Insights into buforin II membrane translocation from molecular dynamics simulations

Buforin II is a histone-derived antimicrobial peptide that readily translocates across lipid membranes without causing significant membrane permeabilization. Previous studies showed that mutating the sole proline of buforin II dramatically decreases its translocation. As well, researchers have proposed that the peptide crosses membranes in a cooperative manner by forming transient toroidal pores. This paper reports molecular dynamics simulations designed to investigate the structure of buforin II upon membrane entry and evaluate whether the peptide is able to form toroidal pore structures. These simulations showed a relationship between protein–lipid interactions and increased structural deformations of the buforin N-terminal region promoted by proline. Moreover, simulations with multiple peptides show how buforin II can embed deeply into membranes and potentially form toroidal pores. Together, these simulations provide structural insight into the translocation process for buforin II in addition to providing more general insight into the role proline can play in antimicrobial peptides.     

Structure and Orientation of the Mammalian Antibacterial Peptide Cecropin P1 within Phospholipid Membranes

Cecropins are positively charged antibacterial peptides that act by permeating the membrane of susceptible bacteria. To gain insight into the mechanism of membrane permeation, the secondary structure and the orientation within phospholipid membranes of the mammalian cecropin P1 (CecP) was studied using attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy and molecular dynamics simulations. The shape and frequency of the amide I and II absorption peaks of CecP within acidic PE/PG multibilayers (phosphatidylethanolamine/phosphatidylglycerol) in a 7:3 (w/w) ratio (a phospholipid composition similar to that of many bacterial membranes), indicated that the peptide is predominantly α-helical. Polarized ATR-FTIR spectroscopy was used to determine the orientation of the peptide relative to the bilayer normal of phospholipid multibilayers. The ATR dichroic ratio of the amide I band of CecP peptide reconstituted into oriented PE/PG phospholipid membranes indicated that the peptide is preferentially oriented nearly parallel to the surface of the lipid membranes. A similar secondary structure and orientation were found when zwitterionic phosphatidylcholine phospho lipids were used. The incorporation of CecP did not significantly change the order parameters of the acyl chains of the multibilayer, further suggesting that CecP does not penetrate the hydrocarbon core of the membranes. Molecular dynamics simulations were used to gain insight into possible effects of transmembrane potential on the orientation of CecP relative to the membrane. The simulations appear to confirm that CecP adopts an orientation parallel to the membrane surface and does not insert into the bilayer in response to acispositive transmembrane voltage difference. Taken together, the results further support a “carpet-like” mechanism, rather than the formation of transmembrane pores, as the mode of action of CecP. According to this model, formation of a layer of peptide monomers on the membrane surface destablizes the phospholipid packing of the membrane leading to its eventual disintegration.     

Biophysical Investigation of the Membrane-Disrupting Mechanism of the Antimicrobial and Amyloid-Like Peptide Dermaseptin S9

Dermaseptin S9 (Drs S9) is an atypical cationic antimicrobial peptide with a long hydrophobic core and with a propensity to form amyloid-like fibrils. Here we investigated its membrane interaction using a variety of biophysical techniques. Rather surprisingly, we found that Drs S9 induces efficient permeabilisation in zwitterionic phosphatidylcholine (PC) vesicles, but not in anionic phosphatidylglycerol (PG) vesicles. We also found that the peptide inserts more efficiently in PC than in PG monolayers. Therefore, electrostatic interactions between the cationic Drs S9 and anionic membranes cannot explain the selectivity of the peptide towards bacterial membranes. CD spectroscopy, electron microscopy and ThT fluorescence experiments showed that the peptide adopts slightly more β-sheet and has a higher tendency to form amyloid-like fibrils in the presence of PC membranes as compared to PG membranes. Thus, induction of leakage may be related to peptide aggregation. The use of a pre-incorporation protocol to reduce peptide/peptide interactions characteristic of aggregates in solution resulted in more α-helix formation and a more pronounced effect on the cooperativity of the gel-fluid lipid phase transition in all lipid systems tested. Calorimetric data together with ²H- and ³¹P-NMR experiments indicated that the peptide has a significant impact on the dynamic organization of lipid bilayers, albeit slightly less for zwitterionic than for anionic membranes. Taken together, our data suggest that in particular in membranes of zwitterionic lipids the peptide binds in an aggregated state resulting in membrane leakage. We propose that also the antimicrobial activity of Drs S9 may be a result of binding of the peptide in an aggregated state, but that specific binding and aggregation to bacterial membranes is regulated not by anionic lipids but by as yet unknown factors.     

