Molecular Dynamics Simulations of Mixed Acidic/Zwitterionic Phospholipid Bilayers

Anionic lipids are key components in the cell membranes. Many cell-regulatory and signaling mechanisms depend upon a complicated interplay between them and membrane-bound proteins. Phospholipid bilayers are commonly used as model systems in experimental or theoretical studies to gain insight into the structure and dynamics of biological membranes. We report here 200-ns-long MD simulations of pure (DMPC and DMPG) and mixed equimolar (DMPC/DMPG, DMPC/DMPS, and DMPC/DMPA) bilayers that each contain 256 lipids. The intra- and intermolecular interaction patterns in pure and mixed bilayers are analyzed and compared. The effect of monovalent ions (Na⁺) on the formation of salt-bridges is investigated. In particular, the number of Na⁺-mediated clusters in the presence of DMPS is higher than with DMPG and DMPA. We observe a preferential clustering of DMPS (and to some extent DMPA) lipids together rather than with DMPC molecules, which can explain the phase separation observed experimentally for DMPC/DMPS and DMPC/DMPA bilayers.     

Differential scanning calorimetric study of the effect of the antimicrobial peptide gramicidin S on the thermotropic phase behavior of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol lipid bilayer membranes

We have studied the effects of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE) and dimyristoyl phosphatidylglycerol (DMPG) by high-sensitivity differential scanning calorimetry. We find that the effect of GS on the lamellar gel to liquid-crystalline phase transition of these phospholipids varies markedly with the structure and charge of their polar headgroups. Specifically, the presence of even large quantities of GS has essentially no effect on the main phase transition of zwitterionic DMPE vesicles, even after repeating cycling through the phase transition, unless these vesicles are exposed to high temperatures, after which a small reduction in the temperature, enthalpy and cooperativity of the gel to liquid-crystalline phase transitions is observed. Similarly, even large amounts of GS produce similar modest decreases in the temperature, enthalpy and cooperativity of the main phase transition of DMPC vesicles, although the pretransition is abolished at low peptide concentrations. However, exposure to high temperatures is not required for these effects of GS on DMPC bilayers to be manifested. In contrast, GS has a much greater effect on the thermotropic phase behavior of anionic DMPG vesicles, substantially reducing the temperature, enthalpy and cooperativity of the main phase transition at higher peptide concentrations, and abolishing the pretransition at lower peptide concentrations as compared to DMPC. Moreover, the relatively larger effects of GS on the thermotropic phase behavior of DMPG vesicles are also manifest without cycling through the phase transition or exposure to high temperatures. Furthermore, the addition of GS to DMPG vesicles protects the phospholipid molecules from the chemical hydrolysis induced by their repeated exposure to high temperatures. These results indicate that GS interacts more strongly with anionic than with zwitterionic phospholipid bilayers, probably because of the more favorable net attractive electrostatic interactions between the positively charged peptide and the negatively charged polar headgroup in such systems. Moreover, at comparable reduced temperatures, GS appears to interact more strongly with zwitterionic DMPC than with zwitterionic DMPE bilayers, probably because of the more fluid character of the former system. In addition, the general effects of GS on the thermotropic phase behavior of zwitterionic and anionic phospholipids suggest that it is located at the polar/apolar interface of liquid-crystalline bilayers, where it interacts primarily with the polar headgroup and glycerol-backbone regions of the phospholipid molecules and only secondarily with the lipid hydrocarbon chains. Finally, the considerable lipid specificity of GS interactions with phospholipid bilayers may prove useful in the design of peptide analogs with stronger interactions with microbial as opposed to eucaryotic membrane lipids.     

