S-thanatin, an analog of
thanatin, was synthesized by substituting the 15th
amino acid of
threonine with
serine, which showed a broad antimicrobial activity against bacteria. We reported earlier that membrane
phospholipid was found to be the target for
S-thanatin with different mechanism from other
antimicrobial peptides. In this study, we have performed its structural characterization by circular dichroism (CD) spectroscopy. The CD analysis showed that
S-thanatin retained its overall conformation beta-sheet in aqueous
buffer, beta-turn in 50%
trifluoroethanol (
TFE) and beta-hairpin in 0.4 mM POPC-LUVs. In
hemolysis assay,
S-thanatin exhibited low hemolytic activity and bacteria selectivity. We investigated the effect of the presence of 33 mol percent
cholesterol on the interactions of the
antimicrobial peptide S-thanatin with
phosphatidylcholine (PC) model membrane systems. The results showed that
S-thanatin was more potent at disrupting
cholesterol-free bacterial than
cholesterol-containing eukaryotic membranes. Thus, in all respects, fluorescence
dye leakage experiments indicated that
cholesterol inhibited the
S-thanatin-induced permeabilization of PC vesicles. Finally, flow cytometry was used to monitor changes in bacterial cell membrane potential and cell membrane integrity, with specific
fluorescent dyes DiBAC(4)(3) and PI. Adding the respiratory
poison CCCP seemed to prevent
peptide-induced membrane damage, which suggested that
S-thanatin acted at the metabolic level on respiratory chain. These findings might explain why
S-thanatin was selective toxicity towards bacteria, but low toxicity towards erythrocytes. It might be due to three factors at least: electrostatic interaction (namely anionic
phospholipids);
cholesterol; respiratory chain.