A Single Molecular Study On The Dynamic Interactions Between Melittin And Cell Membranes

As the first defense of cells,cell membranes separate the interior of cells from the external environments.Moreover,many important biological processes occur at cell membranes,including the endocytosis of particles,cellular uptake of protein/peptide molecules,virus infection,and signal conduction,etc.On the other hand,in recent years the abuse of antibiotics induces drug resistance of bacteria which has become a devastating threat to public health.The antimicrobial peptides(AMPs),which widely exist in the natural world,promise a fundamental solution.AMPs kill bacteria directly by acting on bacterial membranes,leading to poration or disruption of the membrane and content release.As one of the most representative AMPs,melittin kill bacteria in two steps:it binds and accumulates on membrane surface,followed by insertion of peptides and formation of transmembrane pores.However,a high peptide concentration is needed for such function.Some key issues,such as how melittin conquer the high free energy barrier during transition from a surface binding state to a transmembrane insertion state,and how the translocation behavior of peptides in Z direction occurs in membranes,are still largely unknown.Such unknowns hinder the efforts to apply melittin for clinical use.To conquer these issues,in this thesis,we take use of the single-molecular surface-induced fluorescence attenuation(SM-SIFA)method,in combination with computer molecular dynamics simulations,to investigate the dynamic process of melittin-lipid membrane interactions,especially the translocation behavior of peptides in Z direction,on a single molecular level.The microscopic motions of peptides in membranes are observed.For example:(1)We labeled the N-terminus and C-terminus of melittin,respectively,and monitor the peptide-membrane interaction process by SM-SIFA method.It was found that the C-terminus would stay on membrane surface while N-terminus would insert into membrane.(2)During the membrane action process,the N-terminus of melittin performed complicated motion modes,including:a)staying on the membrane surface;b)fluctuating in the upper-leaflet region of bilayer with periodic insertion and extraction behaviors;c)fluctuating across the whole membrane region.Throughout these motions,three types of metastable states of the N-terminus in membrane were observed,i.e.the hydrophobic lipid tail regions of the upper and lower leaflets,and the bilayer center.These microscopic behaviors were in good consistent with the simulation results.Besides,we used the membrane of living cells to wrap graphene oxide(GO)nanoparticles for the development of a novel type of membrane@GO complex particles.Such particles had dual-fluorescence property and well bionic performance.This work helps us to deeply understand the dynamic interaction process between melittin and cell membranes,especially the microscopic translocation behavior of peptides in membranes,on a single molecular level.Moreover,it provides a general method for investigating the membrane action process of molecules such like peptides and proteins.