Interactions of the Australian tree frog antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with lipid model membranes: Differential scanning calorimetric and Fourier transform infrared spectroscopic studies

The interactions of the antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylethanolamine (DMPE) were studied by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. The effects of these peptides on the thermotropic phase behavior of DMPC and DMPG are qualitatively similar and manifested by the suppression of the pretransition, and by peptide concentration-dependent decreases in the temperature, cooperativity and enthalpy of the gel/liquid-crystalline phase transition. However, at all peptide concentrations, anionic DMPG bilayers are more strongly perturbed than zwitterionic DMPC bilayers, consistent with membrane surface charge being an important aspect of the interactions of these peptides with phospholipids. However, at all peptide concentrations, the perturbation of the thermotropic phase behavior of zwitterionic DMPE bilayers is weak and discernable only when samples are exposed to high temperatures. FTIR spectroscopy indicates that these peptides are unstructured in aqueous solution and that they fold into α-helices when incorporated into lipid membranes. All three peptides undergo rapid and extensive H-D exchange when incorporated into D₂O-hydrated phospholipid bilayers, suggesting that they are located in solvent-accessible environments, most probably in the polar/apolar interfacial regions of phospholipid bilayers. The perturbation of model lipid membranes by these peptides decreases in magnitude in the order maculatin 1.1 > aurein 1.2 > citropin 1.1, whereas the capacity to inhibit Acholeplasma laidlawii B growth decreases in the order maculatin 1.1 > aurein 1.2 ≅ citropin 1.1. The higher efficacy of maculatin 1.1 in disrupting model and biological membranes can be rationalized by its larger size and higher net charge. However, despite its smaller size and lower net charge, aurein 1.2 is more disruptive of model lipid membranes than citropin 1.1 and exhibits comparable antimicrobial activity, probably because aurein 1.2 has a higher propensity for partitioning into phospholipid membranes.     

Real-time quantitative analysis of lipid disordering by aurein 1.2 during membrane adsorption, destabilisation and lysis

Effective antimicrobial peptides (AMPs) distinguish between the host and microbial cells, show selective antimicrobial activity and exhibit a fast killing mechanism. Although understanding the structure-function characteristics of AMPs is important, the impact of the peptides on the architecture of membranes with different lipid compositions is also critical in understanding the molecular mechanism and specificity of membrane destabilisation. In this study, the destabilisation of supported lipid bilayers (SLBs) by the AMP aurein 1.2 was quantitatively analysed by dual polarisation interferometry. The lipid bilayers were formed on a planar silicon oxynitride chip, and composed of mixed synthetic lipids, or Escherichiacoli lipid extract. The molecular events leading sequentially from peptide adsorption to membrane lysis were examined in real time by changes in bilayer birefringence (lipid molecular ordering) as a function of membrane-bound peptide mass. Aurein 1.2 bound weakly without any change in membrane ordering at low peptide concentration (5 μM), indicating a surface-associated state without significant perturbation in membrane structure. At 10 μM peptide, marked reversible changes in molecular ordering were observed for all membranes except DMPE/DMPG. However, at 20 μM aurein 1.2, removal of lipid molecules, as determined by mass loss with a concomitant decrease in birefringence during the association phase, was observed for DMPC and DMPC/DMPG SLBs, which indicates membrane lysis by aurein. The membrane destabilisation induced by aurein 1.2 showed cooperativity at a particular peptide/lipid ratio with a critical mass/molecular ordering value. Furthermore, the extent of membrane lysis for DMPC/DMPG was nearly double that for DMPC. However, no lysis was observed for DMPC/DMPG/cholesterol, DMPE/DMPG and E. coli SLBs. The extent of birefringence changes with peptide mass suggested that aurein 1.2 binds to the membrane without inserting through the bilayer and membrane lysis occurs through detergent-like micellisation above a critical P/L ratio. Real-time quantitative analysis of the structural properties of membrane organisation has allowed the membrane destabilisation process to be resolved into multiple steps and provides comprehensive information to determine the molecular mechanism of aurein 1.2 action.     

