Membrane composition determines pardaxin's mechanism of lipid bilayer disruption

Pardaxin is a membrane-lysing peptide originally isolated from the fish Pardachirus marmoratus. The effect of the carboxy-amide of pardaxin (P1a) on bilayers of varying composition was studied using (15)N and (31)P solid-state NMR of mechanically aligned samples and differential scanning calorimetry (DSC). (15)N NMR spectroscopy of [(15)N-Leu(19)]P1a found that the orientation of the peptide's C-terminal helix depends on membrane composition. It is located on the surface of lipid bilayers composed of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and is inserted in lipid bilayers composed of 1,2-dimyristoyl-phosphatidylcholine (DMPC). The former suggests a carpet mechanism for bilayer disruption whereas the latter is consistent with a barrel-stave mechanism. The (31)P chemical shift NMR spectra showed that the peptide significantly disrupts lipid bilayers composed solely of zwitterionic lipids, particularly bilayers composed of POPC, in agreement with a carpet mechanism. P1a caused the formation of an isotropic phase in 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) lipid bilayers. This, combined with DSC data that found P1a reduced the fluid lamellar-to-inverted hexagonal phase transition temperature at very low concentrations (1:50,000), is interpreted as the formation of a cubic phase and not micellization of the membrane. Experiments exploring the effect of P1a on lipid bilayers composed of 4:1 POPC:cholesterol, 4:1 POPE:cholesterol, 3:1 POPC:1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), and 3:1 POPE:POPG were also conducted, and the presence of anionic lipids or cholesterol was found to reduce the peptide's ability to disrupt bilayers. Considered together, these data demonstrate that the mechanism of P1a is dependent on membrane composition.     

Cholesterol interaction attenuates scramblase activity of SCRM-1 in the artificial membrane

Phospholipid (PL) scramblases are single-pass transmembrane protein mediating bidirectional PL translocation. Previously in silico analysis of human PL scramblases, predicted the presence of an uncharacterized cholesterol-binding domain spanning partly in the transmembrane helix as well as in the adjacent extracellular coil. This domain was found to be universally conserved in diverse organisms like Caenorhabditis elegans. In this study, we investigated the saturable cholesterol-binding domain of SCRM-1 using fluorescence sterol binding assay, Stern-Volmer quenching, Förster resonance energy transfer, and CD spectroscopy. We observed high-affinity interaction between cholesterol and SCRM-1. Our results support a previous report, which showed that the cholesterol ordering effect reduced the scramblase activity of hPLSCR1. Considering the presence of a high-affinity binding sequence, we propose that the reduction in activity could partly be due to the cholesterol binding. To validate this, we generated a C-terminal helix (CTH) deletion construct (?CTH SCRM-1) and a point mutation in the putative cholesterol-binding domain I273D SCRM-1. Deletion construct greatly reduced cholesterol affinity along with loss of scramblase activity. In contrast to this, I273D SCRM-1 retained scrambling activity in proteoliposomes containing ~30 mol% cholesterol but lost sterol binding ability. These results suggest that C-terminal helix is crucial for membrane insertion and in the lipid bilayer the scrambling activity of SCRM-1 is modulated through its interaction with cholesterol.     

Cholesterol binding is a prerequisite for the activity of the steroidogenic acute regulatory protein (StAR)

