Melittin is a prototype of the ubiquitous antimicrobial peptides that induce pores in membranes. It is commonly used as a molecular device for membrane permeabilization. Even at concentrations in the nanomolar range, melittin can induce transient pores that allow transmembrane conduction of atomic ions but not leakage of glucose or larger molecules. At micromolar concentrations, melittin induces stable pores allowing transmembrane leakage of molecules up to tens of kilodaltons, corresponding to its antimicrobial activities. Despite extensive studies, aspects of the molecular mechanism for pore formation remain unclear. To clarify the mechanism, one must know the states of the melittin-bound membrane before and after the process. By correlating experiments using giant unilamellar vesicles with those of peptide-lipid multilayers, we found that melittin bound on the vesicle translocated and redistributed to both sides of the membrane before the formation of stable pores. Furthermore, stable pores are formed only above a critical peptide-to-lipid ratio. The initial states for transient and stable pores are different, which implies different mechanisms at low and high peptide concentrations. To determine the lipidic structure of the pore, the pores in peptide-lipid multilayers were induced to form a lattice and examined by anomalous X-ray diffraction. The electron density distribution of lipid labels shows that the pore is formed by merging of two interfaces through a hole. The molecular property of melittin is such that it adsorbs strongly to the bilayer interface. Pore formation can be viewed as the bilayer adopting a lipid configuration to accommodate its excessive interfacial area.toroidal pore | oriented circular dichroism | rhombohedral phase M elittin, the major toxin of the bee venom discovered around 1970 (1), produces a variety of effects on nature membranes, including cell lysis (2), antimicrobial activity (3), and voltage-dependent ion conductance (4). A great number of hostdefense antimicrobial peptides (5, 6) discovered in the past three decades have been found to exhibit similar behavior of melittin (3, 7). Among membrane-active peptides, melittin is perhaps the most extensively studied (8-12). It is widely used for cell and liposome lysis and as a model for pore-forming peptides (7, 13). Its whole and partial amino acid sequences have been incorporated in the designs of synthetic proteins to mimic the property of melittin (14, 15). However, its molecular process and mechanism of activities are still in dispute. It is clear that melittin binds to membranes as monomers but acts on the membrane collectively. Even at concentrations as low as a few nanomoles per liter, melittin can induce transient pores that allow transmembrane conduction of atomic ions but not leakage of glucose or larger molecules (4,7,16). In the micromolar range, melittin induces stable pores allowing transmembrane leakage of molecules up to tens of kilodaltons (13,17,18). At even higher concentrations, it can act as a detergent disin...

Daptomycin is the first approved member of a new structural class of antibiotics, the cyclic lipopeptides. The peptide interacts with the lipid matrix of cell membranes, inducing permeability of the membrane to ions, but its molecular mechanism has been a puzzle. Unlike the ubiquitous membrane-acting host-defense antimicrobial peptides, daptomycin does not induce pores in the cell membranes. Thus, how it affects the permeability of a membrane to ions is not clear. We studied its interaction with giant unilamellar vesicles (GUVs) and discovered a lipid-extracting phenomenon that correlates with the direct action of daptomycin on bacterial membranes observed in a recent fluorescence microscopy study. Lipid extraction occurred only when the GUV lipid composition included phosphatidylglycerol and in the presence of Ca2+ ions, the same condition found to be necessary for daptomycin to be effective against bacteria. Furthermore, it occurred only when the peptide/lipid ratio exceeded a threshold value, which could be the basis of the minimal inhibitory concentration of daptomycin. In this first publication on the lipid extracting effect, we characterize its dependence on ions and lipid compositions. We also discuss possibilities for connecting the lipid extracting effect to the antibacterial activity of daptomycin.

We investigated the physical properties of bacterial cytoplasmic membranes by applying the method of micropipette aspiration to Escherichia coli spheroplasts. We found that the properties of spheroplast membranes are significantly different from that of laboratory-prepared lipid vesicles or that of previously investigated animal cells. The spheroplasts can adjust their internal osmolality by increasing their volumes more than three times upon osmotic downshift. Until the spheroplasts are swollen to their volume limit, their membranes are tensionless. At constant external osmolality, aspiration increases the surface area of the membrane and creates tension. What distinguishes spheroplast membranes from lipid bilayers is that the area change of a spheroplast membrane by tension is a relaxation process. No such time dependence is observed in lipid bilayers. The equilibrium tension-area relation is reversible. The apparent area stretching moduli are several times smaller than that of stretching a lipid bilayer. We conclude that spheroplasts maintain a minimum surface area without tension by a membrane reservoir that removes the excessive membranes from the minimum surface area. Volume expansion eventually exhausts the membrane reservoir; then the membrane behaves like a lipid bilayer with a comparable stretching modulus. Interestingly, the membranes cease to refold when spheroplasts lost viability, implying that the membrane reservoir is metabolically maintained.

Background:Atlastin is large GTPase that catalyzes the homotypic fusion of ER membranes. Results: In vitro and in vivo studies reveal that the C-terminal tail of Atlastin affects its function. Conclusion:The amphipathic C-terminal tail of Atlastin destabilizes lipid bilayers to promote membrane fusion. Significance: Describing the mechanism of Atlastin-mediated fusion is a critical step in our understanding of ER structure formation.

We investigated the phenomena of antimicrobial peptides (AMPs) directly attacking the cytoplasmic membranes of Escherichia coli spheroplasts. We developed a procedure for fluorescence recovery after photobleaching to examine dye leakage through bacterial membranes as AMPs in solution bound to the membranes. We found that the AMP binding did not increase the apparent membrane area of a spheroplast, contrary to the response of a lipid-bilayer vesicle, which always showed a membrane area expansion by AMP binding. The permeability through the bacterial membrane increased in a sigmoidal fashion as the AMP binding increased in time, exhibiting a cooperative behavior of AMPs. The analysis of fluorescence recovery after photobleaching showed that the fluxes of dye molecules into and out of the cell were consistent with diffusion of molecules through a number of pores that increased with binding of AMPs and then saturated to a steady level. We discovered a new, to our knowledge, experimental parameter called the flux rate that characterizes the AMP-induced permeability of dye molecules through bacterial membranes. The phenomena observed in bacterial membranes are consistent with the pore-forming activities of AMPs previously observed in lipid bilayers. The experimental value of the flux rate per pore is much smaller than a theoretical value that assumes no friction for the dye molecule's permeation through the pore. We believe that experimental studies of the flux rate will be useful for further analysis of AMPs' permeabilization mechanisms.