Interaction of the Peptide Antibiotic Alamethicin with Bilayer- and Non-bilayer-Forming Lipids: Influence of Increasing Alamethicin Concentration on the Lipids Supramolecular Structures Angelina Angelova,* ,1,2 Radoslav Ionov,† Michel H. J. Koch,‡ and Gert Rapp‡ , § ‡European Molecular Biology Laboratory, Hamburg Outstation, c/o DESY, Geb. 25A, Notkestr. 85, D-22603 Hamburg, Germany; §Max-Planck-Institut fu ¨ r Kolloid und Grenzfla ¨chenforschung, c/o DESY, Geb. 25F, Notkestr. 85, D-22607 Hamburg, Germany; *College of Sciences “Leonardo da Vinci,” P.O.Box 946, BG-1000 Sofia, Bulgaria; and †Institute of Applied Physics, Technical University, BG-1156 Sofia, Bulgaria Received June 28, 1999, and in revised form January 9, 2000 Incorporation of the helical antimicrobial peptide alamethicin from aqueous phase into hydrated phases of dioleoylphosphatidylethanolamine (DOPE) and dio- leoylphosphatidylcholine (DOPC) was investigated within a range of peptide concentrations and temper- atures by time-resolved synchrotron X-ray diffraction. It was found that alamethicin influences the organiza- tions of the non-bilayer-forming (DOPE) and the bi- layer-forming (DOPC) lipids in different ways. In DOPC, only the bilayer thickness was affected, while in DOPE new phases were induced. At low peptide concentrations (< 1.10 4 M), an inverted hexagonal (H II ) phase was observed as with DOPE dispersions in pure buffer solution. A coexistence of two cubic struc- tures was found at the critical peptide concentration for induction of new lipid/peptide phases. The first one Q 224 (space group Pn3m) was identified within the en- tire temperature region studied (from 1 to 45°C) and was found in coexistence with H II -phase domains. The second lipid/peptide cubic structure was present only at temperatures below 16°C and its X-ray reflections were better fitted by a Q 212 (P4 3 32) space group, rather than by the expected Q 229 (Im3m) space group. At ala- methicin concentrations of 1 mM and higher, a nonla- mellar phase transition from a Q 224 cubic phase into an H II phase was observed. Within the investigated range of peptide concentrations, lamellar structures of two different bilayer periods were established with the bilayer-forming lipid DOPC. They correspond to lipid domains of associated and nonassociated helical pep- tide. The obtained X-ray results suggest that the am- phiphilic alamethicin molecules adsorb from the aque- ous phase at the lipid head group/water interface of the DOPE and DOPC membranes. At sufficiently high (> 1.10 4 M) solution concentrations, the peptide is probably accommodated in the head group region of the lipids thus inducing structural features of mixed lipid/peptide phases. © 2000 Academic Press Key Words: lipid–peptide interactions; lipid cubic phase; inverted hexagonal phase; alamethicin; lipid membranes. In view of the ability of bacteria to develop resistance against antibiotics, such as aminoglycosides, -lac- tams, macrolides, tetracyclines, sulfamides, etc. (1), -helical peptides have recently emerged as a novel class of antimicrobial agents capable of damaging the functions of cytoplasmic and outer cell membranes of bacteria through interaction with their lipids (2–7). The chemotherapeutic efficiency of a particular -heli- cal peptide possessing antibiotic properties is deter- mined by the balance of its antimicrobial and hemo- lytic activities. To avoid toxicity to mammalian cells, it is essential that the antibiotic peptide be able to lyse predominantly bacterial cell membranes rather than mammalian ones. In this relation, studies of the selec- tivity and specificity involved in the molecular mecha- nisms of interaction of antimicrobial helical peptides 1 To whom correspondence should be addressed. E-mail: a-angelova@usa.net. Fax: +359-2-9885349. 2 Visiting EMBL fellow from the Institute of Biophysics, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, BG-1113 Sofia, Bulgaria. 0003-9861/00 $35.00 93 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 378, No. 1, June 1, pp. 93–106, 2000 doi:10.1006/abbi.2000.1696, available online at http://www.idealibrary.com on