Hueih Min Chen

The pathway of cell membrane lysis by the peptide antibiotic cecropin B (CB), which contains both a hydrophobic and an amphipathic α‐helix, was analysed by assessing the morphological changes of Escherichia coli following treatment with the peptide. Exposure of green fluorescent protein (GFP)‐expressing E. coli to CB does not lead to an efflux of GFP. Moreover, transmission electron microscopic (TEM) examination of cecropin B‐treated cells showed that severe swelling precedes cell death and that the outer membrane becomes distended away from the plasma membrane. Using immuno‐gold staining and TEM of E. coli expressing the maltose‐binding protein in the cytoplasm, it was apparent that the protein remains restricted to the cytoplasmic compartment. These observations suggest that CB causes gross disruption of the outer membrane of Gram‐negative bacteria. Circular dichroism measurements of CB in the presence of cell membrane‐mimicking liposomes showed that CB forms secondary structure dependent on the ratio of [lipid]/[peptide]. These observations from this study are important for the future design of custom antimicrobial peptides. Microsc. Res. Tech. 62:423–430, 2003.

Different pathways of bilayer disruption by the structurally related antimicrobial peptides cecropin B, B1 and B3, revealed by surface plasma resonance analysis of immobilized liposomes, differential scanning calorimetry of peptide–large unilamellar vesicle interactions, and light microscopic analysis of peptide‐treated giant unilamellar vesicles, have been identified in this study. Natural cecropin B (CB) has one amphipathic and one hydrophobic α‐helix, whereas cecropins B1 (CB1) and B3 (CB3), which are custom‐designed, chimaeric analogues of CB, possess either two amphipathic or two hydrophobic α‐helices, respectively. Surface plasma resonance analysis of unilamellar vesicles immobilized through a biotin–avidin interaction showed that both CB and CB1 bind to the lipid bilayers at high concentration (>10 µm); in contrast, CB3 induces disintegration of the vesicles at all concentrations tested. Differential scanning calorimetry showed the concentration‐dependent effect of bilayer disruption, based on the different thermotrophic phase behaviours and the shapes of the thermal phase‐transition curves obtained. The kinetics of the lysis of giant unilamellar vesicles observed by microscopy demonstrated that both CB and CB1 effect a continuous process involving loss of integrity followed by coalescence and resolution into smaller vesicles, whereas CB3 induces rapid formation of irregular‐shaped, nonlamellar structures which rapidly disintegrate into twisted, microtubule‐containing debris before being completely destroyed. On the basis of these observations, models by which CB, CB1 and CB3 induce lysis of lipid bilayers are discussed.

In this work, molecular dynamics simulations were used to examine the consequences of a variety of analogs of cecropin A on lipid bilayers. Analog sequences were constructed by replacing either the N- or C-terminal helix with the other helix in native or reverse sequence order, by making palindromic peptides based on both the N- and C-terminal helices, and by deleting the hinge region. The structure of the peptides was monitored throughout the simulation. The hinge region appeared not to assist in maintaining helical structure but help in motion flexibility. In general, the N-terminal helix of peptides was less stable than the C-terminal one during the interaction with anionic lipid bilayers. Sequences with hydrophobic helices tended to regain helical structure after an initial loss while sequences with amphipathic helices were less able to do this. The results suggests that hydrophobic design peptides have a high structural stability in an anionic membrane and are the candidates for experimental investigation.

The solution structure of a custom lytic peptide, cecropin B3 (CB3), having two identical hydrophobic segments on both the N‐ and C‐termini, was investigated by two‐dimensional NMR spectroscopy. The need to determine the structure of this peptide is rooted in its specific ability to lyse lipid layers that have a high content of anionic lipid. The lytic activities of CB3 on cell membranes including cancer cells and bacteria is found to be less than cecropin B1. The results show that CB3 has four discrete segments forming α helical structures. The crumpled structure of CB3 provides evidence for the lysis of the lipid layer being via a pathway that differs from pore formation. The results in this study provide strong clues towards a rational design for a potent antimicrobial and antitumor peptide.