Raman spectroscopic study of the interaction of poly-L-lysine with dipalmitoylphosphatidylglycerol bilayers

The interaction of the basic polypeptide poly-L-lysine with the negatively charged phospholipid dipalmitoylphosphatidylglycerol was studied using Raman spectroscopy. The nature of the interaction appeared to depend on the molar ratio of the constituents. At up to one lysine group per lipid molecule, the bilayer was stabilized by the polypeptide that underwent a conformational transition toward an ordered alpha-helical structure, in which the electrostatic interactions were probably maximal. The stabilization of the bilayer was detected by an increase in both the temperature of the thermotropic transition of the lipid and the interchain vibrational coupling of the methylene C-H vibrations. At higher poly-L-lysine concentration, hydrophobic interactions must have been involved to explain the binding of excess polypeptide. There seemed to be a penetration of poly-L-lysine in the bilayer that increased with the polypeptide concentration. Under these conditions, the chain-packing lattice gradually changed from hexagonal to either orthorhombic or monoclinic symmetry. We believe that this change of structure is associated with the interdigitation of the acyl chains.     

High pressure FTIR study of interaction of melittin with dimyristoylphosphatidyl glycerol bilayers.

Infrared spectra of hydrated dimyristoylphosphatidyl glycerol (DMPG) and of aqueous dispersions of melittin and DMPG at peptide:lipid molar ratios of 1:10 and 1:4 were recorded as a function of pressure from atmospheric to 22 kbar. Spectral features corresponding to vibrations of the amide linkages in melittin and to various functional groups in DMPG (carbonyl, methlylene, phosphate) were monitored in order to investigate the structure and dynamics of melittin:DMPG dispersions. Melittin was found to cause conformational and orientational disordering of the acyl chains in DMPG bilayers. The magnitude of these disorders was higher for higher concentration of melittin in DMPG. The higher concentration of melittin was also found to disrupt the DMPG bilayers through interactions with the lipid head groups. Such disruption may be related to some of the biological properties of melittin.     

Investigation of the interaction between DnaK and DnaJ by surface plasmon resonance spectroscopy.

Hsp70 chaperones assist protein folding through ATP-regulated transient association with substrates. Substrate binding by Hsp70 is controlled by DnaJ co-chaperones which stimulate Hsp70 to hydrolyze ATP and, consequently, to close its substrate binding cavity allowing trapping of substrates. We analyzed the interaction of the Escherichia coli Hsp70 homologue, DnaK, with DnaJ using surface plasmon resonance (SPR) spectroscopy. Resonance signals of complex kinetic characteristics were detected when DnaK was passed over a sensor chip with coupled DnaJ. This interaction was specific as it was not detected with a functionally defective DnaJ mutant protein, DnaJ259, that carries a mutation in the HPD signature motif of the conserved J-domain. Detectable DnaK-DnaJ interaction required ATP hydrolysis by DnaK and was competitively inhibited by chaperone substrates of DnaK. For DnaK mutant proteins with amino acid substitutions in the substrate binding cavity that affect substrate binding, the strength of detected interaction with DnaJ decreased proportionally with increased strength of the substrate binding defects. These findings indicate that the detected response signals resulted from DnaJ and ATP hydrolysis-dependent association of DnaJ as substrate for DnaK. Although not considered as physiologically relevant, this association allowed us to experimentally unravel the mechanism of DnaJ action. Accordingly, DnaJ stimulates ATP hydrolysis only after association of a substrate with the substrate binding cavity of DnaK. Further analysis revealed that this coupling mechanism required the J-domain of DnaJ and was also functional for natural DnaK substrates, and thus is central to the mechanism of action of the DnaK chaperone system.     

Model of interaction between a cardiotoxin and dimyristoylphosphatidic acid bilayers determined by solid-state 31P NMR spectroscopy.