Antimicrobial selectivity based on zwitterionic lipids and underlying balance of interactions

An important feature of antimicrobial peptides is their ability to distinguish pro- from eukaryotic membranes. In vitro experiments on the antimicrobial peptide NK-2 indicate that the discrimination between zwitterionic phosphatidylethanolamine lipids exposed by prokaryotes and phosphatidylcholine lipids exposed by eukaryotes plays an important role. The underlying mechanism is not understood. Here we present molecular dynamics simulations in conjunction with a coarse grained model and thermodynamic integration showing that NK-2 binds more strongly to palmitoyloleoylphosphatidylethanolamine (POPE) than to palmitoyloleoylphosphatidylcholine (POPC) bilayers. Finite size effects on the relative free energy have been corrected for with a method that may also be useful in future studies of the affinities of macromolecules for lipid membranes. Our results support the previous hypothesis that the stronger binding to PE compared to PC arises from a better accessibility of the phosphates of the lipids to the cationic peptide in a sense that a similar number of peptide-lipid salt bridges requires to break more favorable electrostatic headgroup-headgroup interactions for PC relative to PE. The transfer of NK-2 from POPC to POPE is found to lead to a decrease in electrostatic peptide-lipid but an increase in lipid-lipid and ion-lipid interactions, correlating with a dehydration of the lipids and the ions but an increased hydration of the peptide. The increase in affinity of NK-2 for POPE compared to POPC hence arises from a complex interplay of competing interactions. This work opens the perspective to study how the affinity of antimicrobial peptides changes with amino acid sequence and lipid composition.     

Effect of drastic sequence alteration and D-amino acid incorporation on the membrane binding behavior of lytic peptides

The amphipathic alpha-helix is a common motif found in many cell lytic peptides including antimicrobial peptides. We have recently shown that significantly altering the amphipathic structure of a lytic peptide by reshuffling its sequence and/or replacing a few l-amino acids with their D-enantiomers did not significantly affect the antimicrobial activity of the peptides nor their ability to bind and permeate negatively charged (PE/PG) membranes. However, a pronounced effect was observed regarding their hemolytic activity and their ability to bind and permeate zwitterionic (PC/Cho) membranes. To shed light on these findings, here we used surface plasmon resonance (SPR) with mono- and bilayer membranes. We found that the L-amino acid (aa) peptides bound 10-25-fold stronger to PC/Cho bilayers compared with monolayers, whereas the diastereomers bound similarly to both membranes. A two-state reaction model analysis of the data indicated that this difference is due to the insertion of the L-aa peptides into the PC/Cho bilayers, whereas the diastereomers are surface-localized. In contrast, only an approximately 2-fold difference was found with negatively charged membranes. Changes in the amphipathicity markedly affected only the insertion of the L-aa peptides into PC/Cho bilayers. Furthermore, whereas the all-L-aa peptides bound similarly to the PC/Cho and PE/PG membranes, the diastereomers bound approximately 100-fold better to PE/PG compared with PC/Cho membranes, and selectivity was determined only in the first binding step. The effect of the peptides on the lipid order determined by using ATR-FTIR studies supported these findings. Besides shedding light on the mode of action of these peptides, the present study demonstrates SPR as a powerful tool to differentiate between non-cell-selective compared with bacteria-selective peptides, based on differences in their membrane binding behavior.     