Steroidogenesis depends on the delivery of cholesterol from the outer to the inner mitochondrial membrane by StAR (steroidogenic acute regulatory protein). However, the mechanism by which StAR binds to cholesterol and its importance in cholesterol transport are under debate. According to our proposed molecular model, StAR possesses a hydrophobic cavity, which can accommodate one cholesterol molecule. In the bound form, cholesterol interacts with hydrophobic side-chains located in the C-terminal alpha-helix 4, thereby favouring the folding of this helix. To verify this model experimentally, we have characterized the in vitro activity, overall structure, thermodynamic stability and cholesterol-binding affinity of StAR lacking the N-terminal 62 amino acid residues (termed N-62 StAR). This mature form is biologically active and has a well-defined tertiary structure. Addition of cholesterol to N-62 StAR led to an increase in the alpha-helical content and T degrees (melting temperature), indicating the formation of a stable complex. However, the mutation F267Q, which is located in the C-terminal helix interface lining the cholesterol-binding site, reduced the biological activity of StAR. Furthermore, the cholesterol-induced thermodynamic stability and the binding capacity of StAR were significantly diminished in the F267Q mutant. Titration of StAR with cholesterol yielded a 1:1 complex with an apparent K(D) of 3 x 10(-8). These results support our model and indicate that StAR can readily bind to cholesterol with an apparent affinity that commensurates with monomeric cholesterol solubility in water. The proper function of the C-terminal alpha-helix is essential for the binding process.     

Pardaxin-induced apoptosis enhances antitumor activity in HeLa cells

Pardaxin, a pore-forming antimicrobial peptide that encodes 33 amino acids was isolated from the Red Sea Moses sole, Pardachirus mamoratus. In this study, we investigated its antitumor activity in human fibrosarcoma (HT-1080) cells and epithelial carcinoma (HeLa) cells. In vitro results showed that the synthetic pardaxin peptide had antitumor activity in these two types of cancer cells and that 15 μg/ml pardaxin did not lyse human red blood cells. Moreover, this synthetic pardaxin inhibited the proliferation of HT1080 cells in a dose-dependent manner and induced programmed cell death in HeLa cells. DNA fragmentation and increases in the subG1 phase and caspase 8 activities suggest that pardaxin caused HeLa cell death by inducing apoptosis, but had a different mechanism in HT1080 cells.     

The Atlastin C-terminal Tail Is an Amphipathic Helix That Perturbs the Bilayer Structure during Endoplasmic Reticulum Homotypic Fusion

Fusion of tubular membranes is required to form three-way junctions found in reticular subdomains of the endoplasmic reticulum. The large GTPase Atlastin has recently been shown to drive endoplasmic reticulum membrane fusion and three-way junction formation. The mechanism of Atlastin-mediated membrane fusion is distinct from SNARE-mediated membrane fusion, and many details remain unclear. In particular, the role of the amphipathic C-terminal tail of Atlastin is still unknown. We found that a peptide corresponding to the Atlastin C-terminal tail binds to membranes as a parallel α helix, induces bilayer thinning, and increases acyl chain disorder. The function of the C-terminal tail is conserved in human Atlastin. Mutations in the C-terminal tail decrease fusion activity in vitro, but not GTPase activity, and impair Atlastin function in vivo. In the context of unstable lipid bilayers, the requirement for the C-terminal tail is abrogated. These data suggest that the C-terminal tail of Atlastin locally destabilizes bilayers to facilitate membrane fusion.     

Transmembrane helix dynamics of bacterial chemoreceptors supports a piston model of signalling

Transmembrane α-helices play a key role in many receptors, transmitting a signal from one side to the other of the lipid bilayer membrane. Bacterial chemoreceptors are one of the best studied such systems, with a wealth of biophysical and mutational data indicating a key role for the TM2 helix in signalling. In particular, aromatic (Trp and Tyr) and basic (Arg) residues help to lock α-helices into a membrane. Mutants in TM2 of E. coli Tar and related chemoreceptors involving these residues implicate changes in helix location and/or orientation in signalling. We have investigated the detailed structural basis of this via high throughput coarse-grained molecular dynamics (CG-MD) of Tar TM2 and its mutants in lipid bilayers. We focus on the position (shift) and orientation (tilt, rotation) of TM2 relative to the bilayer and how these are perturbed in mutants relative to the wildtype. The simulations reveal a clear correlation between small (ca. 1.5 Å) shift in position of TM2 along the bilayer normal and downstream changes in signalling activity. Weaker correlations are seen with helix tilt, and little/none between signalling and helix twist. This analysis of relatively subtle changes was only possible because the high throughput simulation method allowed us to run large (n = 100) ensembles for substantial numbers of different helix sequences, amounting to ca. 2000 simulations in total. Overall, this analysis supports a swinging-piston model of transmembrane signalling by Tar and related chemoreceptors.     

