Comparison of the Effects of Daptomycin on Bacterial and Model Membranes.

Daptomycin is a phosphatidylglycerol specific, calcium-dependent membrane-active antibiotic that has been approved for the treatment of Gram-positive infections. A recent Bacillus subtilis study found that daptomycin clustered into fluid lipid domains of bacterial membranes and the membrane binding was correlated with dislocation of peripheral membrane proteins and depolarization of membrane potential. In particular, the study disproved the existence of daptomycin ion channels. Our purpose here is to study how daptomycin interacts with lipid bilayers to understand the observed phenomena on bacterial membranes. We performed new types of experiments using aspirated giant vesicles with an ion leakage indicator, making comparisons between daptomycin and ionomycin, performing vesicle-vesicle transfers, and measuring daptomycin binding to fluid phase versus gel phase bilayers and bilayers including cholesterol. Our findings are entirely consistent with the observations for bacterial membranes. In addition, daptomycin is found to cause ion leakage through the membrane only if its concentration in the membrane is over a certain threshold. The ion leakage caused by daptomycin is transient. It occurs only when daptomycin binds the membrane for the first time; afterward, they cease to induce ion leakage. The ion leakage effect of daptomycin cannot be transferred from one membrane to another. The level of membrane binding of daptomycin is reduced in the gel phase versus the fluid phase. Cholesterol also weakens the membrane binding of daptomycin. The combination of membrane concentration threshold and differential binding is significant. This could be a reason why daptomycin discriminates between eukaryotic and prokaryotic cell membranes.

Interplay of entropy and enthalpy in peptide binding to zwitterionic phospholipid membranes as revealed from membrane thinning.

Membrane thinning that resulted from peptide-binding is observed via temperature dependent small-angle X-ray scattering (SAXS). The result reveals a mean thermal thinning rate of 0.038 Å K-1 for the neat unilamellar vesicles (ULVs) of a zwitterionic phospholipid of 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (diC20:1PC) in the temperature range of 285-312 K. The thinning effect promotes greatly the association between a model antimicrobial peptide melittin and the ULV. Scaling the observed isothermal melittin-ULV bilayer thinning to that measured using low-angle X-ray diffraction from the melittin-multilamellar membranes of defined peptide-to-lipid ratios establishes temperature-dependent binding isotherms χb of the peptide-ULV as a function of free peptide concentration in solution. From the binding isotherms, temperature-dependent peptide-membrane binding constant K(T) is extracted on the basis of a modified Gouy-Chapman model. Changes in K(T) follow the linearized van't Hoff equation ln K(T) ∝ -ΔHT-1 with a constant enthalpy change ΔH = 9.6 kcal mol-1, suggesting an entropy-driven binding process prior to membrane pore formation. Correspondingly, a five-fold enhancement of K is observed in the temperature range studied. The peptide-binding strength is found to follow the growth trend of the membrane thermal thinning rate better than the lipid chain length of the three phosphocholine-based ULVs of diCn:1PC with n = 18, 20, and 22.

Molecular State of the Membrane-Active Antibiotic Daptomycin.

Membrane-active antibiotics are potential alternatives to the resistance-prone conventional antibiotics. Daptomycin, a cyclic lipopeptide, is the only membrane-active antibiotic approved by the U.S. Food and Drug Administration so far. The drug interacts with the cytoplasmic membranes of Gram-positive pathogens, causing membrane permeabilization to ions and cell death. The antibiotic activity is calcium-ion dependent and correlates with the target membrane's content of phosphatidylglycerol (PG). For such a complex reaction with membranes, it has been difficult to uncover the molecular process that underlies its antibacterial activity. The role of the cofactor, calcium ions, has been confusing. Many have proposed that calcium ions binding to daptomycin is a precondition for membrane interaction. Here, we report our findings on the molecular state of daptomycin before and after its membrane-binding reaction, particularly at therapeutic concentrations in the low micromolar range. We were able to perform small-angle x-ray scattering at sufficiently low daptomycin concentrations to determine that the molecules are monomeric before membrane binding. By careful circular dichroism (CD) analyses of daptomycin with Ca2+ and PG-containing membranes, we found that there are only two states identifiable by CD, one before and another after membrane binding; all other CD spectra are linear combinations of the two. Before membrane binding, the molecular state of daptomycin as defined by CD is the same with or without calcium ions. We are able to determine the stoichiometric ratios of the membrane-binding reaction. The stoichiometric ratio of daptomycin to calcium is 2:3. The stoichiometric ratio of daptomycin to PG is ∼1:1 if only the PG lipids in the outer leaflets of membranes are accessible to daptomycin.

Membrane-mediated amyloid formation of PrP 106-126: A kinetic study.

