Membrane restructuring by Bordetella pertussis adenylate cyclase toxin, a member of the RTX toxin family

Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium causing whooping cough. ACT is a member of the RTX (repeats in toxin) family of toxins, and like other members in the family, it may bind cell membranes and cause disruption of the permeability barrier, leading to efflux of cell contents. The present paper summarizes studies performed on cell and model membranes with the aim of understanding the mechanism of toxin insertion and membrane restructuring leading to release of contents. ACT does not necessarily require a protein receptor to bind the membrane bilayer, and this may explain its broad range of host cell types. In fact, red blood cells and liposomes (large unilamellar vesicles) display similar sensitivities to ACT. A varying liposomal bilayer composition leads to significant changes in ACT-induced membrane lysis, measured as efflux of fluorescent vesicle contents. Phosphatidylethanolamine (PE), a lipid that favors formation of nonlamellar (inverted hexagonal) phases, stimulated ACT-promoted efflux. Conversely, lysophosphatidylcholine, a micelle-forming lipid that opposes the formation of inverted nonlamellar phases, inhibited ACT-induced efflux in a dose-dependent manner and neutralized the stimulatory effect of PE. These results strongly suggest that ACT-induced efflux is mediated by transient inverted nonlamellar lipid structures. Cholesterol, a lipid that favors inverted nonlamellar phase formation and also increases the static order of phospholipid hydrocarbon chains, among other effects, also enhanced ACT-induced liposomal efflux. Moreover, the use of a recently developed fluorescence assay technique allowed the detection of trans-bilayer (flip-flop) lipid motion simultaneous with efflux. Lipid flip-flop further confirms the formation of transient nonlamellar lipid structures as a result of ACT insertion in bilayers.     

Lipid II induces a transmembrane orientation of the pore-forming peptide lantibiotic nisin

Nisin is an antimicrobial peptide produced by Lactococcus lactis and used as a food preservative in dairy products. The peptide kills Gram-positive bacteria via the permeabilization of the membrane, most probably via pore formation using the cell wall precursor Lipid II as its docking molecule. In this study, site-directed tryptophan spectroscopy was used to determine the topology of nisin in the Lipid II containing membrane, as a start to elucidate the mechanism of targeted pore formation. Three single tryptophan mutants were used, which are viable representatives of the wild-type peptide. The emission spectra of tryptophans located at the N-terminus, the center, and the C-terminus as well as quenching by acrylamide and spin-labeled lipids were investigated using model membrane vesicles composed of DOPC containing 1 mol % Lipid II. Nisin was shown to adopt an orientation where the most probable position of the N-terminus was found to be near the Lipid II headgroup at the bilayer surface, the position of the center of nisin was in the middle of the phospholipid bilayer, and the C-terminus was located near the interface between the headgroups and acyl chain region. These results were used to propose a model for the orientation of nisin in Lipid II containing membranes. Our findings demonstrated that Lipid II changes the overall orientation of nisin in membranes from parallel to perpendicular with respect to the membrane surface. The stable transmembrane orientation of nisin in the presence of Lipid II might allow us to determine the structure of the nisin-Lipid II pores in the lipid bilayer.     

Membrane fusogenic activity of the Alzheimer's peptide A beta(1-42) demonstrated by small-angle neutron scattering

Amyloid-β peptide (Aβ) is considered a triggering agent of Alzheimer's disease. In relation to a therapeutic treatment of the disease, the interaction of Aβ with the cell membrane has to be elucidated at the molecular level to understand its mechanism of action. In previous works, we had ascertained by neutron diffraction on stacked lipid multilayers that a toxic fragment of Aβ is able to penetrate and perturb the lipid bilayer. Here, the influence of Aβ(1-42), the most abundant Aβ form in senile plaques, on unilamellar lipid vesicles of phospholipids is investigated by small-angle neutron scattering. We have used the recently proposed separated form factor method to fit the data and to obtain information about the vesicle diameter and structure of the lipid bilayer and its change upon peptide administration. The lipid membrane parameters were obtained with different models of the bilayer profile. As a result, we obtained an increase in the vesicle radii, indicating vesicle fusion. This effect was particularly enhanced at pH 7.0 and at a high peptide/lipid ratio. At the same time, a thinning of the lipid bilayer occurred. A fusogenic activity of the peptide may have very important consequences and may contribute to cytotoxicity by destabilizing the cell membrane. The perturbation of the bilayer structure suggests a strong interaction and/or insertion of the peptide into the membrane, although its localization remains beyond the limit of the experimental resolution.     

