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...

The condensing effect of cholesterol on phospholipid bilayers was systematically investigated for saturated and unsaturated chains, as a function of cholesterol concentration. X-ray lamellar diffraction was used to measure the phosphate-to-phosphate distances, PtP, across the bilayers. The measured PtP increases nonlinearly with the cholesterol concentration until it reaches a maximum. With further increase of cholesterol concentration, the PtP remains at the maximum level until the cholesterol content reaches the solubility limit. The data in all cases can be quantitatively explained with a simple model that cholesterol forms complexes with phospholipids in the bilayers. The phospholipid molecules complexed with cholesterol are lengthened and this lengthening effect extends into the uncomplexed phospholipids surrounding the cholesterol complexes. This long-range thickening effect is similar to the effect of gramicidin on the thickness of lipid bilayers due to hydrophobic matching.

How antimicrobial peptides form pores in membranes is of interest as a fundamental membrane process. However, the underlying molecular mechanism, which has potential applications in therapeutics, nonviral gene transfer, and drug delivery, has been in dispute. We have resolved this mechanism by observing the time-dependent process of pore formation in individual giant unilamellar vesicles (GUVs) exposed to a melittin solution. An individual GUV first expanded its surface area at constant volume and then suddenly reversed to expanding its volume at constant area. The area expansion, the volume expansion, and the point of reversal all match the results of equilibrium measurements performed on peptide-lipid mixtures. The mechanism includes a negative feedback that makes peptide-induced pores stable with a well defined size, contrary to the suggestion that peptides disintegrate the membrane in a detergent-like manner.antimicrobial peptides ͉ membrane-thinning effect ͉ stable membrane pore ͉ peptide-induced pore ͉ single-membrane experiment M any water-soluble amphipathic peptides spontaneously bind to membranes and form transmembrane pores when the peptide concentrations exceed certain threshold values. Such pore-forming activities are of interest for many reasons. It is the common mode of action used by the ubiquitous antimicrobial peptides (1). A similar mechanism is used by pore-forming proteins, such as the apoptosis regulator Bcl-2-associated X protein (Bax), which activates pore formation in the outer mitochondria membrane to release the apoptotic factor cytochrome c (2). Understanding the relatively simple process of pore formation by small peptides is an important step toward unraveling more complex membranous conformational changes such as membrane fusion (3). Clarifying the pore-forming mechanism will also facilitate its applications, including developing antimicrobial molecules as human therapeutics (1) or smallmolecule agents for nonviral gene transfer and drug delivery. The physical effects caused by the binding of pore-forming peptides to lipid bilayers have been studied by x-ray and neutron diffraction on peptide-lipid mixtures (4-6). The results showed that peptide binding caused membrane thinning and pores appeared only when the thinning reached a critical fraction of the membrane thickness (4-7). However, the equivalent effects have not been demonstrated by kinetic experiments. This is important because pore formation in cell membranes caused by water-soluble peptides typically occurs as a kinetic process. Here, we report the observation of the time behavior of giant unilamellar vesicles (GUVs) exposed to the peptide melittin in solution. The observed time behavior of individual lipid vesicles, although complex, exhibits the physical effects seen in equilibrium experiments, thereby confirming that the mechanism of kinetic pore formation in single membranes is the same as that governing peptide-lipid interactions in the mixtures. It also implies that the same effect is likely to occur in cell membran...

The analytical results indicate that EAT volume is increased in T2DM patients and is associated with unfavourable components of MetS and coronary atherosclerosis. The close anatomical relationship between EAT and the coronary arteries, combined with other evidence indicating that EAT is a biologically active adipokine-secreting tissue, suggest that EAT participates in the pathogenesis of diabetic coronary atherosclerosis.