The interaction of cardiotoxin IIa, a small basic protein extracted from Naja mossambica mossambica venom, with dimyristoylphosphatidic acid (DMPA) membranes has been investigated by solid-state 31P nuclear magnetic resonance spectroscopy. Both the spectral lineshapes and transverse relaxation time values have been measured as a function of temperature for different lipid-to-protein molar ratios. The results indicate that the interaction of cardiotoxin with DMPA gives rise to the complete disappearance of the bilayer structure at a lipid-to-protein molar ratio of 5:1. However, a coexistence of the lamellar and isotropic phases is observed at higher lipid contents. In addition, the number of phospholipids interacting with cardiotoxin increases from about 5 at room temperature to approximately 15 at temperatures above the phase transition of the pure lipid. The isotropic structure appears to be a hydrophobic complex similar to an inverted micellar phase that can be extracted by a hydrophobic solvent. At a lipid-to-protein molar ratio of 40:1, the isotropic structure disappears at high temperature to give rise to a second anisotropic phase, which is most likely associated with the incorporation of the hydrophobic complex inside the bilayer.     

Real-time structural investigation of a lipid bilayer during its interaction with melittin using sum frequency generation vibrational spectroscopy

Interactions between membrane bilayers and peptides/proteins are ubiquitous throughout a cell. To determine the structure of membrane bilayers and the associated peptides/proteins, model systems such as supported lipid bilayers are often used. It has been difficult to directly investigate the interactions between a single membrane bilayer and peptides/proteins without exogenous labeling. In this work we demonstrate that sum frequency generation vibrational spectroscopy can be employed to study the interactions between peptides/proteins and a single lipid bilayer in real time, in situ, and without exogenous labeling. Using melittin and a dipalmitoyl phosphatidylglycerol bilayer as a model system, we monitored the C-H and C-D stretching signals from isotopically symmetric or asymmetric dipalmitoyl phosphatidylglycerol bilayers during their interaction with melittin. It has been found that the extent and kinetics of bilayer perturbation induced by melittin are very sensitive to melittin concentration. Such concentration dependence is correlated to melittin's mode of action. Melittin is found to function via the early and late stage of the carpet model at low and high concentrations, respectively, whereas the toroidal model is probable at intermediate concentrations. This research illustrates the potential of sum frequency generation as a biophysical technique to monitor individual leaflet structure of lipid bilayers in real time during their interactions with biomolecules.     

Investigation of the interaction between DNA and cobalt ferrite nanoparticles by FTIR spectroscopy

The interaction of DNA with nanoparticles of cobalt ferrite powder prepared by the mechano-chemical method was studied. It was shown that CoFe₂O₄ nanoparticles efficiently bind DNA in aqueous solutions (Tris-HCl), forming a bionanocomposite. The adsorption capacity of CoFe₂O₄ nanoparticles for DNA was evaluated to be 5.25 × 10⁻³ mol/m². The desorption of DNA from the surface of the particles was analyzed while changing the pH, the ionic strength, and the chemical content of the medium. The DNA-CoFe₂O₄ nanocomposite was investigated by FTIR spectroscopy. The block of the data allowed one to consider the mechanism of the interaction between a polynucleotide and CoFe₂O₄ nanoparticles and to make the assumption that the binding occurred due to the coordination interaction of the phosphate groups and heterocyclic bases of DNA (oxygen atoms of thymine and guanine) with metal ions on the particle surface. The analysis of the IR spectra showed that binding can lead to the partial destabilization of the DNA structure, with the B conformation of a polynucleotide being preserved.     

Investigation of the interaction of myelin basic protein with phospholipid bilayers using solid-state NMR spectroscopy

Interaction of bovine myelin basic protein and its constituent charge isomers (C1-C3) with phospholipid bilayers was studied using solid-state NMR experiments on model membranes. 31P NMR experiments on multilamellar vesicles and mechanically aligned bilayers were used to measure the degree of protein-induced disorder in the lipid headgroup region while 2H NMR data provided the disorder caused by the protein in the hydrophobic core of the bilayers. Our results suggest that MBP and its charge isomers neither fragment nor significantly disrupt DMPC, POPC, POPC:POPG, and POPE bilayers. These results demonstrate that the MBP-induced fragmentation of POPC bilayers is due to the freeze-thaw cycles used in the preparation of multilamellar vesicles and not due to intrinsic protein-lipid interactions.     