MSI-78, an analogue of the magainin antimicrobial peptides,disrupts lipid bilayer structure via positive curvature strain

In this work, we present the first characterization of the cell lysing mechanism of MSI-78, an antimicrobial peptide. MSI-78 is an amphipathic alpha-helical peptide designed by Genaera Corporation as a synthetic analog to peptides from the magainin family. (31)P-NMR of mechanically aligned samples and differential scanning calorimetry (DSC) were used to study peptide-containing lipid bilayers. DSC showed that MSI-78 increased the fluid lamellar to inverted hexagonal phase transition temperature of 1,2-dipalmitoleoyl-phosphatidylethanolamine indicating the peptide induces positive curvature strain in lipid bilayers. (31)P-NMR of lipid bilayers composed of MSI-78 and 1-palmitoyl-2-oleoyl-phosphatidylethanolamine demonstrated that the peptide inhibited the fluid lamellar to inverted hexagonal phase transition of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, supporting the DSC results, and the peptide did not induce the formation of nonlamellar phases, even at very high peptide concentrations (15 mol %). (31)P-NMR of samples containing 1-palmitoyl-2-oleoyl-phosphatidylcholine and MSI-78 revealed that MSI-78 induces significant changes in the bilayer structure, particularly at high peptide concentrations. At lower concentrations (1-5%), the peptide altered the morphology of the bilayer in a way consistent with the formation of a toroidal pore. Higher concentrations of peptide (10-15%) led to the formation of a mixture of normal hexagonal phase and lamellar phase lipids. This work shows that MSI-78 induces significant changes in lipid bilayers via positive curvature strain and presents a model consistent with both the observed spectral changes and previously published work.     

X-ray studies on the interaction of the antimicrobial peptide gramicidin S with microbial lipid extracts: evidence for cubic phase formation

We have investigated the effect of the interaction of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of model lipid bilayer membranes generated from the total membrane lipids of Acholeplasma laidlawii B and Escherichia coli. The A. laidlawii B membrane lipids consist primarily of neutral glycolipids and anionic phospholipids, while the E. coli inner membrane lipids consist exclusively of zwitterionic and anionic phospholipids. We show that the addition of GS at a lipid-to-peptide molar ratio of 25 strongly promotes the formation of bicontinuous inverted cubic phases in both of these lipid model membranes, predominantly of space group Pn3m. In addition, the presence of GS causes a thinning of the liquid-crystalline bilayer and a reduction in the lattice spacing of the inverted cubic phase which can form in the GS-free membrane lipid extracts at sufficiently high temperatures. This latter finding implies that GS potentiates the formation of an inverted cubic phase by increasing the negative curvature stress in the host lipid bilayer. This effect may be an important aspect of the permeabilization and eventual disruption of the lipid bilayer phase of biological membranes, which appears to be the mechanism by which GS kills bacterial cells and lysis erythrocytes.     

The molecular behavior of a single β‐amyloid inside a dipalmitoylphosphatidylcholine bilayer at three different temperatures: An atomistic simulation study: Aβ interaction with DPPC: Atomistic simulation