A spectroscopic study of the membrane interaction of the antimicrobial peptide Pleurocidin

The cationic amphipathic alpha-helical antibiotic peptide, pleurocidin, from the winter flounder Pleuronectes americanus associates strongly with anionic membranes where it is able to translocate across the membrane, cause dye leakage from vesicles and induce pore like channel conductance. To investigate the mechanism of pleurocidin antibiotic activity in more detail we have applied a variety of spectroscopic techniques to study the interaction of pleurocidin with model membranes. At neutral pH the peptide inserts into membranes containing anionic lipids and, as shown by proton-decoupled 15N solid-state NMR spectroscopy of macroscopically oriented samples, is aligned parallel to the membrane surface. 2H solid-state NMR spectroscopy of chain deuterated phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) lipids in mixed membranes shows that pleurocidin interacts with both the zwitterionic PE and anionic PG but disrupts the lipid acyl chain order of the anionic PG lipids more effectively. At acidic pH the three histidine residues of pleurocidin become protonated and positively charged which does not alter the membrane disrupting effect nor the location of the peptide in the membrane. The results are interpreted in terms of a structural model for pleurocidin inserted into anionic lipid membranes and the implications of our data are discussed in terms of a general mechanism for the antibiotic activity.     

Activity and characterization of a pH-sensitive antimicrobial peptide

Antimicrobial peptides (AMPs) have been an area of great interest, due to the high selectivity of these molecules toward bacterial targets over host cells and the limited development of bacterial resistance to these molecules throughout evolution. Previous work showed that when Histidine was incorporated into the peptide C18G it lost antimicrobial activity. The role of pH on activity and biophysical properties of the peptide was investigated to explain this phenomenon. Minimal inhibitory concentration (MIC) results demonstrated that decreased media pH increased antimicrobial activity. Trichloroethanol (TCE) quenching and red-edge excitation spectroscopy (REES) showed a clear pH dependence on peptide aggregation in solution. Trp fluorescence was used to monitor binding to lipid vesicles and demonstrated the peptide binds to anionic bilayers at all pH values tested, however, binding to zwitterionic bilayers was enhanced at pH 7 and 8 (above the His pKa). Dual Quencher Analysis (DQA) confirmed the peptide inserted more deeply in PC:PG and PE:PG membranes, but could insert into PC bilayers at pH conditions above the His pKa. Bacterial membrane permeabilization assays which showed enhanced membrane permeabilization at pH 5 and 6 but vesicle leakage assays indicate enhanced permeabilization of PC and PC:PG bilayers at neutral pH. The results indicate the ionization of the His side chain affects the aggregation state of the peptide in solution and the conformation the peptide adopts when bound to bilayers, but there are likely more subtle influences of lipid composition and properties that impact the ability of the peptide to form pores in membranes.     

Interactions of the novel antimicrobial peptide buforin 2 with lipid bilayers: proline as a translocation promoting factor

Buforin 2 is an antimicrobial peptide discovered in the stomach tissue of the Asian toad Bufo bufo gargarizans. The 21-residue peptide with +6 net charge shows antimicrobial activity an order of magnitude higher than that of magainin 2, a membrane-permeabilizing antimicrobial peptide from Xenopus laevis [Park, C. B., Kim, M. S., and Kim, S. C. (1996) Biochem. Biophys. Res. Commun. 218, 408-413]. In this study, we investigated the interactions of buforin 2 with phospholipid bilayers in comparison with magainin 2 to obtain insight into the mechanism of action of buforin 2. Equipotent Trp-substituted peptides were used to fluorometrically monitor peptide-lipid interactions. Circular dichroism measurements showed that buforin 2 selectively bound to liposomes composed of acidic phospholipids, assuming a secondary structure similar to that in trifluoroethanol/water, which is an amphipathic helix distorted around Pro(11) with a flexible N-terminal region [Yi, G. S., Park, C. B., Kim, S. C., and Cheong, C. (1996) FEBS Lett. 398, 87-90]. Magainin 2 induced the leakage of a fluorescent dye entrapped within lipid vesicles coupled to lipid flip-flop. These results have been interpreted as the formation of a peptide-lipid supramolecular complex pore [Matsuzaki, K. (1998) Biochim. Biophys. Acta 1376, 391-400]. Buforin 2 exhibited much weaker membrane permeabilization activity despite its higher antimicrobial activity. In contrast, buforin 2 was more efficiently translocated across lipid bilayers than magainin 2. These results suggested that the ultimate target of buforin 2 is not the membrane but intracellular components. Furthermore, buforin 2 induced no lipid flip-flop, indicating that the mechanism of translocation of buforin 2 is different from that of magainin 2. The role of Pro was investigated by use of a P11A derivative of buforin 2. The derivation caused a change to magainin 2-like secondary structure and membrane behavior. Pro(11) was found to be a very important structural factor for the unique properties of buforin 2.     