Structural and thermodynamic analyses of the interaction between melittin and lipopolysaccharide

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is the very first site of interactions with the antimicrobial peptides. In this work, we have determined a solution conformation of melittin, a well-known membrane active amphiphilic peptide from honey bee venom, by transferred nuclear Overhauser effect (Tr-NOE) spectroscopy in its bound state with lipopolysaccharide. The LPS bound conformation of melittin is characterized by a helical structure restricted only to the C-terminus region (residues A15-R24) of the molecule. Saturation transfer difference (STD) NMR studies reveal that several C-terminal residues of melittin including Trp19 are in close proximity with LPS. Isothermal titration calorimetry (ITC) data demonstrates that melittin binding to LPS or lipid A is an endothermic process. The interaction between melittin and lipid A is further characterized by an equilibrium association constant (Ka) of 2.85 x 10(6) M(-1) and a stoichiometry of 0.80, melittin/lipid A. The estimated free energy of binding (delta G0), -8.8 kcal mol(-1), obtained from ITC experiments correlates well with a partial helical structure of melittin in complex with LPS. Moreover, a synthetic peptide fragment, residues L13-Q26 or mel-C, derived from the C-terminus of melittin has been found to contain comparable outer membrane permeabilizing activity against Escherichia coli cells. Intrinsic tryptophan fluorescence experiments of melittin and mel-C demonstrate very similar emission maxima and quenching in presence of LPS micelles. The Red Edge Excitation Shift (REES) studies of tryptophan residue indicate that both peptides are located in very similar environment in complex with LPS. Collectively, these results suggest that a helical conformation of melittin, at its C-terminus, could be an important element in recognition of LPS in the outer membrane.     

Structural and thermodynamic analyses of the interaction between melittin and lipopolysaccharide

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is the very first site of interactions with the antimicrobial peptides. In this work, we have determined a solution conformation of melittin, a well-known membrane active amphiphilic peptide from honey bee venom, by transferred nuclear Overhauser effect (Tr-NOE) spectroscopy in its bound state with lipopolysaccharide. The LPS bound conformation of melittin is characterized by a helical structure restricted only to the C-terminus region (residues A15-R24) of the molecule. Saturation transfer difference (STD) NMR studies reveal that several C-terminal residues of melittin including Trp19 are in close proximity with LPS. Isothermal titration calorimetry (ITC) data demonstrates that melittin binding to LPS or lipid A is an endothermic process. The interaction between melittin and lipid A is further characterized by an equilibrium association constant (K_a) of 2.85 × 10⁶ M^− 1 and a stoichiometry of 0.80, melittin/lipid A. The estimated free energy of binding (ΔG⁰), − 8.8 kcal mol^− 1, obtained from ITC experiments correlates well with a partial helical structure of melittin in complex with LPS. Moreover, a synthetic peptide fragment, residues L13-Q26 or mel-C, derived from the C-terminus of melittin has been found to contain comparable outer membrane permeabilizing activity against Escherichia coli cells. Intrinsic tryptophan fluorescence experiments of melittin and mel-C demonstrate very similar emission maxima and quenching in presence of LPS micelles. The Red Edge Excitation Shift (REES) studies of tryptophan residue indicate that both peptides are located in very similar environment in complex with LPS. Collectively, these results suggest that a helical conformation of melittin, at its C-terminus, could be an important element in recognition of LPS in the outer membrane.     

Fluorescence-quenching-resolved spectra of melittin in lipid bilayers

The interaction of bee venom melittin with dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles has been studied by means of fluorescence quenching of the single tryptophan residue of the protein, at lipid-to-peptide ratio, Ri = 50 and at high ionic strength (2 M NaCl). The method of fluorescence-quenching-resolved spectra (FQRS), applied in this study with potassium iodide as a quencher, enabled us to decompose the tryptophan emission spectrum of liposome-bound melittin into components, at temperatures above as well as below the main phase transition temperature (Tt) of DMPC. One of the two resolved spectra exhibits maximum at 342 and 338 nm for experiments above and below Tt, respectively, and is similar to the maximum of tryptophan emission found for tetrameric melittin in solution (340 nm). This spectrum is characterized by the Stern-Volmer quenching constant, Ksv, of about 4 M-1 and it represents the fraction of melittin molecules whose tryptophan residues are exposed to the solvent to a degree comparable with tetrameric species in solution. The other spectrum component, corresponding to the quencher-inaccessible fraction of tryptophan molecules (Ksv = 0 M-1) has its maximum blue-shifted up to 15 nm, indicating a decrease in polarity of the environment. For experiments above Tt, the blue spectrum component revealed the excitation-wavelength dependence, originating probably from the relaxation processes between the excited tryptophan molecules and lipid polar head groups. We conclude that melittin bound to DMPC liposomes exists in two lipid-associated forms; one, with tryptophan residues exposed to the solvent and the other, penetrating the membrane interior, with tryptophan residues located in close proximity to the phospholipid polar head groups of the outer vesicle lipid layer. We also discuss our data with current models of melittin-bilayer interactions.     

