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 synthesized. These peptides have particular sequence characteristics, with CB1 having two amphipathic alpha-helical segments and CB3 having two hydrophobic alpha-helical segments. These differences were exploited for a study of their efficacy in breaking up liposomes, which had different combinations of phosphatidic acid (PA) and phosphatidylcholine (PC), and a study of their lipid binding ability. Binding and nonbinding lysis actions of CB1 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 CB1 and CB3 with liposomes were compared. These observations indicate a larger restriction of the motion of the spin-labeled chains in the presence of CB1. Plots of the effective order parameter at the various probe positions (chain flexibility gradient) versus the peptide-lipid ratio further suggested that the lysis action of CB1 is related to its capacity to bind to the lipid bilayers. In contrast, there is no evidence of binding for CB3. To augment these findings, four spin-labeled peptides, C8SL-CB1, C32SL-CB1, C5SL-CB3, and C30SL-CB3, were also examined for their binding to and their state of aggregation within the lipid bilayers. Association isotherms of the peptides were measured for liposomes containing two molar fractions of PA (0.25 and 0.75). The membrane binding of the CB1 peptides exhibited a cooperative behavior, whereas the association isotherm of CB3 revealed binding to the lipid only for beta = 0.75 liposomes. To further identify the location of CB1 in the lipid bilayers, measurements of the collision rate with chromium oxalate in solution were conducted. Results from ESR power saturation measurements suggested that the NH(2)-terminal alpha-helix of CB1 is located on the surface of the lipid bilayers, whereas the COOH-terminal alpha-helix of CB1 is embedded below the surface of the lipid bilayers. These conclusions were further supported by the observed relationship between the partition distribution of peptides bound to liposomes at different PA/PC ratios and the amounts of free peptides. Based on the above observations, possible mechanisms of the bilayer lysis induced by CB1 and CB3 on liposomes of different composition are discussed.
Hung
S C
SC
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Wang
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W
Chan
S I
SI
Chen
H M
HM
D000595:Amino Acid Sequence; D000818:Animals; D000890:Anti-Infective Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D004578:Electron Spin Resonance Spectroscopy; D066298:In Vitro Techniques; D019476:Insect Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology*; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D012996:Solutions; D013113:Spin Labels; D014867:Water
D000890:Anti-Infective Agents; D019476:Insect Proteins; D008051:Lipid Bilayers; D008081:Liposomes; D012996:Solutions; D013113:Spin Labels; C115150:cecropin B1 protein, insect; C115151:cecropin B3 protein, insect; D014867:Water