Solid-state NMR investigation of the selective perturbation of lipid bilayers by the cyclic antimicrobial peptide RTD-1

RTD-1 is a cyclic beta-hairpin antimicrobial peptide isolated from rhesus macaque leukocytes. Using (31)P, (2)H, (13)C, and (15)N solid-state NMR, we investigated the interaction of RTD-1 with lipid bilayers of different compositions. (31)P and (2)H NMR of uniaxially oriented membranes provided valuable information about how RTD-1 affects the static and dynamic disorder of the bilayer. Toward phosphatidylcholine (PC) bilayers, RTD-1 causes moderate orientational disorder independent of the bilayer thickness, suggesting that RTD-1 binds to the surface of PC bilayers without perturbing its hydrophobic core. Addition of cholesterol to the POPC membrane does not affect the orientational disorder. In contrast, binding of RTD-1 to anionic bilayers containing PC and phosphatidylglycerol lipids induces much greater orientational disorder without affecting the dynamic disorder of the membrane. These correlate with the selectivity of RTD-1 for anionic bacterial membranes as opposed to cholesterol-rich zwitterionic mammalian membranes. Line shape simulations indicate that RTD-1 induces the formation of micrometer-diameter lipid cylinders in anionic membranes. The curvature stress induced by RTD-1 may underlie the antimicrobial activity of RTD-1. (13)C and (15)N anisotropic chemical shifts of RTD-1 in oriented PC bilayers indicate that the peptide adopts a distribution of orientations relative to the magnetic field. This is most likely due to a small fraction of lipid cylinders that change the RTD-1 orientation with respect to the magnetic field. Membrane-bound RTD-1 exhibits narrow line widths in magic-angle spinning spectra, but the sideband intensities indicate rigid-limit anisotropies. These suggest that RTD-1 has a well-defined secondary structure and is likely aggregated in the membrane. These structural and dynamical features of RTD-1 differ significantly from those of PG-1, a related beta-hairpin antimicrobial peptide.     

Structural contributions to the intracellular targeting strategies of antimicrobial peptides

The interactions of cationic amphipathic antimicrobial peptides (AMPs) with anionic biological membranes have been the focus of much research aimed at improving the activity of such compounds in the search for therapeutic leads. However, many of these peptides are thought to have other polyanions, such as DNA or RNA, as their ultimate target. Here a combination of fluorescence and circular dichroism (CD) spectroscopies has been used to assess the structural properties of amidated versions of buforin II, pleurocidin and magainin 2 that support their varying abilities to translocate through bacterial membranes and bind to double stranded DNA. Unlike magainin 2 amide, a prototypical membrane disruptive AMP, buforin II amide adopts a poorly helical structure in membranes closely mimicking the composition of Gram negative bacteria, such as Escherichia coli, and binds to a short duplex DNA sequence with high affinity, ultimately forming peptide-DNA condensates. The binding affinities of the peptides to duplex DNA are shown to be related to the structural changes that they induce. Furthermore, CD also reveals the conformation of the bound peptide buforin II amide. In contrast with a synthetic peptide, designed to adopt a perfect amphipathic α-helix, buforin II amide adopts an extended or polyproline II conformation when bound to DNA. These results show that an α-helix structure is not required for the DNA binding and condensation activity of buforin II amide.     

Utilizing ESEEM spectroscopy to locate the position of specific regions of membrane-active peptides within model membranes