The behavior of a single Aβ40 molecule within a dipalmitoylphosphatidylcholine (DPPC) bilayer was studied by all‐atom molecular dynamics simulations. The effect of membrane structure was investigated on Aβ40 behavior, secondary structure, and insertion depth. Simulations were performed at three temperatures (323, 310, and 300 K) to probe three different bilayer fluidities. Results show that at all above temperatures, the peptide contains two short helices, coil, bend, and turn structures. At 300 K, the peptide contains a region with β structure in C‐terminal region. Our results also show that Aβ decreases the bilayer thickness and the order of lipids in its vicinity which leads to water insertion into the bilayer and concomitant increase in the local fluidity. The peptide remains embedded in the bilayer at all temperatures, and become inserted into the bilayer up to several residues at 323 and 310 K. At 310 and 300 K, the dominant interaction energy between Aβ and bilayer changes from electrostatic to van der Waals. It can be proposed that at higher temperatures (e.g., 323 K), Lys28 and the C‐terminal region of the peptide play the role of two anchors that keep Aβ inside the top leaflet. This study demonstrates that Aβ molecule can perturb the integrity of cellular membranes. Proteins 2017; 85:1298–1310. © 2017 Wiley Periodicals, Inc.     

Lipid dependence of diadinoxanthin solubilization and de-epoxidation in artificial membrane systems resembling the lipid composition of the natural thylakoid membrane

In the present study, the solubility and enzymatic de-epoxidation of diadinoxanthin (Ddx) was investigated in three different artificial membrane systems: (1) Unilamellar liposomes composed of different concentrations of the bilayer forming lipid phosphatidylcholine (PC) and the inverted hexagonal phase (H_II phase) forming lipid monogalactosyldiacylglycerol (MGDG), (2) liposomes composed of PC and the H_II phase forming lipid phosphatidylethanolamine (PE), and (3) an artificial membrane system composed of digalactosyldiacylglycerol (DGDG) and MGDG, which resembles the lipid composition of the natural thylakoid membrane. Our results show that Ddx de-epoxidation strongly depends on the concentration of the inverted hexagonal phase forming lipids MGDG or PE in the liposomes composed of PC or DGDG, thus indicating that the presence of inverted hexagonal structures is essential for Ddx de-epoxidation. The difference observed for the solubilization of Ddx in H_II phase forming lipids compared with bilayer forming lipids indicates that Ddx is not equally distributed in the liposomes composed of different concentrations of bilayer versus non-bilayer lipids. In artificial membranes with a high percentage of bilayer lipids, a large part of Ddx is located in the membrane bilayer. In membranes composed of equal proportions of bilayer and H_II phase forming lipids, the majority of the Ddx molecules is located in the inverted hexagonal structures. The significance of the pigment distribution and the three-dimensional structure of the H_II phase for the de-epoxidation reaction is discussed, and a possible scenario for the lipid dependence of Ddx (and violaxanthin) de-epoxidation in the native thylakoid membrane is proposed.     

Lipid dependence of diadinoxanthin solubilization and de-epoxidation in artificial membrane systems resembling the lipid composition of the natural thylakoid membrane

In the present study, the solubility and enzymatic de-epoxidation of diadinoxanthin (Ddx) was investigated in three different artificial membrane systems: (1) Unilamellar liposomes composed of different concentrations of the bilayer forming lipid phosphatidylcholine (PC) and the inverted hexagonal phase (H(II) phase) forming lipid monogalactosyldiacylglycerol (MGDG), (2) liposomes composed of PC and the H(II) phase forming lipid phosphatidylethanolamine (PE), and (3) an artificial membrane system composed of digalactosyldiacylglycerol (DGDG) and MGDG, which resembles the lipid composition of the natural thylakoid membrane. Our results show that Ddx de-epoxidation strongly depends on the concentration of the inverted hexagonal phase forming lipids MGDG or PE in the liposomes composed of PC or DGDG, thus indicating that the presence of inverted hexagonal structures is essential for Ddx de-epoxidation. The difference observed for the solubilization of Ddx in H(II) phase forming lipids compared with bilayer forming lipids indicates that Ddx is not equally distributed in the liposomes composed of different concentrations of bilayer versus non-bilayer lipids. In artificial membranes with a high percentage of bilayer lipids, a large part of Ddx is located in the membrane bilayer. In membranes composed of equal proportions of bilayer and H(II) phase forming lipids, the majority of the Ddx molecules is located in the inverted hexagonal structures. The significance of the pigment distribution and the three-dimensional structure of the H(II) phase for the de-epoxidation reaction is discussed, and a possible scenario for the lipid dependence of Ddx (and violaxanthin) de-epoxidation in the native thylakoid membrane is proposed.     