DNA protection by aminothiols: study of the cysteamine - DNA interaction by vibrational spectroscopy

Infrared linear dichroism measurements and Roman scattering spectra show that the cysteamine molecule binds strongly to the DNA stabilizing the double helix in a B geometry conformation. The B→A conformational transition is not observed for a cysteamine/DNA ratio of one cysteamine molecule per two phosphate sites. No evidence of interaction has been found between the radioprotector and the DNA bases. A model is proposed in which the cysteamine molecule is bound by its two ends through electrostatic interaction to two consecutive phosphate groups along the same DNA strand.     

Morphological behavior of lipid bilayers induced by melittin near the phase transition temperature

Morphological changes of DMPC, DLPC, and DPPC bilayers containing melittin (lecithin/melittin molar ratio of 10:1) around the gel-to-liquid crystalline phase transition temperatures (Tc) were examined by a variety of biophysical methods. First, giant vesicles with the diameters of approximately 20 microm were observed by optical microscopy for melittin-DMPC bilayers at 27.9 degrees C. When the temperature was lowered to 24.9 degrees C (Tc = 23 degrees C for the neat DMPC bilayers), the surface of vesicles became blurred and dynamic pore formation was visible in the microscopic picture taken at different exposure times. Phase separation and association of melittin molecules in the bilayers were further detected by fluorescent microscopy and mass spectrometry, respectively. These vesicles disappeared completely at 22.9 degrees C. It was thus found that the melittin-lecithin bilayers reversibly undergo their fusion and disruption near the respective Tcs. The fluctuation of lipids is, therefore, responsible for the membrane fusion above the Tc, and the association of melittin molecules causes membrane fragmentation below the Tc. Subsequent magnetic alignments were observed by solid-state (31)P NMR spectra for the melittin-lecithin vesicles at a temperature above the respective Tcs. On the other hand, additional large amplitude motion induced by melittin at a temperature near the Tc breaks down the magnetic alignment.     

Probing the interaction of Arg9Cys mutated phospholamban with phospholipid bilayers by solid-state NMR spectroscopy

Phospholamban (PLB) is a 52 amino acid integral membrane protein that interacts with the sarcoplasmic reticulum Ca^2 + ATPase (SERCA) and helps to regulate Ca^2 + flow. PLB inhibits SERCA impairing Ca^2 + translocation. The inhibition can be relieved upon phosphorylation of PLB. The Arg9 to Cys (R9C) mutation is a loss of function mutation with reduced inhibitory potency. The effect R9C PLB has on the membrane surface and the hydrophobic region dynamics was investigated by ³¹P and ²H solid-state NMR spectroscopy in multilamellar vesicles (MLVs). The ³¹P NMR spectra indicate that, like the phosphorylated PLB (P-PLB), the mutated R9C-PLB protein has significantly less interaction with the lipid bilayer headgroup when compared to wild-type PLB (WT-PLB). Similar to P-PLB, R9C-PLB slightly decreases ³¹P T₁ values in the lipid headgroup region. ²H S_CD order parameters of ²H nuclei along the lipid acyl chain decrease less dramatically for R9C-PLB and P-PLB when compared to WT-PLB. The results suggest that R9C-PLB interacts less with the membrane surface and hydrophobic region than WT-PLB. Detachment of the cytoplasmic domain of R9C-PLB from the membrane surface could be related to its loss of function.     