Activation and molecular recognition of the GPCR rhodopsin - insights from time-resolved fluorescence depolarisation and single molecule experiments

The cytoplasmic surface of the G-protein coupled receptor (GPCR) rhodopsin is a key element in membrane receptor activation, molecular recognition by signalling molecules, and receptor deactivation. Understanding of the coupling between conformational changes in the intramembrane domain and the membrane-exposed surface of the photoreceptor rhodopsin is crucial for the elucidation of the molecular mechanism in GPCR activation. As little is known about protein dynamics, particularly the conformational dynamics of the cytoplasmic surface elements on the nanoseconds timescale, we utilised time-resolved fluorescence anisotropy experiments and site-directed fluorescence labelling to provide information on both, conformational space and motion. We summarise our recent advances in understanding rhodopsin dynamics and function using time-resolved fluorescence depolarisation and single molecule fluorescence experiments, with particular focus on the amphipathic helix 8, lying parallel to the cytoplasmic membrane surface and connecting transmembrane helix 7 with the long C-terminal tail.     

Dynamical structure of the short multifunctional peptide BP100 in membranes

BP100 is a multifunctional membrane-active peptide of only 11 amino acids, with a high antimicrobial activity, an efficient cell-penetrating ability, and low hemolytic side-effects. It forms an amphiphilic α-helix, similar to other antimicrobial peptides like magainin. However, BP100 is very short and thus unlikely to form membrane-spanning pores as proposed for longer peptides as a mechanism of action. We thus studied the conformation, membrane alignment and dynamical behavior of BP100 in lipid bilayers (DMPC/DMPG), using oriented circular dichroism (OCD) and solid-state ¹⁹F and ¹⁵N NMR. According to OCD and ¹⁵N NMR, the BP100 helix is oriented roughly parallel to the membrane surface, but these methods yield no information on the azimuthal alignment angle or the dynamics of the molecule. To address these questions, a systematic ¹⁹F NMR analysis was performed, which was not straightforward for this short peptide. Only a limited number of positions could be ¹⁹F-labeled, all of which are located on one face of the helix, which was found to lead to artifacts in the data analysis. It was nevertheless possible to reconcile the ¹⁹F NMR data with the OCD and ¹⁵N NMR data by using an advanced dynamical model, in which peptide mobility is described by fluctuating tilt and azimuthal angles with Gaussian distributions. ¹⁹F NMR thus confirmed the regular α-helical conformation of BP100, revealed its azimuthal angle, and described its high mobility in the membrane. Furthermore, the very sensitive ¹⁹F NMR experiments showed that the alignment of BP100 does not vary with peptide concentration over a peptide-to-lipid molar ratio from 1:10 to 1:3000.     

Antimicrobial Effects of Helix D-derived Peptides of Human Antithrombin III

Antithrombin III (ATIII) is a key antiproteinase involved in blood coagulation. Previous investigations have shown that ATIII is degraded by Staphylococcus aureus V8 protease, leading to release of heparin binding fragments derived from its D helix. As heparin binding and antimicrobial activity of peptides frequently overlap, we here set out to explore possible antibacterial effects of intact and degraded ATIII. In contrast to intact ATIII, the results showed that extensive degradation of the molecule yielded fragments with antimicrobial activity. Correspondingly, the heparin-binding, helix d-derived, peptide FFFAKLNCRLYRKANKSSKLV (FFF21) of human ATIII, was found to be antimicrobial against particularly the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. Fluorescence microscopy and electron microscopy studies demonstrated that FFF21 binds to and permeabilizes bacterial membranes. Analogously, FFF21 was found to induce membrane leakage of model anionic liposomes. In vivo, FFF21 significantly reduced P. aeruginosa infection in mice. Additionally, FFF21 displayed anti-endotoxic effects in vitro. Taken together, our results suggest novel roles for ATIII-derived peptide fragments in host defense.     