Folding is not required for bilayer insertion: replica exchange simulations of an alpha-helical peptide with an explicit lipid bilayer

We implement the replica exchange molecular dynamics algorithm to study the interactions of a model peptide (WALP-16) with an explicitly represented DPPC membrane bilayer. We observe the spontaneous, unbiased insertion of WALP-16 into the DPPC bilayer and its folding into an alpha-helix with a transbilayer orientation. The free energy surface suggests that the insertion of the peptide into the DPPC bilayer precedes secondary structure formation. Although the peptide has some propensity to form a partially helical structure in the interfacial region of the DPPC/water system, this state is not a productive intermediate but rather an off-pathway trap for WALP-16 insertion. Equilibrium simulations show that the observed insertion/folding pathway mirrors the potential of mean force (PMF). Calculation of the enthalpic and entropic contributions to this PMF show that the surface bound conformation of WALP-16 is significantly lower in energy than other conformations, and that the insertion of WALP-16 into the bilayer without regular secondary structure is enthalpically unfavorable by 5-10 kcal/mol/residue. The observed insertion/folding pathway disagrees with the dominant conceptual model, which is that a surface-bound helix is an obligatory intermediate for the insertion of alpha-helical peptides into lipid bilayers. In our simulations, the observed insertion/folding pathway is favored because of a large (>100 kcal/mol) increase in system entropy that occurs when the unstructured WALP-16 peptide enters the lipid bilayer interior. The insertion/folding pathway that is lowest in free energy depends sensitively on the near cancellation of large enthalpic and entropic terms. This suggests the possibility that intrinsic membrane peptides may have a diversity of insertion/folding behaviors depending on the exact system of peptide and lipid under consideration.     

Mixtures of a series of homologous hydrophobic peptides with lipid bilayers: a simple model system for examining the protein-lipid interface

The interactions of several members of a homologous series of peptides with the phospholipid bilayer have been examined by using fluorescence and deuterium NMR spectroscopy, differential scanning calorimetry, and measurements of water-to-bilayer partition coefficients. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers and tripeptides of the form Ala-X-Ala-O-tert-butyl are used as a model system to probe the influence of amino acid side-chain substitution on the insertion of peptides into membranes and the behavior of peptide/bilayer mixtures. Tripeptides with X = Gly, Ala, Phe, and Trp have been examined. All of the tripeptides are water soluble, and all partition into DMPC bilayer vesicles to some extent. The Gly-containing peptide is the least soluble and the Trp-containing peptide the most soluble in the bilayer. The extent of perturbation of the bilayer structure induced by the peptides parallels their bilayer solubility: the Gly and Ala peptides act as simple impurities while peptides containing bulky aromatic rings cause a phase separation. Changes in the fluorescence properties of the Trp analogue upon incorporation into the bilayer indicate that the Trp side chain is probably immersed in the hydrocarbon region of the bilayer. Peptides of this form should serve as easily modifiable model systems with which to examine details of how the bilayer environment affects peptide conformation, as well as how hydrophobic peptides affect the bilayer structure.     

Inverted micellar intermediates and the transitions between lamellar, cubic, and inverted hexagonal lipid phases. II. Implications for membrane-membrane interactions and membrane fusion.