Intramembrane Water Associated with TOAC Spin-Labeled Alamethicin: Electron Spin-Echo Envelope Modulation by D2O

Alamethicin is a 20-residue, hydrophobic, helical peptide, which forms voltage-sensitive ion channels in lipid membranes. The helicogenic, nitroxyl amino acid TOAC was substituted isosterically for Aib at residue positions 1, 8, or 16 in a F50/5 alamethicin analog to enable EPR studies. Electron spin-echo envelope modulation (ESEEM) spectroscopy was used to investigate the water exposure of TOAC-alamethicin introduced into membranes of saturated or unsaturated diacyl phosphatidylcholines that were dispersed in D₂O. Echo-detected EPR spectra were used to assess the degree of assembly of the peptide in the membrane, via the instantaneous diffusion from intermolecular spin-spin interactions. The profile of residue exposure to water differs between membranes of saturated and unsaturated lipids. In monounsaturated dioleoyl phosphatidylcholine, D₂O-ESEEM intensities decrease from TOAC¹ to TOAC⁸ and TOAC¹⁶ but not uniformly. This is consistent with a transmembrane orientation for the protoassembled state, in which TOAC¹⁶ is located in the bilayer leaflet opposite to that of TOAC¹ and TOAC⁸. Relative to the monomer in fluid bilayers, assembled alamethicin is disposed asymmetrically about the bilayer midplane. In saturated dimyristoyl phosphatidylcholine, the D₂O-ESEEM intensity is greatest for TOAC⁸, indicating a more superficial location for alamethicin, which correlates with the difference in orientation between gel- and fluid-phase membranes found by conventional EPR of TOAC-alamethicin in aligned phosphatidylcholine bilayers. Increasing alamethicin/lipid ratio in saturated phosphatidylcholine shifts the profile of water exposure toward that with unsaturated lipid, consistent with proposals of a critical concentration for switching between the two different membrane-associated states.     

Structure and fluctuations of charged phosphatidylserine bilayers in the absence of salt

Using x-ray diffraction and NMR spectroscopy, we present structural and material properties of phosphatidylserine (PS) bilayers that may account for the well documented implications of PS headgroups in cell activity. At 30 degrees C, the 18-carbon monounsaturated DOPS in the fluid state has a cross-sectional area of 65.3 A(2) which is remarkably smaller than the area 72.5 A(2) of the DOPC analog, despite the extra electrostatic repulsion expected for charged PS headgroups. Similarly, at 20 degrees C, the 14-carbon disaturated DMPS in the gel phase has an area of 40.8 A(2) vs. 48.1 A(2) for DMPC. This condensation of area suggests an extra attractive interaction, perhaps hydrogen bonding, between PS headgroups. Unlike zwitterionic lipids, stacks of PS bilayers swell indefinitely as water is added. Data obtained for osmotic pressure versus interbilayer water spacing for fluid phase DOPS are well fit by electrostatic interactions calculated for the Gouy-Chapman regime. It is shown that the electrostatic interactions completely dominate the fluctuational pressure. Nevertheless, the x-ray data definitively exhibit the effects of fluctuations in fluid phase DOPS. From our measurements of fluctuations, we obtain the product of the bilayer bending modulus K(C) and the smectic compression modulus B. At the same interbilayer separation, the interbilayer fluctuations are smaller in DOPS than for DOPC, showing that B and/or K(C) are larger. Complementing the x-ray data, (31)P-chemical shift anisotropy measured by NMR suggest that the DOPS headgroups are less sensitive to osmotic pressure than DOPC headgroups, which is consistent with a larger K(C) in DOPS. Quadrupolar splittings for D(2)O decay less rapidly with increasing water content for DOPS than for DOPC, indicating greater perturbation of interlamellar water and suggesting a greater interlamellar hydration force in DOPS. Our comparisons between bilayers of PS and PC lipids with the same chains and the same temperature enable us to focus on the effects of these headgroups on bilayer properties.     

Interactions of the Australian tree frog antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with lipid model membranes: differential scanning calorimetric and Fourier transform infrared spectroscopic studies

