Fungicidal effect of antimicrobial peptide, PMAP-23, isolated from porcine myeloid against Candida albicans

The antifungal activity and mechanism of a 23-mer peptide, PMAP-23, derived from pig myeloid was investigated. PMAP-23 displayed strong antifungal activity against yeast and mold. To investigate the antifungal mechanism of PMAP-23, fluorescence activated flow cytometry and confocal laser scanning microscopy were performed. Candida albicans treated with PMAP-23 showed higher fluorescence intensity by propidium iodide(PI) staining, which was similar to that of Melittin than untreated cells. Confocal microscopy showed that the peptide was located in the plasma membrane. The action of peptides against fungal cell membranes was examined by treating prepared protoplasts of C. albicans with the peptide and lipid vesicle titration test. The result showed that the peptide prevented the regeneration of fungal cell walls and induced release of the fluorescent dye trapped in the artificial membrane vesicles, indicating that the peptide exerts its antifungal activity by acting on the plasma lipid membrane.     

Antinematodal effect of antimicrobial peptide, PMAP-23, isolated from porcine myeloid against Caenorhabditis elegans.

The antinematodal activity and mechanism of a 23-mer antimicrobial peptide, PMAP-23, derived from pig myeloid was investigated. PMAP-23 displayed a strong antinematodal activity against the eggs and worms of Caenorhabditis elegans. To investigate the antinematodal mechanism of PMAP-23, fluorescence activated flow cytometry and confocal laser scanning microscopy were performed. C. elegans treated with PMAP-23 showed higher fluorescence intensity by propidium iodide (PI) staining than normal cells. Confocal microscopy showed that the peptide was localized in the egg's shell and cell membrane. The action of the peptide against C. elegans membranes was examined by testing the membrane disrupting activity using liposome (PC/PS; 3:1, w/w). The result suggests that PMAP-23 may exert its antinematodal activity by disrupting the structure of the cell membrane via pore formation or via direct interaction with the lipid bilayers.     

C-terminal amidation of PMAP-23: translocation to the inner membrane of Gram-negative bacteria

PMAP-23 is a member of the cathelicidin family derived from pig myeloid cells and has potent antimicrobial activity. Amidation of the carboxyl terminus (C-terminus) of an antimicrobial peptide generally enhances its structural stability and antimicrobial activity or decreases its cytotoxicity. The aim of the present study was to investigate the effect of amidation on the mode of action in PMAP-23. Irrespective of amidation, PMAP-23 adopts a helix–hinge–helix structure in a membrane-mimetic environment. The antibacterial activities of PMAP-23C, which had a free C-terminus, and PMAP-23N, which had an amidated C-terminus, were similar against Gram-negative bacteria, reflecting a similar ability to neutralize lipopolysaccharide. However, PMAP-23N assumed a perpendicular orientation across the outer to the inner leaflet of the bacterial inner membrane, while PMAP-23C was orientated parallel to the lipid bilayer, as determined by following the blue shift in tryptophan fluorescence, as well as calcein release from liposomes and SYTOX Green uptake assays. These results suggest that N-terminal amidation of PMAP-23 provides structural stability and increases the peptide’s cationic charge, facilitating translocation into the bacterial inner membrane.     

Effects of tryptophan residues of porcine myeloid antibacterial peptide PMAP-23 on antibiotic activity.

PMAP-23 is a 23-residue antimicrobial peptide from porcine myeloid cells. In order to determine the effects of two Trp residues in positions 7 and 21 of PMAP-23 on antibacterial activity and phospholipid vesicle interacting property, two analogues in which Ala is substituted for Trp residue in position 7 or 21 were synthesized. A(21)-PMAP-23 exhibited reduced antibacterial activity and phospholipid vesicle disrupting activity when compared to those of PMAP-23 and A(7)-PMAP-23. PMAP-23 readily interacted with model lipid membrane and induced membrane destabilization. Therefore antibacterial activity induced by PMAP-23 is due to the interaction of cell membrane with peptide followed by membrane perturbation. A significant structural change on the SDS micelle was not found by Ala substitution of the Trp residue of PMAP-23. Also, there is a good correlation between hydrophobic interaction on RP-HPLC, expressed as retention time on RP-HPLC, and antibacterial activity. The vesicle titration experiment indicated that Trp residues located at near C-terminus are accessible to hydrophobic tail of phospholipid vesicle. This result suggests that the C-terminal end of PMAP-23 penetrates into the lipid bilayer in the course of the interaction with phospholipid membranes and is important for its antibacterial activity.     