The Vps4 C-terminal helix is a critical determinant for assembly and ATPase activity and has elements conserved in other members of the meiotic clade of AAA ATPases

Sorting of membrane proteins into intralumenal endosomal vesicles, multivesicular body (MVB) sorting, is critical for receptor down regulation, antigen presentation and enveloped virus budding. Vps4 is an AAA ATPase that functions in MVB sorting. Although AAA ATPases are oligomeric, mechanisms that govern Vps4 oligomerization and activity remain elusive. Vps4 has an N-terminal microtubule interacting and trafficking domain required for endosome recruitment, an AAA domain containing the ATPase catalytic site and a beta domain, and a C-terminal alpha helix positioned close to the catalytic site in the 3D structure. Previous attempts to identify the role of the C-terminal helix have been unsuccessful. Here, we show that the C-terminal helix is important for Vps4 assembly and ATPase activity in vitro and function in vivo, but not endosome recruitment or interactions with Vta1 or ESCRT-III. Unlike the beta domain, which is also important for Vps4 assembly, the C-terminal helix is not required in vivo for Vps4 homotypic interaction or dominant-negative effects of Vps4-E233Q, carrying a mutation in the ATP hydrolysis site. Vta1 promotes assembly of hybrid complexes comprising Vps4-E233Q and Vps4 lacking an intact C-terminal helix in vitro. Formation of catalytically active hybrid complexes demonstrates an intersubunit catalytic mechanism for Vps4. One end of the C-terminal helix lies in close proximity to the second region of homology (SRH), which is important for assembly and intersubunit catalysis in AAA ATPases. We propose that Vps4 SRH function requires an intact C-terminal helix. Co-evolution of a distinct Vps4 SRH and C-terminal helix in meiotic clade AAA ATPases supports this possibility.     

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.     

Membrane interface composition drives the structure and the tilt of the single transmembrane helix protein PMP1: MD studies

PMP1, a regulatory subunit of the yeast plasma membrane H⁺-ATPase, is a single transmembrane helix protein. Its cytoplasmic C-terminus possesses several positively charged residues and interacts with phosphatidylserine lipids as shown through both ¹H- and ²H-NMR experiments. We used all-atom molecular dynamics simulations to obtain atomic-scale data on the effects of membrane interface lipid composition on PMP1 structure and tilt. PMP1 was embedded in two hydrated bilayers, differing in the composition of the interfacial region. The neutral bilayer is composed of POPC (1-palmitoyl-2-oleoyl-3-glycero-phosphatidylcholine) lipids and the negatively charged bilayer is composed of POPC and anionic POPS (1-palmitoyl-2-oleoyl-3-glycero-phosphatidylserine) lipids. Our results were consistent with NMR data obtained previously, such as a lipid sn-2 chain lying on the W28 aromatic ring and in the groove formed on one side of the PMP1 helix. In pure POPC, the transmembrane helix is two residues longer than the initial structure and the helix tilt remains constant at 6 ± 3°. By contrast, in mixed POPC-POPS, the initial helical structure of PMP1 is stable throughout the simulation time even though the C-terminal residues interact strongly with POPS headgroups, leading to a significant increase of the helix tilt within the membrane to 20 ± 5°.     