The interactions of the antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylethanolamine (DMPE) were studied by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. The effects of these peptides on the thermotropic phase behavior of DMPC and DMPG are qualitatively similar and manifested by the suppression of the pretransition, and by peptide concentration-dependent decreases in the temperature, cooperativity and enthalpy of the gel/liquid-crystalline phase transition. However, at all peptide concentrations, anionic DMPG bilayers are more strongly perturbed than zwitterionic DMPC bilayers, consistent with membrane surface charge being an important aspect of the interactions of these peptides with phospholipids. However, at all peptide concentrations, the perturbation of the thermotropic phase behavior of zwitterionic DMPE bilayers is weak and discernable only when samples are exposed to high temperatures. FTIR spectroscopy indicates that these peptides are unstructured in aqueous solution and that they fold into alpha-helices when incorporated into lipid membranes. All three peptides undergo rapid and extensive H-D exchange when incorporated into D(2)O-hydrated phospholipid bilayers, suggesting that they are located in solvent-accessible environments, most probably in the polar/apolar interfacial regions of phospholipid bilayers. The perturbation of model lipid membranes by these peptides decreases in magnitude in the order maculatin 1.1>aurein 1.2>citropin 1.1, whereas the capacity to inhibit Acholeplasma laidlawii B growth decreases in the order maculatin 1.1>aurein 1.2 congruent with citropin 1.1. The higher efficacy of maculatin 1.1 in disrupting model and biological membranes can be rationalized by its larger size and higher net charge. However, despite its smaller size and lower net charge, aurein 1.2 is more disruptive of model lipid membranes than citropin 1.1 and exhibits comparable antimicrobial activity, probably because aurein 1.2 has a higher propensity for partitioning into phospholipid membranes.     

The role of electrostatic interactions in the membrane binding of melittin

The binding of melittin and the C-terminally truncated analogue of melittin (21Q) to a range of phospholipid bilayers was studied using surface plasmon resonance (SPR). The phospholipid model membranes included zwitterionic dimyristylphosphatidylcholine (DMPC) and dimyristylphosphatidylethanolamine (DMPE), together with mixtures DMPC/dimyristylphosphatidylglycerol (DMPG), DMPC/DMPG/cholesterol and DMPE/DMPG. Melittin bound rapidly to all membrane mixtures, whereas 21Q, which has a reduced charge, bound much more slowly on the DMPC and DMPC/DMPG mixtures reflecting the role of the initial electrostatic interaction. The loss of the cationic residues also significantly decreased the binding of 21Q with DMPC/DMPG/Cholesterol, DMPE and DMPE/DMPG. The role of electrostatics was also highlighted with NaCl in the buffer, which affected the way melittin bound to the different membranes, causing a more uniform, concentration dependant increase in response. The biosensor results were correlated with the conformation of the peptides determined by circular dichroism analysis, which indicated that high α-helicity was associated with high binding affinity. Overall, the results demonstrate that the positively charged residues at the C-terminus of melittin play an essential role in membrane binding, that modulation of peptide charge influences selectivity of binding to different phospholipids and that manipulation of the cationic regions of antimicrobial peptides can be used to modulate membrane selectivity.     

Modulation of gramicidin channel conformation and organization by hydrophobic mismatch in saturated phosphatidylcholine bilayers

The matching of hydrophobic lengths of integral membrane proteins and the surrounding lipid bilayer is an important factor that influences both structure and function of integral membrane proteins. The ion channel gramicidin is known to be uniquely sensitive to membrane properties such as bilayer thickness and membrane mechanical properties. The functionally important carboxy terminal tryptophan residues of gramicidin display conformation-dependent fluorescence which can be used to monitor gramicidin conformations in membranes [S.S. Rawat, D.A. Kelkar, A. Chattopadhyay, Monitoring gramicidin conformations in membranes: a fluorescence approach, Biophys. J. 87 (2004) 831-843]. We have examined the effect of hydrophobic mismatch on the conformation and organization of gramicidin in saturated phosphatidylcholine bilayers of varying thickness utilizing the intrinsic conformation-dependent tryptophan fluorescence. Our results utilizing steady state and time-resolved fluorescence spectroscopic approaches, in combination with circular dichroism spectroscopy, show that gramicidin remains predominantly in the channel conformation and gramicidin tryptophans are at the membrane interfacial region over a range of mismatch conditions. Interestingly, gramicidin conformation shifts toward non-channel conformations in extremely thick gel phase membranes although it is not excluded from the membrane. In addition, experiments utilizing self quenching of tryptophan fluorescence indicate peptide aggregation in thicker gel phase membranes.     

