Mechanism Of Pore Formation In Liposome Surface By Antimicrobial Peptides Melittin

Antimicrobial peptides (AMP) represent a class of compounds to combat antibiotic resistance to microor-ganisms, neutralize biological warfare agents, and as topical antimicrobial agents. AMP kills microbialcells through insertion and permeabilization of the cytoplasmic membranes. It is important to pre-dict the efficacy of AMP at low concentration to circumvent their toxicity. Leakage of fluorescentdyes (calcein, FD4and FD20) of different molecular weights entrapped within1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/cholesterol mixed liposomes by AMP melittin indicated (i) a criticalmelittin concentration for pore formation and (ii) a lag time for pore formation above this critical con-centration. The lag time decreased with an increase in melittin concentration and was in the orderFD20> FD4> calcein.%α helix of melittin increased when exposed to liposome with this increase beingmore pronounced at higher concentrations eventually reaching an asymptotic value. The rate of dyeleakage following the lag time was found to be larger at higher melittin concentration. A simplifiedmathematical model for nucleation and growth of pores formed by an aggregate of melittin in lipidbilayer is proposed to predict the variation of rate of dye leakage with melittin concentration whichagreed fairly well with the data for calcein and seems to suggest a toroidal mechanism of pore formationwith participation of large number of phospholipid heads.By means of a new attempt of data ananlysis, Confocal microscopy images clearly indicate the disruption of the liposome by Melittin above a threshold concentration.The key of the new method is the segmentation of the fullscreen image of liposomes.The fluorescence intensity is meared of each micro-circle region and show the dye flow direction which suggest a position of the pore formation in the surface of the liposomes.Evolution of fluorescence intensity due to leakage of dyes ofdifferent sizes from liposome by melittin of different concentrations indicated a critical melittin concentration for pore formationand a lag time above this critical concentration which was largerfor lower melittin concentration as well as for larger size of dyemolecule. The rate of dye leakage following the lag time was foundto be larger at higher melittin concentration. A mathematicalmodel for nucleation and growth of pores formed by an aggregate of melittin in lipid bilayer is proposed. The difference in the lagtime for dye leakage for the three dyes of different sizes is shownto be due to the difference in the time of growth of pores from thecritical pore size to the size of the molecule.Model prediction ofvariation of dye leakage rate with melittin concentration agreedfairly well with the experimental data for calcein and seems tosuggest a toroidal mechanism of pore formation with participationof large number of phospholipid heads.