Three-dimensional structure/hydrophobicity of latarcins specifies their mode of membrane activity

Latarcins, linear peptides from the Lachesana tarabaevi spider venom, exhibit a broad-spectrum antimicrobial activity, likely acting on the bacterial cytoplasmic membrane. We study their spatial structures and interaction with model membranes by a combination of experimental and theoretical methods to reveal the structure-activity relationship. In this work, a 26 amino acid peptide, Ltc1, was investigated. Its spatial structure in detergent micelles was determined by (1)H nuclear magnetic resonance (NMR) and refined by Monte Carlo simulations in an implicit water-octanol slab. The Ltc1 molecule was found to form a straight uninterrupted amphiphilic helix comprising 8-23 residues. A dye-leakage fluorescent assay and (31)P NMR spectroscopy established that the peptide does not induce the release of fluorescent marker nor deteriorate the bilayer structure of the membranes. The voltage-clamp technique showed that Ltc1 induces the current fluctuations through planar membranes when the sign of the applied potential coincides with the one across the bacterial inner membrane. This implies that Ltc1 acts on the membranes via a specific mechanism, which is different from the carpet mode demonstrated by another latarcin, Ltc2a, featuring a helix-hinge-helix structure with a hydrophobicity gradient along the peptide chain. In contrast, the hydrophobic surface of the Ltc1 helix is narrow-shaped and extends with no gradient along the axis. We have also disclosed a number of peptides, structurally homologous to Ltc1 and exhibiting similar membrane activity. This indicates that the hydrophobic pattern of the Ltc1 helix and related antimicrobial peptides specifies their activity mechanism. The latter assumes the formation of variable-sized lesions, which depend upon the potential across the membrane.     

Interaction of fluorescently labeled pardaxin and its analogues with lipid bilayers.

Fluorescence measurements were used to monitor the interaction of the neurotoxin pardaxin and its analogues with membranes. Eight peptides were selectively labeled with the fluorophore 7-nitrobenz-2-oxa-1,3-diazole-4-yl, either at their N-terminal or at their C-terminal. No detectable changes in membrane permeability or hemolytic activity were observed upon modification. Upon the titration of solutions containing the different peptides with small unilamellar vesicles, the fluorescent emission spectra of 7-nitrobenz-2-oxa-1,3-diazole-4-yl-labeled pardaxin and its analogues, but not those of control peptides, displayed blue shifts in addition to enhanced intensities upon relocation of the probe to a more apolar environment. The results revealed that the N terminus of pardaxin is buried within the lipid bilayer while the C terminus is located at the bilayer's surface. Binding isotherms were obtained from the observed increases in the fluorescence emission yields, from which surface partition constants, in the range of 10(4) M-1, were in turn derived. The existence of an aggregation process was suggested by the shape of the binding isotherms. Furthermore, the results show good correlation between the incidence of aggregation and the ability of the different analogues to induce the release of relatively large molecules from vesicles. As such, our results suggest that the mechanism of pore formation employed by pardaxin and its analogues could be described by the "barrel stave" model.     

A mechanistic role of Helix 8 in GPCRs: Computational modeling of the dopamine D2 receptor interaction with the GIPC1–PDZ-domain

Helix-8 (Hx8) is a structurally conserved amphipathic helical motif in class-A GPCRs, adjacent to the C-terminal sequence that is responsible for PDZ-domain-recognition. The Hx8 segment in the dopamine D2 receptor (D2R) constitutes the C-terminal segment and we investigate its role in the function of D2R by studying the interaction with the PDZ-containing GIPC1 using homology models based on the X-ray structures of very closely related analogs: the D3R for the D2R model, and the PDZ domain of GIPC2 for GIPC1–PDZ. The mechanism of this interaction was investigated with all-atom unbiased molecular dynamics (MD) simulations that reveal the role of the membrane in maintaining the helical fold of Hx8, and with biased MD simulations to elucidate the energy drive for the interaction with the GIPC1–PDZ. We found that it becomes more favorable energetically for Hx8 to adopt the extended conformation observed in all PDZ–ligand complexes when it moves away from the membrane, and that C-terminus palmitoylation of D2R enhanced membrane penetration by the Hx8 backbone. De-palmitoylation enables Hx8 to move out into the aqueous environment for interaction with the PDZ domain. All-atom unbiased MD simulations of the full D2R–GIPC1-PDZ complex in sphingolipid/cholesterol membranes show that the D2R carboxyl C-terminus samples the region of the conserved GFGL motif located on the carboxylate-binding loop of the GIPC1–PDZ, and the entire complex distances itself from the membrane interface. Together, these results outline a likely mechanism of Hx8 involvement in the interaction of the GPCR with PDZ-domains in the course of signaling.     