Interaction of the neuropeptide met-enkephalin with zwitterionic and negatively charged bicelles as viewed by 31P and 2H solid-state NMR

The interaction of the neuropeptide methionine-enkephalin (Menk) with bicelles was investigated by solid-state NMR. Bicelles composed of dimyristoylphosphatidylcholine (DMPC) and dicaproylphosphatidylcholine (DCPC) were modified to investigate the effect of the lipid headgroup and electrostatic charges on the association with Menk. A total of 10 mol % of DMPC was replaced by zwitterionic phosphatidylethanolamine (DMPE), anionic phosphatidylglycerol (DMPG), or phosphatidylserine (DMPS). The preparation of DMPE-doped bicelles (Bic/PE) is reported for the first time. The (31)P and (2)H NMR results revealed changes in the lipid dynamics when Menk interacts with the bicellar systems. (2)H NMR experiments showed a disordering effect of Menk on the lipid chains in all the bicelles except Bic/PG, whereas the study of the choline headgroups indicated a decreased order of the lipids only in Bic/PE and Bic/PG. Our results suggest that the insertion depth of Menk into bicelles is modulated by their composition, more specifically by the balance between hydrophobic and electrostatic interactions. Menk would be buried at the lipid polar/apolar interface, the depth of penetration into the hydrophobic membrane core following the scaling Bic > Bic/PE > Bic/PS at the slightly acidic pH used in this study. The peptide would not insert into the bilayer core of Bic/PG and would rather remain at the surface.     

Dynamic properties of gramicidin A in phospholipid membranes

The flexibility of the tryptophan side chains of gramicidin A and the rotational diffusion of the peptide in methanolic solution and in three membrane systems were studied with deuterium nuclear magnetic resonance (NMR). Gramicidin A was selectively deuterated at the aromatic ring systems of its four tryptophan side chains. In methanolic solution, the tryptophan residues remained immobile and served as a probe for the overall rotation of the peptide. The experimentally determined rotational correlation time of tau c = 0.6 X 10(-9) s was consistent with the formation of gramicidin A dimers. For gramicidin A incorporated into bilayer membranes, quite different results were obtained depending on the chemical and physical nature of the lipids employed. When mixed with 1-palmitoyl-sn-glycero-3-phosphocholine (LPPC) at a stoichiometric lipid:peptide ratio of 4:1, gramicidin A induced the formation of stable bilayer membranes in which the lipids were highly fluid. In contrast, the gramicidin A molecules of this membrane remained completely static over a large temperature interval, suggesting strong protein-protein interactions. The peptide molecules appeared to form a rigid two-dimensional lattice in which the interstitial spaces were filled with fluidlike lipids. When gramicidin A was incorporated into bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) above the lipid phase transition, the deuterium NMR spectra were motionally narrowed, indicating large-amplitude rotational fluctuations. From the measurement of the quadrupole echo relaxation time, a rotational correlation time of 2 X 10(-7) s was estimated, leading to a membrane viscosity of 1-2 P if the rotational unit was assumed to be a gramicidin A dimer. (ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphatidylethanolamine enhances the concentration-dependent exchange of phospholipids between bilayers

It has previously been demonstrated that lipid exchange between phosphatidylcholine vesicles, at higher concentrations, is characterized by a second-order concentration-dependent exchange process in addition to the first-order process operative at lower concentrations (Jones, J. D., & Thompson, T. E. (1989) Biochemistry 28, 129-134). Furthermore, it was demonstrated that the second-order process occurs as a result of an enhancement of the first-order desorption process, possibly resulting from attractive interactions between a potentially desorbing lipid molecule and a transiently apposed bilayer (Jones, J. D., & Thompson, T. E. (1990) Biochemistry 29, 1593-1600). In this work we have studied the exchange of [3H]dimyristoylphosphatidylcholine (DMPC) between large vesicles of the compositions 100% DMPC, 70/30 (mol/mol) DMPC/dimyristoylphosphatidylethanolamine (DMPE), and 68.25/30/1.75 (mol/mol/mol) DMPC/DMPE/dimyristoylphosphatidylglycerol (DMPG). The second-order exchange process is enhanced by 100-fold or more in vesicles containing 30 mol % DMPE relative to 100% DMPC and is reduced or eliminated by the addition of 1.75% of the anionic lipid DMPG. These effects can be achieved by alterations in the equilibrium bilayer separation of 5 A or less. The results are in accord with the model of Jones and Thompson and indicate that relatively low concentrations of PE in a PC bilayer can have significant effects on bilayer surface properties and on potential interactions between bilayers.     