Results of a kinetic model of thermotropic L alpha----HII phase transitions are used to predict the types and order-of-magnitude rates of interactions between unilamellar vesicles that can occur by intermediates in the L alpha----HII phase transition. These interactions are: outer monolayer lipid exchange between vesicles; vesicle leakage subsequent to aggregation; and (only in systems with ratios of L alpha and HII phase structural dimensions in a certain range or with unusually large bilayer lateral compressibilities) vesicle fusion with retention of contents. It was previously proposed that inverted micellar structures mediate membrane fusion. These inverted micellar structures are thought to form in all systems with such transitions. However, I show that membrane fusion probably occurs via structures that form from these inverted micellar intermediates, and that fusion should occur in only a sub-set of lipid systems that can adopt the HII phase. For single-component phosphatidylethanolamine (PE) systems with thermotropic L alpha----HII transitions, lipid exchange should be observed starting at temperatures several degrees below TH and at all higher temperatures, where TH is the L alpha----HII transition temperature. At temperatures above TH, the HII phase forms between apposed vesicles, and eventually ruptures them (leakage). In most single-component PE systems, fusion via L alpha----HII transition intermediates should not occur. This is the behavior observed by Bentz, Ellens, Lai, Szoka, et al. in PE vesicle systems. Fusion is likely to occur under circumstances in which multilamellar samples of lipid form the so-called "inverted cubic" or "isotropic" phase. This is as observed in the mono-methyl DOPE system (Ellens, H., J. Bentz, and F. C. Szoka. 1986. Fusion of phosphatidylethanolamine containing liposomes and the mechanism of the L alpha-HII phase transition. Biochemistry. In press.) In lipid systems with L alpha----HII transitions driven by cation binding (e.g., Ca2+-cardiolipin), fusion should be more frequent than in thermotropic systems.     

Mechanism of lipid bilayer disruption by the human antimicrobial peptide,LL-37

LL-37 is an amphipathic, alpha-helical, antimicrobial peptide. (15)N chemical shift and (15)N dipolar-shift spectroscopy of site-specifically labeled LL-37 in oriented lipid bilayers indicate that the amphipathic helix is oriented parallel to the surface of the bilayer. This surface orientation is maintained in both anionic and zwitterionic bilayers and at different temperatures and peptide concentrations, ruling out a barrel-stave mechanism for bilayer disruption by LL-37. In contrast, electrostatic factors, the type of lipid, and the presence of cholesterol do affect the extent to which LL-37 perturbs the lipids in the bilayer as observed with (31)P NMR. The (31)P spectra also show that micelles or other small, rapidly tumbling membrane fragments are not formed in the presence of LL-37, excluding a detergent-like mechanism. LL-37 does increase the lamellar to inverted hexagonal phase transition temperature of both PE model lipid systems and Escherichia coli lipids, demonstrating that it induces positive curvature strain in these environments. These results support a toroidal pore mechanism of lipid bilayer disruption by LL-37.     

Limiting an antimicrobial peptide to the lipid-water interface enhances its bacterial membrane selectivity: a case study of MSI-367

In a minimalist design approach, a synthetic peptide MSI-367 [(KFAKKFA)(3)-NH(2)] was designed and synthesized with the objective of generating cell-selective nonlytic peptides, which have a significant bearing on cell targeting. The peptide exhibited potent activity against both bacteria and fungi, but no toxicity to human cells at micromolar concentrations. Bacterial versus human cell membrane selectivity of the peptide was determined via membrane permeabilization assays. Circular dichroism investigations revealed the intrinsic helix propensity of the peptide, β-turn structure in aqueous buffer and extended and turn conformations upon binding to lipid vesicles. Differential scanning calorimetry experiments with 1,2-dipalmitoleoyl-sn-glycero-3-phosphatidylethanolamine bilayers indicated the induction of positive curvature strain and repression of the fluid lamellar to inverted hexagonal phase transition by MSI-367. Results of isothermal titration calorimetry (ITC) experiments suggested the possibility of formation of specific lipid-peptide complexes leading to aggregation. (2)H nuclear magnetic resonance (NMR) of deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) multilamellar vesicles confirmed the limited effect of the membrane-embedded peptide at the lipid-water interface. (31)P NMR data indicated changes in the lipid headgroup orientation of POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine lipid bilayers upon peptide binding. Membrane-embedded and membrane-inserted states of the peptide were observed via sum frequency generation vibrational spectroscopy. Circular dichroism, ITC, and (31)P NMR data for Escherichia coli lipids agree with the hypothesis that strong electrostatic lipid-peptide interactions embrace the peptide at the lipid-water interface and provide the basis for bacterial cell selectivity.     