Membrane-active peptides participate in many cellular processes, and therefore knowledge of their mode of interaction with phospholipids is essential for understanding their biological function. Here we present a new methodology based on electron spin-echo envelope modulation to probe, at a relatively high resolution, the location of membrane-bound lytic peptides and to study their effect on the water concentration profile of the membrane. As a first example, we determined the location of the N-terminus of two membrane-active amphipathic peptides, the 26-mer bee venom melittin and a de novo designed 15-mer D,L-amino acid amphipathic peptide (5D-L9K6C), both of which are antimicrobial and bind and act similarly on negatively charged membranes. A nitroxide spin label was introduced to the N-terminus of the peptides and measurements were performed either in H2O solutions with deuterated model membranes or in D2O solutions with nondeuterated model membranes. The lipids used were dipalmitoyl phosphatidylcholine (DPPC) and phosphatidylglycerol (PG), (DPPC/PG (7:3 w/w)), egg phosphatidylcholine (PC) and PG (PC/PG (7:3 w/w)), and phosphatidylethanolamine (PE) and PG (PE/PG, 7:3w/w). The modulation induced by the 2H nuclei was determined and compared with a series of controls that produced a reference "ruler". Actual estimated distances were obtained from a quantitative analysis of the modulation depth based on a simple model of an electron spin situated at a certain distance from the bottom of a layer with homogeneously distributed deuterium nuclei. The N-terminus of both peptides was found to be in the solvent layer in both the DPPC/PG and PC/PG membranes. For PE/PG, a further displacement into the solvent was observed. The addition of the peptides was found to change the water distribution in the membrane, making it "flatter" and increasing the penetration depth into the hydrophobic region.     

Molecular dynamics simulations of palmitoyloleoylphosphatidylglycerol bilayers

Phosphatidylglycerol (PG) is one of the important components of biological membranes, but there is a paucity of experimental data to test the accuracy of molecular dynamics (MD) simulations. This work consists of testing the accuracy of the CHARMM36 (C36) lipid force field on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) lipid bilayers. MD simulations of POPG lipid bilayers are compared to recently available X-ray and neutron scattering and deuterium NMR measurements. Overall, the C36 lipid force field accurately represents the X-ray and neutron form factors, bilayer and hydrocarbon thicknesses and chain deuterium order parameters. The surface area per lipid from MD simulations with C36 (67.7 ± 0.2 Å²) is in excellent agreement with the experimentally determined value of 66.0 ± 1.3 Å². C36 outperforms the lipid force field developed by Berger et al. [15] and suggests that past studies with this force field may result in lateral areas that are too small. Moreover, our studies give some insight into the structural model used in experiments and suggest that the functional form for the head group may not be Gaussian-like. Based on our simulations, the POPG lipid in the C36 lipid force field is well parameterised and can be used for other PG lipids and membrane models with mixed lipids.     

Membrane perturbation by the antimicrobial peptide PMAP-23: A fluorescence and molecular dynamics study

Several bioactive peptides exert their biological function by interacting with cellular membranes. Structural data on their location inside lipid bilayers are thus essential for a detailed understanding of their mechanism of action. We propose here a combined approach in which fluorescence spectroscopy and molecular dynamics (MD) simulations were applied to investigate the mechanism of membrane perturbation by the antimicrobial peptide PMAP-23. Fluorescence spectra, depth-dependent quenching experiments, and peptide-translocation assays were employed to determine the location of the peptide inside the membrane. MD simulations were performed starting from a random mixture of water, lipids and peptide, and following the spontaneous self-assembly of the bilayer. Both experimental and theoretical data indicated a peptide location just below the polar headgroups of the membrane, with an orientation essentially parallel to the bilayer plane. These findings, together with experimental results on peptide-induced leakage from large and giant vesicles, lipid flip-flop and peptide exchange between vesicles, support a mechanism of action consistent with the “carpet” model. Furthermore, the atomic detail provided by the simulations suggested the occurrence of an additional, more specific and novel mechanism of bilayer destabilization by PMAP-23, involving the unusual insertion of charged side chains into the hydrophobic core of the membrane.     

The modulating effects of lipids on purified rat liver Golgi galactosyltransferase