Biophysical properties of membrane lipids of anammox bacteria: I. Ladderane phospholipids form highly organized fluid membranes

Anammox bacteria that are capable of anaerobically oxidizing ammonium (anammox) with nitrite to nitrogen gas produce unique membrane phospholipids that comprise hydrocarbon chains with three or five linearly condensed cyclobutane rings. To gain insight into the biophysical properties of these ‘ladderane’ lipids, we have isolated a ladderane phosphatidylcholine and a mixed ladderane phosphatidylethanolamine/phosphatidylglycerol lipid fraction and reconstituted these lipids in different membrane environments. Langmuir monolayer experiments demonstrated that the purified ladderane phospholipids form fluid films with a relatively high lipid packing density. Fluid-like behavior was also observed for ladderane lipids in bilayer systems as monitored by cryo-electron microscopy on large unilamellar vesicles (LUVs) and epi-fluorescence microscopy on giant unilamellar vesicles (GUVs). Analysis of the LUVs by fluorescence depolarization revealed a relatively high acyl chain ordering in the hydrophobic region of the ladderane phospholipids. Micropipette aspiration experiments were applied to study the mechanical properties of ladderane containing lipid bilayers and showed a relatively high apparent area compressibility modulus for ladderane containing GUVs, thereby confirming the fluid and acyl chain ordered characteristics of these lipids. The biophysical findings in this study support the previous postulation that dense membranes in anammox cells protect these microbes against the highly toxic and volatile anammox metabolites.     

The membrane insertion of helical antimicrobial peptides from the N-terminus of Helicobacter pylori ribosomal protein L1

The interaction of two helical antimicrobial peptides, HPA3 and HPA3P with planar supported lipid membranes was quantitatively analysed using two complementary optical biosensors. The peptides are analogues of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1). The binding of these two peptide analogues to zwitterionic dimyristoyl-phosphatidylcholine (DMPC) and negatively charged membranes composed of DMPC/dimyristoylphosphatidylglycerol (DMPG) (4:1) was determined using surface plasmon resonance (SPR) and dual polarisation interferometry (DPI). Using SPR analysis, it was shown that the proline substitution in HPA3P resulted in much lower binding for both zwitterionic and anionic membranes than HPA3. Structural changes in the planar DMPC and DMPC/DMPG (4:1) bilayers induced by the binding of both Hp(2-20) analogues were then resolved in real-time with DPI. The overall process of peptide-induced changes in membrane structure was analysed by the real-time changes in bound peptide mass as a function of bilayer birefringence. The insertion of both HPA3 and HPA3P into the supported lipid bilayers resulted in a decrease in birefringence with increasing amounts of bound peptide which reflects a decrease in the order of the bilayer. The binding of HPA3 to each membrane was associated with a higher level of bound peptide and greater membrane lipid disordering and a faster and higher degree of insertion into the membrane than HPA3P. Furthermore, the binding of both HPA3 and HPA3P to negatively charged DMPC/DMPG bilayers also leads to a greater disruption of the lipid ordering. These results demonstrate the geometrical changes in the membrane upon peptide insertion and the extent of membrane structural changes can be obtained quantitatively. Moreover, monitoring the effect of peptides on a structurally characterised bilayer has provided further insight into the role of membrane structure changes in the molecular basis of peptide selectivity and activity and may assist in defining the mode of antimicrobial action.     