Structural and thermodynamic aspects of the interaction between heparan sulfate and analogues of melittin

Melittin is an amphipathic cationic peptide derived from honeybee venom with well-known cytolytic and antimicrobial properties. When coupled to cationic polymers or lipid molecules, it forms conjugates with high transfection efficiency and low toxicity with promising applications in gene therapy. A first step in the internalization of melittin and its conjugates is their binding to the cell surface, a reaction likely to involve heparan sulfate proteoglycans (HSPG). In the present work, we characterize the binding equilibrium of heparan sulfate (HS) with two melittin analogues, [Cys(1)]melittin (mel-SH) and retro-inverso [Cys(1)]melittin (ri-mel-SH). The terminal cysteine found in these peptides replaces the N-terminal glycine present in native melittin and allows covalent binding to other molecules. Isothermal titration calorimetry (ITC) reveals a high affinity of each melittin analogue to HS. Association constants of 4.7 x 10(4) and 3.5 x 10(5) M(-)(1) are found at physiological ionic strength and 15 degrees C for ri-mel-SH and mel-SH, respectively. The reaction enthalpy measured under these conditions is DeltaH(degrees)pep= 4.2 kcal/mol for ri-mel-SH and DeltaH(degrees)pep= 1.1 kcal/mol for mel-SH. The peptide-to-HS stoichiometry is approximately 20 for ri-mel-SH and approximately 14 for mel-SH under the same conditions. Temperature dependence studies using ri-mel-SH (mel-SH) show that DeltaH(degrees)pep decreases in magnitude upon increase in temperature, which results in a molar heat capacity of DeltaH(degrees)pep= -322 cal mol(-)(1) K(-)(1) (-45 cal mol(-)(1) K(-)(1)). Such a negative heat capacity change is not expected for a purely electrostatic interaction and indicates that hydrophobic and other interactions are also involved in the binding equilibrium. Salt dependence studies of the binding constants confirm that nonelectrostatic forces are an important component of the HS-melittin interaction. Binding to HS induces conformational changes in both peptides, with ri-mel-SH showing a 6-fold increase of the alpha-helix content when incubated with HS under saturation conditions.     

Different states of self-association of melittin in phospholipid bilayers

Melittin is known to self-associate as tetramers in solutions of high ionic strength. Here, an N-bromosuccinimide oxidized-Trp₁₉ melittin is prepared. This derivative can act as an acceptor of the fluorescence of native melittin and is used in order to observe a possible self-association of melittin in phospholipid bilayers.Resonance energy transfer was shown to occur in solutions of high ionic strength, showing that oxidized melittin can associate with native melittin.In phospholipid bilayers, no association is detected in the absence of NaCl. In its presence, an equilibrium between monomeric melittin and oligomeric species is observed. These species are not dimers, but any other degree of association may account for our experimental results. Significant differences in characteristic transfer efficiency reveal differences in the structure of these oligomers according to the length or state of phospholipids (fluid or at the transition temperature). These bound complexes are also different from the soluble hetero-oligomer.Some models of bound complexes are proposed which may explain the leakage and the further disruption of vesicles or cells induced by melittin.Abbreviations NBS N-bromosuccinimide - NATA N-acetyl tryptophanamide - DMPC dimyristoyl phosphatidylcholine - DPPC dipalmitoyl phosphatidylcholine - PG phosphatidylglycerol - EPC egg phosphatidylcholine - O-melittin oxindole-melittin - RET resonance energy transfer - EDTA ethylene diamine tetracetic acid - Mel melittin     

Stopped-flow fluorometric study of the interaction of melittin with phospholipid bilayers: importance of the physical state of the bilayer and the acyl chain length.

Stopped-flow fluorometry has been employed to study the effects of melittin, the major protein component of bee venom, on dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) small unilamellar vesicles (SUVs) on the millisecond time scale, before melittin-induced vesicle fusion takes place. Use is made of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), which is an oriented fluorescent probe that anchors itself to the bilayer-water interface and is aligned parallel to the normal to the bilayer surface; its fluorescence anisotropy reports on the "fluidity" of the bilayer. For DMPC bilayers, melittin is found to decrease their fluidity only at their melting transition temperature. This perturbation appears to be exerted almost instantaneously on the millisecond time scale of the measurements, as deduced from the fact that its rate is comparable to that obtained by following the change in the fluorescence of the single tryptophan residue of melittin upon inserting itself into the bilayer. The perturbation is felt in the bilayer over a distance of at least 50 A, with measurements of transfer of electronic energy indicating that the protein is not sequestered in the neighborhood of TMA-DPH. The length of the acyl chains is found to be an important physical parameter in the melittin-membrane interaction: unlike the case of DMPC SUVs, melittin does not alter the fluidity of DPPC SUVs and has a considerably greater affinity for them. These results are discussed in terms of the concept of elastic distortion of the lipids, which results from a mismatch between the protein and the acyl chains that are attempting to accommodate it. Melittin is also found to cause a small (approximately 10%) enhancement in the total fluorescence intensity of TMA-DPH, which is interpreted as indicating a reduction in the degree of hydration of the bilayer.     

