Abstract
A natural antibacterial peptide, cecropin B (CB), and designed analogs, CB-1 and CB-3, were synthesized. The three peptides have different structural characteristics, with CB having one hydrophobic and one amphipathic alpha-helix, CB-1 having two amphipathic alpha-helices, and CB-3 having two hydrophobic alpha-helices. These differences were used as the rationale for a study of their efficacy in breaking liposomes with different combinations of phosphatidic acid and phosphatidylcholine. Biosensor binding measurements and encapsulating dye leakage studies showed that the higher binding affinity of CB and CB-1 to the polar heads of lipids is not necessary for the peptides to be more effective at lysing lipid bilayers, especially when liposomes have a higher phosphatidic acid content. Kinetic studies, by intrinsic and extrinsic fluorescence stopped-flow measurements, revealed two transitional steps in liposome breakage by CB and CB-1, although only one kinetic step was found for CB-3. Circular dichroism stopped-flow measurements, monitoring the formation of secondary structure in the peptides, found one kinetic step for the interaction of all of the peptides with the liposomes. Also, the alpha-helical motif of the peptides was maintained after interacting with the liposomes. Based on these results, the mechanisms of liposome lysis by CB, CB-1, and CB-3 are discussed.
Highlights
Antibacterial peptide groups such as the magainins [1], defensins [2], and cecropins [3] are found widely in the animal kingdom
To investigate further the role of the composition of the membranes and the physical characteristics of antibacterial/ antimalignant (aBaM) peptides in cell lysis we have studied the action of cecropin B (CB) and two designed analogs on membranes consisting of varying ratios of phosphatidic acid (PA) and phosphatidylcholine (PC)
Transmission Electron Microscopy (TEM) Morphological Observations of Liposomes Affected by CB, CB-1, and CB-3—To confirm the existence and observe the morphology of liposomes, with or without peptides, negative stain TEM was used
Summary
Antibacterial peptide groups such as the magainins [1], defensins [2], and cecropins [3] are found widely in the animal kingdom. Differences in the construction of the outer leaflet of the cell membrane, such as the lipopolysaccharides found on Gram-negative bacteria, the microvilli on cancer cells [20], or the greater anionic nature of some membranes, such as tumor cells [21], might influence this. It has been shown, for example, that the binding affinity between bactericidal permeability-increasing protein and lipopolysaccharide is a preliminary step to the protein enhancing the permeability of bacteria [22]. The NH2-terminal helix (or region) is amphipathic with a strongly basic face, and the COOH-terminal helix (or region) is largely hydrophobic
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