PrP 106-126 conserves the pathogenic and physicochemical properties of the Scrapie isoform of the prion protein. PrP 106-126 and other amyloidal proteins are capable of inducing ion permeability through cell membranes, and this property may represent the common primary mechanism of pathogenesis in the amyloid-related degenerative diseases. However, for many amyloidal proteins, despite numerous phenomenological observations of their interactions with membranes, it has been difficult to determine the molecular mechanisms by which the proteins cause ion permeability. One approach that has not been undertaken is the kinetic study of protein-membrane interactions. We found that the reaction time constant of the interaction between PrP 106-126 and membranes is suitable for such studies. The kinetic experiment with giant lipid vesicles showed that the membrane area first increased by peptide binding but then decreased. The membrane area decrease was coincidental with appearance of extramembranous aggregates including lipid molecules. Sometimes, the membrane area would increase again followed by another decrease. The kinetic experiment with small vesicles was monitored by circular dichroism for peptide conformation changes. The results are consistent with a molecular simulation following a simple set of well-defined rules. We deduced that at the molecular level the formation of peptide amyloids incorporated lipid molecules as part of the aggregates. Most importantly the amyloid aggregates desorbed from the lipid bilayer, consistent with the macroscopic phenomena observed with giant vesicles. Thus we conclude that the main effect of membrane-mediated amyloid formation is extraction of lipid molecules from the membrane. We discuss the likelihood of this effect on membrane ion permeability.

Process of inducing pores in membranes by melittin.

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.

Peptide-induced bilayer thinning structure of unilamellar vesicles and the related binding behavior as revealed by X-ray scattering.

We have studied the bilayer thinning structure of unilamellar vesicles (ULV) of a phospholipid 1,2-dierucoyl-sn-glycero-3-phosphocholine (di22:1PC) upon binding of melittin, a water-soluble amphipathic peptide. Successive thinning of the ULV bilayers with increasing peptide concentration was monitored via small-angle X-ray scattering (SAXS). Results suggest that the two leaflets of the ULV of closed bilayers are perturbed and thinned asymmetrically upon free peptide binding, in contrast to the centro-symmetric bilayer thinning of the substrate-oriented multilamellar membranes (MLM) with premixed melittin. Moreover, thinning of the melittin-ULV bilayer associates closely with peptide concentration in solution and saturates at ~4%, compared to the ~8% maximum thinning observed for the correspondingly premixed peptide-MLM bilayers. Linearly scaling the thinning of peptide-ULV bilayers to that of the corresponding peptide-MLM of a calibrated peptide-to-lipid ratio, we have deduced the number of bound peptides on the ULV bilayers as a function of free peptide concentration in solution. The hence derived X-ray-based binding isotherm allows extraction of a low binding constant of melittin to the ULV bilayers, on the basis of surface partition equilibrium and the Gouy-Chapman theory. Moreover, we show that the ULV and MLM bilayers of di22:1PC share a same thinning constant upon binding of a hydrophobic peptide alamethicin; this result supports the linear scaling approach used in the melittin-ULV bilayer thinning for thermodynamic binding parameters of water-soluble peptides.

Mechanism and kinetics of pore formation in membranes by water-soluble amphipathic peptides.

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.

The bound states of amphipathic drugs in lipid bilayers: study of curcumin.

Drug-membrane interactions are well known but poorly understood. Here we describe dual measurements of membrane thickness change and membrane area change due to the binding of the amphipathic drug curcumin. The combined results allowed us to analyze the binding states of a drug to lipid bilayers, one on the water-membrane interface and another in the hydrocarbon region of the bilayer. The transition between the two states is strongly affected by the elastic energy of membrane thinning (or, equivalently, area stretching) caused by interfacial binding. The data are well described by a two-state model including this elastic energy. The binding of curcumin follows a common pattern of amphipathic peptides binding to membranes, suggesting that the binding states of curcumin are typical for amphipathic drugs.

Energetics of pore formation induced by membrane active peptides.

Antimicrobial peptides are known to form pores in cell membranes. We study this process in model bilayers of various lipid compositions. We use two of the best-studied peptides, alamethicin and melittin, to represent peptides making two types of pores, that is, barrel-stave pores and toroidal pores. In both cases, the key control variable is the concentration of the bound peptides in the lipid bilayers (expressed in the peptide-lipid molar ratio, P/L). The method of oriented circular dichroism (OCD) was used to monitor the peptide orientation in bilayers as a function of P/L. The same samples were scanned by X-ray diffraction to measure the bilayer thickness. In all cases, the bilayer thickness decreases linearly with P/L and then levels off after P/L exceeds a lipid-dependent critical value, (P/L)*. OCD spectra showed that the helical peptides are oriented parallel to the bilayers as long as P/L < (P/L)*, but as P/L increases over (P/L)*, an increasing fraction of peptides changed orientation to become perpendicular to the bilayer. We analyzed the data by assuming an internal membrane tension associated with the membrane thinning. The free energy containing this tension term leads to a relation explaining the P/L-dependence observed in the OCD and X-ray diffraction measurements. We extracted the experimental parameters from this thermodynamic relation. We believe that they are the quantities that characterize the peptide-lipid interactions related to the mechanism of pore formation. We discuss the meaning of these parameters and compare their values for different lipids and for the two different types of pores. These experimental parameters are useful for further molecular analysis and are excellent targets for molecular dynamic simulation studies.