Effect of Membrane Composition on Antimicrobial Peptides Aurein 2.2 and 2.3 From Australian Southern Bell Frogs

The effects of hydrophobic thickness and the molar phosphatidylglycerol (PG) content of lipid bilayers on the structure and membrane interaction of three cationic antimicrobial peptides were examined: aurein 2.2, aurein 2.3 (almost identical to aurein 2.2, except for a point mutation at residue 13), and a carboxy C-terminal analog of aurein 2.3. Circular dichroism results indicated that all three peptides adopt an α-helical structure in the presence of a 3:1 molar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPC/DMPG), and 1:1 and 3:1 molar mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG). Oriented circular dichroism data for three different lipid compositions showed that all three peptides were surface-adsorbed at low peptide concentrations, but were inserted into the membrane at higher peptide concentrations. The ³¹P solid-state NMR data of the three peptides in the DMPC/DMPG and POPC/POPG bilayers showed that all three peptides significantly perturbed lipid headgroups, in a peptide or lipid composition-dependent manner. Differential scanning calorimetry results demonstrated that both amidated aurein peptides perturbed the overall phase structure of DMPC/DMPG bilayers, but perturbed the POPC/POPG chains less. The nature of the perturbation of DMPC/DMPG bilayers was most likely micellization, and for the POPC/POPG bilayers, distorted toroidal pores or localized membrane aggregate formation. Calcein release assay results showed that aurein peptide-induced membrane leakage was more severe in DMPC/DMPG liposomes than in POPC/POPG liposomes, and that aurein 2.2 induced higher calcein release than aurein 2.3 and aurein 2.3-COOH from 1:1 and 3:1 POPC/POPG liposomes. Finally, DiSC₃5 assay data further delineated aurein 2.2 from the others by showing that it perturbed the lipid membranes of intact S. aureus C622 most efficiently, whereas aurein 2.3 had the same efficiency as gramicidin S, and aurein 2.3-COOH was the least efficient. Taken together, these data show that the membrane interactions of aurein peptides are affected by the hydrophobic thickness of the lipid bilayers and the PG content.     

Effect of arbutin on the dipole potential and area per lipid of ester and ether phosphatidylcholine and phosphatidyl ethanolamine monolayers

The present results report for the first time a systematic study of the effect of arbutin on the dipole potential of lipid membranes. The dipole potential and the area per lipid were measured in monolayers of dimyristoylphosphatidylcholine (DMPC), 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (dietherPC), dimyristoylphosphatidylethanolamine (DMPE) and 1,2-di-O-tetradecyl-sn-glycero-3-phosphoethanolamine (dietherPE), spread on aqueous solutions of different concentrations of arbutin. The decrease of the dipole potential of DMPC, both in condensed and expanded monolayers, is parallel to an increase in the area per lipid. In contrast, for dietherPC, the area per lipid is not affected, in spite of the fact that arbutin is also able to decrease the dipole potential in a less drastic extent. In the case of DMPE, the response is similar to that observed with dietherPC: the dipole potential decreases, while the area per lipid remains unchanged. However, when the carbonyl groups are absent in phosphatidylethanolamine derivatives such as the dietherPE, the dipole potential is not affected by arbutin, with a small decrease in the area. The effect of arbutin on the dipole potential differs from that of sucrose, trehalose and phloretin and is congruent with previous results obtained by FTIR on its interaction with the CO groups. Arbutin binding is interpreted in terms of the exposure to water of the phosphate and carbonyl groups at the membrane interface of the different monolayers.     

Bilayer interactions of pHLIP, a peptide that can deliver drugs and target tumors

The pH-dependent insertion of pHLIP across membranes is proving to be a useful property for targeting acidic tissues or tumors and delivering drugs attached to its C-terminus. It also serves as a model peptide for studies of protein insertion into membranes, so further elucidation of the insertion mechanism of pHLIP and its features is desirable. We examine how the peptide perturbs a model phosphatidylcholine membrane and how it associates with the lipid bilayer using an array of fluorescence techniques, including fluorescence anisotropy measurements of TMA-DPH anchored in bilayers, quenching of pHLIP fluorescence by brominated lipids and acrylamide, and measurements of energy transfer between aromatic residues of pHLIP and TMA-DPH. When pHLIP is bound to the surface of bilayers near neutral pH, the membrane integrity is preserved whereas the elastic properties of bilayers are changed as reported by an increase of membrane viscosity. When it is inserted, there is little perturbation of the lipids. The results also suggest that pHLIP can bind to the membrane surface in a shallow or a deep mode depending on the phase state of the lipids. Using parallax analysis, the change of the penetration depth of pHLIP was estimated to be 0.4 Å from the bilayer center and 2.8 Å from the membrane surface after the liquid-to-gel phase transition.     

