Melittin-Biomembranes Interaction Pathway Regulated By Membrane Component

Antimicrobial peptides are the natural immunity tools against bacteria in animals with high biomedical research value.Melittin is a typical cationic amphiphilic antimicrobial peptide,which can nonspecifically target bacteria and cause bacterial death.Based on the LUVs(Large Unilamellar Vesicles),GUVs(Giant Unilamellar Vesicles),and tethered vesicle platform,we studied the membrane leakage induced by melittin using the fluorescence spectroscopy and microscopy.To understand the bacteria target of melittin,it is necessary to understand the interaction mechanism between melittin and various biomembranes.It is expected to fundamentally solve the destructive threat of drug-resistant bacteria by means of antimicrobial peptides to target coated bacteria.In the first chapter,we outlined the properties of biofilms,including lipid diversity,fluidity,and the comparisons of membrane structure between prokaryotes and eukaryotes.It provided a good overview of bacterial lipid membrane activity of antimicrobial peptides.At the same time,the influence of membrane lipid structure on the physical and chemical properties of membrane was proposed,and it was found that membrane lipid structure affected the biological conformation,activity and localization of membrane proteins.Secondly,we explained the basic antibacterial mechanism of antimicrobial peptides,and the basic information of the antimicrobial peptide model in this study.Analyzing the research progress of the interactions between peptides and lipid membrane.In the second chapter,we mainly introduced the specific biofilm model used in the research work,as well as basic preparation process.Meanwhile,we obtained more suitable preparation technology for experimental research through optimizing.Expounding the characterization methods and three main experimental techniques in the process of membrane osmotic peptide’s experiment in detail.Melittin binding and pore formation on lipid membrane are complex and inter-related,which can be affected by many factors.A lot of researches have been devoted to studying the membranes leakage influenced by an individual factor.In the third chapter,we investigated the interactions mechanism between melittin and mono-component and multi-component phospholipid model membranes using fluorescence spectroscopy method.The mono-component vesicle experiments showed that the mechanisms of melittin acting on biomembranes with various phase and electrical properties was significantly different.Many barrel-stave holes form in the zwitterionic liquid membranes and the leakage is efficient.Only a few unstable U-shaped pores form in zwitterionic gel membranes and the leakage rate is lower.And two interaction stages were observed in the negatively charged membranes,i.e.parallel peptide binding and membrane thinning-cleavage,and the leakage rate is the lowest.Additionally,for the multi-component membranes,the leakage is determined by the lipids showing strong affinity to the peptides.This study deepens the understanding of the interactions between peptides and biomembranes.And it provides a reference for the peptide-related controlled release drug-delivery systems.Lipid types and structures affect the biological conformation,activity and localization of membrane proteins.The effects caused by phosphatidylserine(PS)on bacterial membrane are still unclear.In the fourth chapter,we explored the interactions between PS membranes(DOPC/DOPS and DOPC/DPPS)and melittin based on the tethered single vesicle platform and fluorescence microscopy.It is found that the melittin-induced response in saturated PS membranes was higher than that in unsaturated PS membranes.This reveals that the cone configuration of melittin is prone to binding to lipid membrane containing saturated PS.Furthermore,we used SMH(Shai-Matsuzaki-Huang)model to analyze the interactions from melittin at different concentrations.We used four main factors to analyze the membrane-peptide interaction kinetics.Melittin caused the saturated PS membranes to form carpet pores and burst,which is the result of the micelle-favored negative spontaneous curvature of the saturated phospholipids.And melittin caused pore and carpet formation in unsaturated PS membranes,which is the result of the low bending rigidity of unsaturated PS membranes and the reduced energy barrier for pore formation.Lastly,we also observed the curvature-dependent membrane-melittin interactions.This study deepens the understanding of the interactions mechanism between melittins and multi-component Biomembranes.Finally,we summarize the thesis and present outlooks for the future work based on some experimental results.