Membrane lysis by the antibacterial peptides cecropins B1 and B3: A spin-label electron spin resonance study on phospholipid bilayers

Custom antibacterial peptides, cecropins B1 (CB1) and B3 (CB3), were synthe sized. These peptides have particular sequence characteristics, with CB1 ha ving two amphipathic alpha-helical segments and CB3 having two hydrophobic alpha-helical segments. These differences were exploited for a study of the ir efficacy in breaking up liposomes, which had different combinations of p hosphatidic acid (PA) and phosphatidylcholine (PC), and a study of their li pid binding ability. Binding and nonbinding lysis actions of CBI and CB3 on liposomes were examined further by electron spin resonance(ESR). The spin- labeled lipids 5'SL-PC, 7'SL-PC, 10'SL-PC, 12'SL-PC, and 16'SL-PC were used as probes. The ESR spectra revealed larger outer hyperfine splittings (2A( max)) for CB1 when the interactions of CBI and CB3 with liposomes were comp ared. These observations indicate a larger restriction of the motion of the spin-labeled chains in the presence of GB1. Plots of the effective order p arameter at the various probe positions (chain flexibility gradient) versus the peptide-lipid ratio further suggested that the lysis action of CBI is related to its capacity to bind to the lipid bilayers. In contrast, there i s no evidence of binding for CB3. To augment these findings, four spin-labe led peptides, C8SL-CB1, C32SL-CB1, C5SL-CB3; and C30SL-CB3, were also exami ned for their binding to and their state of aggregation within the lipid bi layers. Association isotherms of the peptides were measured for liposomes c ontaining two molar fractions of PA (0.25 and 0.75). The membrane binding o f the CBI peptides exhibited a cooperative behavior, whereas the associatio n isotherm of CB3 revealed binding to the lipid only for beta = 0.75 liposo mes. To further identify the location of CB1 in the lipid bilayers, measure ments of the collision rate with chromium oxalate in solution were conducte d. Results from ESR power saturation measurements suggested that the NH2-te rminal alpha-helix of CB1 is located on the surface of the lipid bilayers, whereas the COOH-terminal a-helix of GB1 is embedded below the surface of t he lipid bilayers. These conclusions were further supported by the observed relationship between the partition distribution of peptides bound to lipos omes at different PA/PC ratios and the amounts of free peptides. Based on t he above observations, possible mechanisms of the bilayer lysis induced by CBI and CB3 an liposomes of different composition are discussed.