Galactosyltransferase was purified from rat liver Golgi membranes. The Triton X-100, used to solubilize the enzyme was removed immediately prior to the lipid interaction studies. In lipid vesicles, prepared from a variety of phosphatidylcholines (PCs), including egg PC, DOPC, DMPC, DPPC and DSPC, the ability of the lipids to stimulate the enzyme decreased in the order egg PC greater than DOPC greater than DMPC greater than DPPC greater than DSPC, i.e. the lower the transition temperature (Tc) the greater the stimulation of the enzyme. A second, neutral lipid, phosphatidylethanolamine was used to permit a comparison of the effect of a different head group of the same net charge at neutral pH. The PEs included, egg PE, soy PE, Pl-PE, PE(PC) and DPPE in order of increasing Tc. The effect of the PEs was opposite to that of the PCs, i.e. the higher the Tc, the greater the stimulation of the enzyme. In fact egg PE and soy PE which have the lowest Tc values were inhibitory. Thus the modulation of the Golgi membrane galactosyltransferase by these lipids was different from that reported earlier for the bovine milk galactosyltransferase. The effects of two acidic lipids, egg phosphatidic acid (PA) and egg phosphatidylglycerol (PG) were studied also. Both totally inhibited the enzyme even at low concentrations of lipid, however, the PA was more effective than PG. In mixtures of neutral lipid (PC) and acidic lipid (PA or PG), the effect of the acidic lipid dominated. Even in the presence of excess PC, total inhibition of the enzyme was observed. It was concluded that the enzyme bound the acidic lipid preferentially to itself. The choice of the lipids allowed us to make several direct comparisons concerning the effect of the nature of the lipid head group on the activity of the enzyme. For example PE(PC), egg PA and egg PG would have fatty acid chains identical to egg PC since these three lipids are all prepared by modification of egg PC. As well, DPPE differs from DPPC only by nature of the head group. These comparisons indicated that not only the net charge but also chemical nature of the head group were important in the lipid modulation of Golgi galactosyltransferase.     

The importance of bacterial membrane composition in the structure and function of aurein 2.2 and selected variants

For cationic antimicrobial peptides to become useful therapeutic agents, it is important to understand their mechanism of action. To obtain high resolution data, this involves studying the structure and membrane interaction of these peptides in tractable model bacterial membranes rather than directly utilizing more complex bacterial surfaces. A number of lipid mixtures have been used as bacterial mimetics, including a range of lipid headgroups, and different ratios of neutral to negatively charged headgroups. Here we examine how the structure and membrane interaction of aurein 2.2 and some of its variants depend on the choice of lipids, and how these models correlate with activity data in intact bacteria (MICs, membrane depolarization). Specifically, we investigated the structure and membrane interaction of aurein 2.2 and aurein 2.3 in 1:1 cardiolipin/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (CL/POPG) (mol/mol), as an alternative to 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)/POPG and a potential model for Gram positive bacteria such as S. aureus. The structure and membrane interaction of aurein 2.2, aurein 2.3, and five variants of aurein 2.2 were also investigated in 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)/POPG (mol/mol) lipids as a possible model for other Gram positive bacteria, such as Bacillus cereus. Solution circular dichroism (CD) results demonstrated that the aurein peptides adopted α-helical structure in all lipid membranes examined, but demonstrated a greater helical content in the presence of POPE/POPG membranes. Oriented CD and 31P NMR results showed that the aurein peptides had similar membrane insertion profiles and headgroup disordering effects on POPC/POPG and CL/POPG bilayers, but demonstrated reduced membrane insertion and decreased headgroup disordering on mixing with POPE/POPG bilayers at low peptide concentrations. Since the aurein peptides behaved very differently in POPE/POPG membrane, minimal inhibitory concentrations (MICs) of the aurein peptides in B. cereus strain C737 were determined. The MIC results indicated that all aurein peptides are significantly less active against B. cereus than against S. aureus and S. epidermidis. Overall, the data suggest that it is important to use a relevant model for bacterial membranes to gain insight into the mode of action of a given antimicrobial peptide in specific bacteria.     

Structural study of the β-hairpin marine antimicrobial peptide arenicin-2 in PC/PG lipid bilayers by fourier transform infrared spectroscopy

Arenicin-2 is a 21-residue β-hairpin antimicrobial peptide isolated from the marine lugworm Arenicola marina. The structure of this cationic peptide in partly charged lipid membrane made of PC/PG (7: 3) was studied by FTIR, CD, and Trp fluorescence spectroscopies. FTIR spectra of arenicin in amide I region were analyzed using curve-fitting and second derivative procedures. The FTIR data for the peptide in PC/PG liposomes were compared with the data obtained in anionic SDS micelles where arenicin forms a dimer stabilized by parallel association of two β-hairpins according to previous NMR spectroscopy studies [Ovchinnikova et al., Biopolymers, 2007, vol. 89, pp. 455–464; Shenkarev et al., Biochemistry, 2011, vol. 50, pp. 6255–6265]. The results obtained in present work indicate that arenicin forms the dimeric structure in partly charged PC/PG lipid membrane. This finding is discussed in relation to interpretation of low-conducting pores observed for arenicin in negatively charged membranes.     