Insight into the impact of environments on structure of chimera C3 of human β-defensins 2 and 3 from molecular dynamics simulations

C3 is a chimera from human β-defensins 2 and 3 and possesses higher antimicrobial activity compared with its parental molecules, so it is an attractive candidate for clinical application of antimicrobial peptides. In continuation with the previous studies, molecular dynamics (MD) simulations were carried out for further investigating the effect of ambient environments (temperature and bacterial membrane) on C3 dynamics. Our results reveal that C3 has higher flexibility, larger intensity of motion, and more relevant secondary structural changes at 363 K to adapt the high temperature and maintain its antimicrobial activity, comparison with it at 293 K; when C3 molecule associates with the bacterial membrane, it slightly fluctuates and undergoes local conformational changes; in summary, C3 molecule demonstrates stable conformations under these environments. Furthermore, MD results analysis show that the hydrophobic contacts, the hydrogen bonds, and disulfide bonds in the peptide are responsible for maintaining its stable conformation. In addition, our simulation shows that C3 peptides can make anionic lipids clustered in the bacterial membrane; it means that positive charges and pronounced regional cationic charge density of C3 are most key factors for its antimicrobial activity.     

Possible role of a PXXP central hinge in the antibacterial activity and membrane interaction of PMAP-23, a member of cathelicidin family

Cathelicidins are essential components of the innate immune system of mammals, providing them a weapon against microbial invasion. PMAP-23 adopting a helix-hinge-helix structure with a central PXXP motif is a member of the cathelicidin family and has potent killing activities against a broad spectrum of microbial organisms. Although the antimicrobial effect of PMAP-23 is believed to be mediated by membrane disruption, many details of this event remain unclear. Here, we try to characterize the interaction between PMAP-23 and membrane phospholipids, focusing on the function of the central PXXP motif. PMAP-PA, in which the Pro residues were substituted by Ala, had significantly more alpha-helical content than PMAP-23, but was less amphipathic and more damaging to human erythrocytes and zwitterionic liposomes. The observed differences in the structures and biological activities of PMAP-23 and PMAP-PA confirmed the functional importance of the central hinge PXXP motif, which enables PMAP-23 to adopt a well-defined amphipathic conformation along its entire length and to have selective antimicrobial activity. CD and Trp fluorescence studies using fragments corresponding to the two helical halves of PMAP-23 revealed that the N-terminal half binds to anionic phospholipids and is more stable than the C-terminal half. In addition, Trp fluorescence quench analyses revealed that the C-terminal helix inserts more deeply into the hydrophobic region of the membrane than the N-terminal helix. Finally, observations made using biosensor technology enabled us to distinguish between the membrane binding and insertion steps, substantiating a proposed kinetic mode of the peptide-membrane interaction in which PMAP-23 first attaches to the membrane via the N-terminal amphipathic helix, after which bending and/or swiveling of the PXXP motif enables insertion of the C-terminal helix into the lipid bilayer.     

Solid-state NMR investigation of the membrane-disrupting mechanism of antimicrobial peptides MSI-78 and MSI-594 derived from magainin 2 and melittin

