A BP100 Peptide Dimer is More Efficient Than the Monomer for Permeabilizing Model Membranes

BP100 (H-KKLFKKILKYL-NH₂) is a short antimicrobial peptide with high efficiency and selectivity towards bacterial membranes. To further improve the antimicrobial activity, a dimeric BP100 (BP100-D) containing two BP100 chains (H-CAAAKKLFKKILKYL-NH₂) linked by a disulfide bond at the N-terminus was obtained. Large Unilamellar Vesicles composed of different proportions of zwitterionic POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) and anionic POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)) were used as membrane models. POPC and POPG were used as models for eucaryotic and bacterial membranes, respectively. Circular dichroism and Nuclear Magnetic Resonance (H¹-NMR) showed that BP100-D has a random coil structure in aqueous solution and acquires an alpha helix structure in contact with membranes with POPG content varying from 10 to 100%. In contradistinction to BP100, BP100-D seems to develop less alpha helix conformation when in contact with POPC membranes. The analysis of zeta potential and size of LUVs by Dynamic Light Scattering (DLS) showed an increase in the zeta potential of LUVs containing POPC, POPC:POPG 50:50, and POPG upon the addition of the positively charged BP100-D. The increase in zeta potential was accompanied by an increase in size of POPG-containing LUVs, while no increase in size was observed in the POPC LUVs. Optical microscopy of Giant Unilamellar Vesicles (GUVs) revealed that BP100-D can permeabilize POPC:POPG 50:50 GUVs at peptide concentrations starting at 3µM and causes rapid vesicle rupture at 5µM. These values represent a 10-fold decrease in the concentration of peptide required for GUV permeabilization by BP100. Fluorescence binding assay showed a sharp increase in the fluorescence of the BP100-D tyrosine’s upon the addition of POPC:POPG LUVs. The increase in fluorescence and the lipid concentration required for maximum binding correlate with the POPG content. Our work has characterized several features of the BP100-D/ model membranes' interaction. Our results show a strong dependence on membrane surface charge on peptide-induced vesicle aggregation and permeabilization; however, in contrast to BP100, BP100-D also strongly binds to POPC LUVs and causes rapid bursting of POPC GUVs. Overall, our data show that BP100-D is more efficient than BP100 in permeabilizing model membranes, although with lower selectivity.