Effect of Pro-to-Nala Substitution on Structure and Bacterial Selectivity of a Designed Antimicrobial Peptide P18

P18 (KWKLFKKIPKFLHLAKKF-NH2), a novel a-helical antimicrobial peptide, was designed from cecropin A-magainin 2 hybrid peptide. It has strong antimicrobial activity against Gram-negative and Gram-positive bacteria, but relatively weak hemolytic activity, indicating that P18 has good bacterial selectivity. In this study, to investigate the influence of substituting Pro of P18 with Ala-peptoid residue (Nala) on the structure and bacterial selectivity, we synthesized P18-Nala, in which Pro9 in P18 was replaced by Nala. P18-Nala was as potent as P18 in antibacterial activity (MIC: 0.5-2.0 yM), whereas less toxic to human red blood cells than P18, indicating P18-Nala is more selective to bacterial cells than P18. Circular dichroism analysis revealed that P18-Nala had lower a-helicity than P18 in membrane mimicking conditions such as 50% TFE or 30 mM SDS micelles, suggesting Nala provides more structural flexibility than proline. Tryptophan fluorescence spectroscopy showed that the blue shift in the fluorescence emission maximum of P18-Nala was much smaller in zwitterionic EYPC liposomes than in negatively charged EYPG liposomes. These results suggested that the improvement in bacterial selectivity of P18-Nala correlated with its less permeability to zwitterionic phospholipids. Taken together, our results showed that Pro-to-Nala substitution (P18-Nala) in P18 increased the peptide s structural flexibility and the selectivity to negatively charged phospholipids, resulting in improving bacterial selectivity.