The mechanism of membrane interaction of two amphipathic antimicrobial peptides, MSI-78 and MSI-594, derived from magainin-2 and melittin, is presented. Both the peptides show excellent antimicrobial activity. The 8-anilinonaphthalene-1-sulfonic acid uptake experiment using Escherichia coli cells suggests that the outer membrane permeabilization is mainly due to electrostatic interactions. The interaction of MSI-78 and MSI-594 with lipid membranes was studied using 31P and 2H solid-state NMR, circular dichroism, and differential scanning calorimetry techniques. The binding of MSI-78 and MSI-594 to the lipid membrane is associated with a random coil to alpha-helix structural transition. MSI-78 and MSI-594 also induce the release of entrapped dye from POPC/POPG (3:1) vesicles. Measurement of the phase-transition temperature of peptide-DiPoPE dispersions shows that both MSI-78 and MSI-594 repress the lamellar-to-inverted hexagonal phase transition by inducing positive curvature strain. 15N NMR data suggest that both the peptides are oriented nearly perpendicular to the bilayer normal, which infers that the peptides most likely do not function via a barrel-stave mechanism of membrane-disruption. Data obtained from 31P NMR measurements using peptide-incorporated POPC and POPG oriented lamellar bilayers show a disorder in the orientation of lipids up to a peptide/lipid ratio of 1:20, and the formation of nonbilayer structures at peptide/lipid ratio>1:8. 2H-NMR experiments with selectively deuterated lipids reveal peptide-induced disorder in the methylene units of the lipid acyl chains. These results are discussed in light of lipid-peptide interactions leading to the disruption of membrane via either a carpet or a toroidal-type mechanism.     

Self-assembly of a simple membrane protein: coarse-grained molecular dynamics simulations of the influenza M2 channel

The transmembrane (TM) domain of the M2 channel protein from influenza A is a homotetrameric bundle of α-helices and provides a model system for computational approaches to self-assembly of membrane proteins. Coarse-grained molecular dynamics (CG-MD) simulations have been used to explore partitioning into a membrane of M2 TM helices during bilayer self-assembly from lipids. CG-MD is also used to explore tetramerization of preinserted M2 TM helices. The M2 helix monomer adopts a membrane spanning orientation in a lipid (DPPC) bilayer. Multiple extended CG-MD simulations (5 × 5 μs) were used to study the tetramerization of inserted M2 helices. The resultant tetramers were evaluated in terms of the most populated conformations and the dynamics of their interconversion. This analysis reveals that the M2 tetramer has 2× rotationally symmetrical packing of the helices. The helices form a left-handed bundle, with a helix tilt angle of ∼16°. The M2 helix bundle generated by CG-MD was converted to an atomistic model. Simulations of this model reveal that the bundle's stability depends on the assumed protonation state of the H37 side chains. These simulations alongside comparison with recent x-ray (3BKD) and NMR (2RLF) structures of the M2 bundle suggest that the model yielded by CG-MD may correspond to a closed state of the channel.     

Membrane Binding, Structure, and Localization of Cecropin-Mellitin Hybrid Peptides: A Site-Directed Spin-Labeling Study

The interaction of antimicrobial peptides with membranes is a key factor in determining their biological activity. In this study we have synthesized a series of minimized cecropin-mellitin hybrid peptides each containing a single cysteine residue, modified the cysteine with the sulfhydryl-specific methanethiosulfonate spin-label, and used electron paramagnetic resonance spectroscopy to measure membrane-binding affinities and determine the orientation and localization of peptides bound to membranes that mimic the bacterial cytoplasmic membrane. All of the peptides were unstructured in aqueous solution but underwent a significant conformational change upon membrane binding that diminished the rotational mobility of the attached spin-label. Apparent partition coefficients were similar for five of the six constructs examined, indicating that location of the spin-label had little effect on peptide binding as long as the attachment site was in the relatively hydrophobic C-terminal domain. Depth measurements based on accessibility of the spin-labeled sites to oxygen and nickel ethylenediaminediacetate indicated that at high lipid/peptide ratios these peptides form a single α-helix, with the helical axis aligned parallel to the bilayer surface and immersed ~5 Å below the membrane-aqueous interface. Such a localization would provide exposure of charged/polar residues on the hydrophilic face of the amphipathic helix to the aqueous phase, and allow the nonpolar residues along the opposite face of the helix to remain immersed in the hydrophobic phase of the bilayer. These results are discussed with respect to the mechanism of membrane disruption by antimicrobial peptides.