Structural effects of the antimicrobial peptide maculatin 1.1 on supported lipid bilayers

The interactions of the antimicrobial peptide maculatin 1.1 (GLFGVLAKVAAHVVPAIAEHF-NH₂) with model phospholipid membranes were studied by use of dual polarisation interferometry and neutron reflectometry and dimyristoylphosphatidylcholine (DMPC) and mixed DMPC–dimyristoylphosphatidylglycerol (DMPG)-supported lipid bilayers chosen to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC bilayers concentration-dependent binding and increasing perturbation of bilayer order by maculatin were observed. By contrast, in mixed DMPC–DMPG bilayers, maculatin interacted more strongly and in a concentration-dependent manner with retention of bilayer lipid order and structure, consistent with pore formation. These results emphasise the importance of membrane charge in mediating antimicrobial peptide activity and emphasise the importance of using complementary methods of analysis in probing the mode of action of antimicrobial peptides.     

Anionic phospholipids inhibit apolipoprotein E--low-density lipoprotein receptor interactions

Apolipoprotein E (apoE) is a ligand for members of the low-density lipoprotein receptor (LDLR) family. Lipid-free apoE is not recognized by LDLR, yet interaction with lipid confers receptor recognition properties. Although lipid interaction is known to induce a conformational change in apoE, it is not known if the lipid composition of the resulting complex influences binding. Using reconstituted lipoprotein particles of apoE3 N-terminal (NT) domain and dimyristoylphosphatidylcholine (DMPC), maximal LDLR binding was observed at DMPC:apoE3-NT ratios >2.5:1 (w/w). ApoE3-NT lipid particles prepared with egg sphingomyelin were functional as LDLR ligands while complexes formed with the anionic phospholipids dimyristoylphosphatidylglycerol or dimyristoylphosphatidylserine (DMPS) were not. In the case of apoE3-NT, lipid particles comprised of a mixture of DMPC and DMPS, a DMPS concentration dependent inhibition of LDLR binding activity was observed. Thus, in addition to affecting apoE conformational status, the lipid composition of ligand particles can modulate LDLR binding activity.     

Effect of Lipid Head Groups on Double-Layered Two-Dimensional Crystals Formed by Aquaporin-0

Aquaporin-0 (AQP0) is a lens-specific water channel that also forms membrane junctions. Reconstitution of AQP0 with dimyristoyl phosphatidylcholine (DMPC) and E. coli polar lipids (EPL) yielded well-ordered, double-layered two-dimensional (2D) crystals that allowed electron crystallographic structure determination of the AQP0-mediated membrane junction. The interacting tetramers in the two crystalline layers are exactly in register, resulting in crystals with p422 symmetry. The high-resolution density maps also allowed modeling of the annular lipids surrounding the tetramers. Comparison of the DMPC and EPL bilayers suggested that the lipid head groups do not play an important role in the interaction of annular lipids with AQP0. We now reconstituted AQP0 with the anionic lipid dimyristoyl phosphatidylglycerol (DMPG), which yielded a mixture of 2D crystals with different symmetries. The different crystal symmetries result from shifts between the two crystalline layers, suggesting that the negatively charged PG head group destabilizes the interaction between the extracellular AQP0 surfaces. Reconstitution of AQP0 with dimyristoyl phosphatidylserine (DMPS), another anionic lipid, yielded crystals that had the usual p422 symmetry, but the crystals showed a pH-dependent tendency to stack through their cytoplasmic surfaces. Finally, AQP0 failed to reconstitute into membranes that were composed of more than 40% dimyristoyl phosphatidic acid (DMPA). Hence, although DMPG, DMPS, and DMPA are all negatively charged lipids, they have very different effects on AQP0 2D crystals, illustrating the importance of the specific lipid head group chemistry beyond its mere charge.