Trypsin induced destabilization of liposomes composed of dioleoylphosphatidylethanolamine and glycophorin

Destabilization of liposomes composed of phosphatidylethanolamine (PE) and purified glycophorin of human erythrocytes was studied with the release of an entrapped fluorescent dye, calcein. Proteolytic cleavage of liposomes by trypsin induced a rapid increase of turbidity and the leakage of calcein from the liposomes. Kinetic experiments indicated that the destabilization was a second order reaction, i.e. it required liposome collision. Using N-(7-nitro-2,1,3-benzoxadiazol-4-yl) PE as a fluorescent probe for the formation of hexagonal phase of PE, tryptic digestion of the liposomes resulted in a higher tendency of the PE bilayer to transform into the hexagonal phase. We propose that hexagonal (or inverted micellar) structures are involved in the trypsin induced liposome destabilization.     

Role of transmembrane pH gradient and membrane binding in nisin pore formation.

Nisin is a cationic antimicrobial peptide that belongs to the group of lantibiotics. It is thought to form oligomeric pores in the target membrane by a mechanism that requires the transmembrane electrical potential delta psi and that involves local pertubation of the lipid bilayer structure. Here we show that nisin does not form exclusively voltage-dependent pores: even in the absence of a delta psi, nisin is able to dissipate the transmembrane pH gradient (delta pH) in sensitive Lactococcus lactis cells and proteoliposomes. The rate of dissipation increases with the magnitude of the delta pH. Nisin forms pores only when the delta pH is inside alkaline. The efficiency of delta psi-induced pore formation is strongly affected by the external pH, whereas delta pH-induced pore formation is rather insensitive to the external pH. Nisin(1-12), an amino-terminal fragment of nisin, and (des-deltaAla5)-(nisin(1-32) amide have a strongly reduced capacity to dissipate the delta psi and delta pH in cytochrome c oxidase proteoliposomes and L. lactis cells. Both variants bind with reduced efficiency to liposomes containing negatively charged phospholipids, suggesting that both ring A and rings C to E play a role in membrane binding. Nisin(1-12) competes with nisin for membrane binding and antagonizes pore formation. These findings are consistent with the wedge model of nisin-induced pore formation.     

Interaction of peptides corresponding to mitochondrial presequences with membranes.

The transport of the F1-ATPase beta-subunit precursor into mitochondria is dependent upon a presequence at its amino terminus. Within the mitochondrial membrane translocation site the potential amphiphilic character of the presequence region may be necessary to stabilize binding to the mitochondrial inner membrane. To better understand its role in protein import, the interaction of the F1 beta-presequence with lipid membranes was measured using circular dichroism and surface tensiometry. These studies reveal that a 20-residue peptide containing the F1 beta-presequence binds to phospholipid vesicles (Kd = 4.5-6.0 x 10(-8)M and adopts a predominantly alpha-helical structure. Although the presequence peptide binds avidly to lipids, it does not appear to penetrate deeply into the bilayer to perturb a reporter probe in the membrane interior. Compared with the effect of the peptides with demonstrated membrane insertion and lytic properties, the F1-beta-presequence appears to displace phospholipid head groups but not insert deeply into the bilayer. High concentrations (greater than 50 microM) of presequence peptides are required to noticibly perturb import of the full length F1 alpha- or F1 beta-subunit precursors. Thus, the F1 beta-presequence alone is not sufficient to efficiently compete for import but may require a protein context or a minimal length to assist insertion into the transport site. These observations are discussed in light of the different requirements for import of various presequence containing precursors into mitochondria.     

Effect of N-terminal acetylation on lytic activity and lipid-packing perturbation induced in model membranes by a mastoparan-like peptide