Modulation of interaction forces between bilayers exposing short-chained ethylene oxide headgroups.

The use of liposomes as drug delivery systems has been limited by their rapid clearance from circulation by the mononuclear phagocyte system. Recent studies have found that circulation times can be greatly enhanced by incorporating a small amount of modified lipids whose headgroups are derivatized with a bulky water soluble polymeric chain of poly ethylene oxide. We report here a systematic study using the Surface Forces Apparatus to measure directly the interactions between two phosphatidyl ethanolamine lipid bilayers, exposing this polymeric headgroup at different concentrations in the bilayer. We found that the force becomes repulsive at all separations and that the thickness of the steric barrier could be controlled easily by adjusting the concentration of the modified lipids. Equilibrium force profiles were measured that were reversible and largely insensitive to changes in electrolyte concentration and temperature. The results have enabled the Dolan and Edwards theory for the steric forces of low coverage polymer surfaces and the Alexander de Gennes theory for high coverage surfaces to be tested, and both were found to apply. We conclude that these simple theories can be used to model the interactions of surprisingly short segments and, hence, apply to such systems as lipids with bulky headgroups and liposomes containing a sterically stabilizing polymer.     

Conformation and dynamics of melittin bound to magnetically oriented lipid bilayers by solid-state (31)P and (13)C NMR spectroscopy

The conformation and dynamics of melittin bound to the dimyristoylphosphatidylcholine (DMPC) bilayer and the magnetic orientation in the lipid bilayer systems were investigated by solid-state (31)P and (13)C NMR spectroscopy. Using (31)P NMR, it was found that melittin-lipid bilayers form magnetically oriented elongated vesicles with the long axis parallel to the magnetic field above the liquid crystalline-gel phase transition temperature (T(m) = 24 degrees C). The conformation, orientation, and dynamics of melittin bound to the membrane were further determined by using this magnetically oriented lipid bilayer system. For this purpose, the (13)C NMR spectra of site-specifically (13)C-labeled melittin bound to the membrane in the static, fast magic angle spinning (MAS) and slow MAS conditions were measured. Subsequently, we analyzed the (13)C chemical shift tensors of carbonyl carbons in the peptide backbone under the conditions where they form an alpha-helix and reorient rapidly about the average helical axis. Finally, it was found that melittin adopts a transmembrane alpha-helix whose average axis is parallel to the bilayer normal. The kink angle between the N- and C-terminal helical rods of melittin in the lipid bilayer is approximately 140 degrees or approximately 160 degrees, which is larger than the value of 120 degrees determined by x-ray diffraction studies. Pore formation was clearly observed below the T(m) in the initial stage of lysis by microscope. This is considered to be caused by the association of melittin molecules in the lipid bilayer.     

Similarities in melittin functional group reactivities during self-association and association with lipid bilayers.

Competitive labeling of melittin over a range of concentrations in the presence and absence of liposomes provides a series of "snapshots" of the chemical reactivities of melittin's intrinsic nucleophiles. Distinct trends in apparent reactivities were observed for the Gly-1 alpha-amino group and the epsilon-amino groups of Lys-7 and Lys-21 and -23, over a range of concentrations, providing evidence for different forms of associated melittin in solution. The monomer-tetramer transition can be followed, in accord with structural details derived from X-ray crystallography. The reactivity behavior of the alpha-amino group of Gly-1 and the epsilon-amino groups of Lys-21 and Lys-23 suggests these groups undergo similar perturbations in their microenvironments during the monomer-tetramer transition in free solution. Similar changes in reactivity behavior occur upon association of melittin monomers with bilayer-forming lipids. Together, these findings suggest that the local environments of the N- and C-terminal segments have similar physicochemical properties in both the solution tetramer and the lipid-associated complex. The concentration dependence of the chemical properties of melittin is correlated with surface accessibility calculations which are used to provide a framework for interpretation. Aspects of several previously proposed models of membrane lysis can be accounted for by concentration-dependent properties of melittin.