INFLUENCE OF THE ANGLE SUBTENDED BY THE POSITIVELY CHARGED HELIX FACEON THE MEMBRANE-ACTIVITY OF AMPHIPATHIC, ANTIBACTERIAL PEPTIDES

To investigate the influence of the angle subtended by the positively charged helix face on membrane activity, six amphipathic alpha-helical peptides with angles between 80 degrees and 180 degrees, but with ret ained hydrophobicity, hydrophobic moment, and positive overall charge, were designed starting from the sequence of the antibacterial peptide magainin 2. CD investigations revealed that all analogs are in an alp ha-helical conformation in vesicle suspension. The ability of the pept ides to induce dye release from negatively charged phosphatidylglycero l (PG) vesicles decreased with increasing angle. However, peptides wit h a large angle of positively charged residues (140-180 degrees) exhib ited a considerably higher permeabilizing activity at zwitterionic pho sphatidylcholine (PC) and mixed PC/PG (3:1) vesicles than analogs with a small angle (80-120 degrees). In addition, analogs with large angle s were more active in antibacterial and hemolytic assays. The antibact erial specificity of these analogs was decreased. Binding investigatio ns showed that peptide binding is favored by a large angle and a high content of negatively charged phospholipid. In contrast, a small angle and a low negative membrane charge enhanced the membrane-permeabilizi ng efficiency of the bound peptide fraction. All analogs stabilized th e bilayer phase of phosphatidylethanolamine over the inverted hexagona l phase. Therefore, a class L mechanism of permeabilization can be exc luded. Furthermore, the analogs do not act by the induction of positiv e curvature strain or by a ''carpet-like'' mechanism. Our results are in accordance with a pore mechanism: The membrane-permeabilizing effic iency of analogs with enhanced angle of positively charged residues is reduced due to electrostatic repulsion between adjacent helices withi n the pore, thus resulting in a decreased pore-forming probability and /or pore destabilization.