L1A (IDGLKAIWKKVADLLKNT-NH2) is a peptide that displays a selective antibacterial activity to Gram-negative bacteria without being hemolytic. Its lytic activity in anionic lipid vesicles was strongly enhanced when its N-terminus was acetylated (ac-L1A). This modification seems to favor the perturbation of the lipid core of the bilayer by the peptide, resulting in higher membrane lysis. In the present study, we used lipid monolayers and bilayers as membrane model systems to explore the impact of acetylation on the L1A lytic activity and its correlation with lipid-packing perturbation. The lytic activity investigated in giant unilamellar vesicles (GUVs) revealed that the acetylated peptide permeated the membrane at higher rates compared with L1A, and modified the membrane's mechanical properties, promoting shape changes. The peptide secondary structure and the changes in the environment of the tryptophan upon adsorption to large unilamellar vesicles (LUVs) were monitored by circular dichroism (CD) and red-edge excitation shift experiments (REES), respectively. These experiments showed that the N-terminus acetylation has an important effect on both, peptide secondary structure and peptide insertion into the bilayer. This was also confirmed by experiments of insertion into lipid monolayers. Compression isotherms for peptide/lipid mixed films revealed that ac-L1A dragged lipid molecules to the more disordered phase, generating a more favorable environment and preventing the lipid molecules from forming stiff films. Enthalpy changes in the main phase transition of the lipid membrane upon peptide insertion suggested that the acetylated peptide induced higher impact than the non-acetylated one on the thermotropic behavior of anionic vesicles.     

The activation energy for insertion of transmembrane alpha-helices is dependent on membrane composition

The physical mechanisms that govern the folding and assembly of integral membrane proteins are poorly understood. It appears that certain properties of the lipid bilayer affect membrane protein folding in vitro, either by modulating helix insertion or packing. In order to begin to understand the origin of this effect, we investigate the effect of lipid forces on the insertion of a transmembrane alpha-helix using a water-soluble, alanine-based peptide, KKAAAIAAAAAIAAWAAIAAAKKKK-amide. This peptide binds to preformed 1,2-dioleoyl-l-alpha-phosphatidylcholine (DOPC) vesicles at neutral pH, but spontaneous transmembrane helix insertion directly from the aqueous phase only occurs at high pH when the Lys residues are de-protonated. These results suggest that the translocation of charge is a major determinant of the activation energy for insertion. Time-resolved measurements of the insertion process at high pH indicate biphasic kinetics with time constants of ca 30 and 430 seconds. The slower phase seems to correlate with formation of a predominantly transmembrane alpha-helical conformation, as determined from the transfer of the tryptophan residue to the hydrocarbon region of the membrane. Temperature-dependent measurements showed that insertion can proceed only above a certain threshold temperature and that the Arrhenius activation energy is of the order of 90 kJ mol(-1). The kinetics, threshold temperature and the activation energy change with the mole fraction of 1,2-dioleoyl-l-alpha-phosphatidylethanolamine (DOPE) introduced into the DOPC membrane. The activation energy increases with increasing DOPE content, which could reflect the fact that this lipid drives the bilayer towards a non-bilayer transition and increases the lateral pressure in the lipid chain region. This suggests that folding events involving the insertion of helical segments across the bilayer can be controlled by lipid forces.     

Stability of an ion channel in lipid bilayers: implicit solvent model calculations with gramicidin

Gramicidin is a helical peptide, 15 residues in length, which dimerizes to form ion-conducting channels in lipid bilayers. Here we report calculations of its free energy of transfer from the aqueous phase into bilayers of different widths. The electrostatic and nonpolar contributions to the desolvation free energy were calculated using implicit solvent models, in which gramicidin was described in atomic detail and the hydrocarbon region of the membrane was described as a slab of hydrophobic medium embedded in water. The free energy penalties from the lipid perturbation and membrane deformation effects, and the entropy loss associated with gramicidin immobilization in the bilayer, were estimated from a statistical thermodynamic model of the bilayer. The calculations were carried out using two classes of experimentally observed conformations: a head-to-head dimer of two single-stranded (SS) beta-helices and a double-stranded (DS) intertwined double helix. The calculations showed that gramicidin is likely to partition into the bilayer in all of these conformations. However, the SS conformation was found to be significantly more stable than the DS in the bilayer, in agreement with most of the experimental data. We tested numerous transmembrane and surface orientations of gramicidin in bilayers of various widths. Our calculations indicate that the most favorable orientation is transmembrane, which is indeed to be expected from a channel-forming peptide. The calculations demonstrate that gramicidin insertion into the membrane is likely to involve a significant deformation of the bilayer to match the hydrophobic width of the peptide (22 A), again in good agreement with experimental data. Interestingly, deformation of the bilayer was induced by all of the gramicidin conformations.