Anionic phospholipid interactions with the potassium channel KcsA: simulation studies

Molecular dynamics (MD) simulations have been used to unmask details of specific interactions of anionic phospholipids with intersubunit binding sites on the surface of the bacterial potassium channel KcsA. Crystallographic data on a diacyl glycerol fragment at this site were used to model phosphatidylethanolamine (PE), or phosphatidylglycerol (PG), or phosphatidic acid (PA) at the intersubunit binding sites. Each of these models of a KcsA-lipid complex was embedded in phosphatidyl choline bilayer and explored in a 20 ns MD simulation. H-bond analysis revealed that in terms of lipid-protein interactions PA > PG > PE and revealed how anionic lipids (PG and PA) bind to a site provided by two key arginine residues (R(64) and R(89)) at the interface between adjacent subunits. A 27 ns simulation was performed in which KcsA (without any lipids initially modeled at the R(64)/R(89) sites) was embedded in a PE/PG bilayer. There was a progressive specific increase over the course of the simulation in the number of H-bonds of PG with KcsA. Furthermore, two specific PG binding events at R(64)/R(89) sites were observed. The phosphate oxygen atoms of bound PG formed H-bonds to the guanidinium group of R(89), whereas the terminal glycerol H-bonded to R(64). Overall, this study suggests that simulations can help identify and characterize sites for specific lipid interactions on a membrane protein surface.     

Lipid composition regulates the conformation and insertion of the antimicrobial peptide maculatin 1.1

Antimicrobial peptides interact with cell membranes and their selectivity is contingent on the nature of the constituent lipids. Eukaryotic and bacterial membranes are comprised of different proportions of a range of lipid species with different physical properties. Hence, characterisation of antimicrobial peptides with respect to the magnitude of their interactions with model membranes of different lipid types is needed. Maculatin 1.1 is a short antimicrobial peptide secreted from the skin of several Australian tree-frog species. Circular dichroism spectroscopy (CD) was used to explore the interaction of maculatin 1.1 with a wide range of model membrane systems of different head group and acyl chain characteristics. For neutral phosphatidylcholine (PC), unlike anionic phospholipids, the magnitude of the peptide interactions was dependent on the length and degree of saturation of the constituent acyl chains. Oriented circular dichroism (OCD) data indicated that helical structure was likely promoted by peptide insertion into the hydrophobic core of PC bilayers. The addition of cholesterol (30% mol/mol) tended to decrease the membrane interaction of maculatin 1.1. Anionic lipids locked maculatin 1.1 via electrostatic interactions onto the surface of oriented bilayers as seen in OCD spectra. Furthermore, increasing the membrane curvature by reducing the vesicle radii only slightly reduced the proportion of helical structure in all systems by approximately 10%. The peptide-lipid interaction was strongly dependent on both the lipid chain length and head group, which highlights the importance of the lipid composition used to mimic different cell types. This article is part of a Special Issue entitled: Membrane protein structure and function.     

On the role of anionic lipids in charged protein interactions with membranes

We investigate the role of anionic lipids in the binding to, and subsequent movement of charged protein groups in lipid membranes, to help understand the role of membrane composition in all membrane-active protein sequences. We demonstrate a small effect of phosphatidylglycerol (PG) lipids on the ability of an arginine (Arg) side chain to bind to, and cross a lipid membrane, despite possessing a neutralizing charge. We observe similar membrane deformations in lipid bilayers composed of phosphatidylcholine (PC) and PC/PG mixtures, with comparable numbers of water and lipid head groups pulled into the bilayer hydrocarbon core, and prohibitively large ~20 kcal/mol barriers for Arg transfer across each bilayer, dropping by just 2-3 kcal/mol due to the binding of PG lipids. We explore the causes of this small effect of introducing PG lipids and offer an explanation in terms of the limited membrane interaction for the choline groups of PC lipids bound to the translocating ion. Our calculations reveal a surprising lack of preference for Arg binding to PG lipids themselves, but a small increase in interfacial binding affinity for lipid bilayers containing PG lipids. These results help to explain the nature of competitive lipid binding to charged protein sequences, with implications for a wide range of membrane binding domains and cell perturbing peptides.