicin; Computer Simulation; Hypocreales; Lipid Bilayers; Models, Chemical; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular; Peptaibols; Peptides; Phosphorus Isotopes; Phosphorylcholine; Protein Structure, Secondary; Protons; Tandem Mass Spectrometry; X-Ray Diffraction

An automated approach to peptaibols using microwave-assisted solid-phase peptide synthesis is demonstrated with a combination of HBTU and acid fluoride mediated couplings for normal and alpha,alpha-dialkylated amino acids, respectively. The method is utilized for the automated synthesis of several full-length peptaibols, including alamethicin, tylopeptin, ampullosporin, bergofungin, cervinin, trikoningin, trichogin, and peptaibolin, reducing both synthesis time and costs significantly as compared to other approaches. Furthermore, the use of noncommercially available reagents is minimized.

Topics: Alamethicin; Amino Acid Sequence; Amino Acids; Chromatography, High Pressure Liquid; Fluorenes; Microwaves; Molecular Sequence Data; Peptaibols; Peptides; Peptides, Cyclic