Effects of positively charged arginine residues on membrane pore forming activity of Rev-NIS peptide in bacterial cells.

Here, we investigated antibacterial effects of Rev-NIS and suggested the role of positively charged amino acids on membrane pore forming activity of the peptide in bacterial cells, by synthesizing two analogs, Anal R and Anal S. Based on the amphipathic property of Rev-NIS, Anal R and Anal S were designed by substituting E(1) and L(3) to R and L(3) to S, respectively. The circular dichroism (CD) spectroscopy showed that Anal R and Anal S have the same conformation of Rev-NIS, with a significant fraction of helical structure. In succession, the antibacterial susceptibility testing showed that Rev-NIS and its analogs possessed significant activities (Anal R>Rev-NIS>Anal S), without hemolytic effects, against bacterial pathogens including antibiotics-resistant strains. Moreover, the membrane studies, 3,3'-dipropylthiadicarbocyanine iodide (diSC(3)5) staining and FITC-dextran (FD) leakage assay demonstrated that the analogs as well as Rev-NIS acted on the bacterial membranes and potently made pores, with the hydrodynamic radius between 1.4nm and 2.3nm. Especially, Anal R made larger pores than other peptides, with the radius between 2.3nm and 3.3nm. These results also corresponded to the result of antibacterial susceptibility testing. In summary, this study indicates that the two arginine residues are more influential than the hydrophobicity or the helicity, regarding the molecular activity of the peptide, and finally suggests that Anal R peptide may be applied to novel antibacterial agents.