Experimental And Simulation Study On The Membrane Permeation Activity Of Antibacterial Peptides Based On Cellular Imitating Membrane System

Antimicrobial peptides have broad-spectrum antibacterial properties,while they also have various physiological properties such as antiviral,antitumor,and immunomodulation.Compared with antibiotics,antimicrobial peptides,by virtue of their unique membrane permeating activity mechanism,have a great advantage of not easily generating drug resistance and have a promising application prospect.In depth exploration of the response of antimicrobial peptide membrane penetration activity under different membrane physical properties is an important prerequisite for antibacterial peptides to be widely used.Unfortunately,the molecular mechanisms of action for these are not fully understood at present.Exploring the mechanism of antimicrobial peptide membrane permeating activity based on a cell mimetic membrane model can avoid the influence of potentially active cellular processes.Here,in this paper,melittin and magainin 2 are used as antimicrobial peptide models,and giant unilamellar vesicles(GUVs)can be directly observed and manipulated under a light microscope,meanwhile combined with molecular dynamics simulation,to specifically study the phospholipid electrical properties and membrane potentials for antimicrobial peptide membrane penetration activity by leaking the vesicles induced by antimicrobial peptides and analysis of parameters such as molecular dynamics simulation trajectory RMSD and RMSF.The targeting of melittin to the cell membrane is nonspecific,and the study of using melittin to construct a drug loaded system to attack tumor cells is emerging.In the third chapter,we take GUVs of two different electrical phospholipid components,POPC and POPC/POPS(7:3),respectively,as the targeting membrane of melittin to obtain the specific response of melittin under two different electrical phospholipid stimuli in mock normal and mock tumor cells.The study concluded that vesicle leakage experiments induced by low concentrations of melittin showed that neutral phospholipid vesicles divulged in the pore mode,and negatively charged phospholipid vesicles divulged in the bursting mode;The experiments on vesicle leakage induced by high concentrations of melittin showed that negatively charged phospholipids could delay the effect of melittin action relative to neutral phospholipids;Melittin tryptophan residue fluorescence spectra indicated that the amount of melittin adsorbed on the vesicle membrane surface as well as leakage pattern was dependent on phospholipid electrical property.In addition,the model of the penetration effect of melittin on different electrical phospholipid membranes was inferred.The targeting of magainin 2 to the cell membrane is specific and mainly relies on electrostatic driving to associate with it.The effect of membrane potential on plasma membrane damage produced by magainin 2 is an important but ill defined process.In Chapter 4,to characterize the effect of membrane potential on the membrane permeance activity of magainin2,we used GUVs composed of DOPG/DOPC/gramicidin A(40:60:0.01)as the membrane targeting antimicrobial peptide,in which gramicidin A was used to substitute for protein ion channels in the cell membrane,allowing GUVs to generate different membrane potentials by changing the concentration of K~+inside and outside the vesicles.Found that in the presence of membrane potential,magainin 2 can induce GUVs to divulge in a pore mode with high efficiency and that the pore formation rate constant of GUVs is related to the membrane potential,which increases with the membrane negative potential,also suggesting that the presence of membrane negative potential is beneficial for the expression of magainin 2membrane permeation activity.On the basis of the experimental findings and on the basis of the two-state model mechanism of action of antimicrobial peptides,the influence of membrane potential on this process is further described.Combining molecular dynamics simulations with experiments has become an important means of studying complex biological processes.In the fifth chapter of this paper,we simulated the application of melittin on POPC and pops phospholipid bilayers and the penetration of melittin across POPC and POPC/POPS(7:3)phospholipid bilayers,respectively,and comparatively studied the effect of phospholipid electrical properties on the membrane penetration activity of melittin.Through the analysis of simulated trajectories it was found that melittin would shift on the phospholipid membrane when it adsorbed to the phospholipid membrane in a parallel conformation,and the magnitude of the shift was related to phospholipid electrical property,and the structure of melittin on the negatively charged phospholipid bilayer was more stable.In addition,we found that melittin is more difficult to cross the POPC/POPS phospholipid bilayer than the more rigid POPC phospholipid bilayer.Finally,we summarize the conclusions of the research contents of this paper obtained and provide an outlook on future research interests.