Partitioning of 2,6-Bis(1H-Benzimidazol-2-yl)pyridine fluorophore into a phospholipid bilayer: complementary use of fluorescence quenching studies and molecular dynamics simulations

Successful use of fluorescence sensing in elucidating the biophysical properties of lipid membranes requires knowledge of the distribution and location of an emitting molecule in the bilayer. We report here that 2,6-bis(1H-benzimidazol-2-yl)pyridine (BBP), which is almost non-fluorescent in aqueous solutions, reveals a strong emission enhancement in a hydrophobic environment of a phospholipid bilayer, making it interesting for fluorescence probing of water content in a lipid membrane. Comparing the fluorescence behavior of BBP in a wide variety of solvents with those in phospholipid vesicles, we suggest that the hydrogen bonding interactions between a BBP fluorophore and water molecules play a crucial role in the observed “light switch effect”. Therefore, the loss of water-induced fluorescence quenching inside a membrane are thought to be due to deep penetration of BBP into the hydrophobic, water-free region of a bilayer. Characterized by strong quenching by transition metal ions in solution, BBP also demonstrated significant shielding from the action of the quencher in the presence of phospholipid vesicles. We used the increase in fluorescence intensity, measured upon titration of probe molecules with lipid vesicles, to estimate the partition constant and the Gibbs free energy (ΔG) of transfer of BBP from aqueous buffer into a membrane. Partitioning BBP revealed strongly favorable ΔG, which depends only slightly on the lipid composition of a bilayer, varying in a range from − 6.5 to − 7.0 kcal/mol. To elucidate the binding interactions of the probe with a membrane on the molecular level, a distribution and favorable location of BBP in a POPC bilayer were modeled via atomistic molecular dynamics (MD) simulations using two different approaches: (i) free, diffusion-driven partitioning of the probe molecules into a bilayer and (ii) constrained umbrella sampling of a penetration profile of the dye molecule across a bilayer. Both of these MD approaches agreed with regard to the preferred location of a BBP fluorophore within the interfacial region of a bilayer, located between the hydrocarbon acyl tails and the initial portion of the lipid headgroups. MD simulations also revealed restricted permeability of water molecules into this region of a POPC bilayer, determining the strong fluorescence enhancement observed experimentally for the membrane-partitioned form of BBP.     

Structural studies of porcine myeloid antibacterial peptide PMAP-23 and its analogues in DPC micelles by NMR spectroscopy.

PMAP-23 is a cathelicidin-derived antimicrobial peptide identified from porcine leukocytes. PMAP-23 was reported to show potent antimicrobial activity against Gram-negative and Gram-positive bacteria without hemolytic activity. To study the structure-antibiotic activity relationships of PMAP-23, two analogues by replacing Trp with Ala were synthesized and their tertiary structures bound to DPC micelles have been studied by NMR spectroscopy. PMAP-23 has two alpha-helices, one from Arg1 to Arg10 in the N-terminal region and the other from Phe18 to Arg23 in the C-terminal region. PMAP-1 (Trp(7)-->Ala) shows similar structure to PMAP-23, while PMAP-2 (Trp(21)-->Ala) has a random structure in the C-terminus. PMAP-2 was found to show less antibacterial and vesicle-disrupting activities than PMAP-23 and PMAP-1 [J. H. Kang, S. Y. Shin, S. Y. Jang, K. L. Kim, and K.-S. Hahm (1999) Biochem. Biophys. Res. Commun. 264, 281-286]. Trp(21) in PMAP-23 which induces an alpha-helical structure in the second alpha-helix is essential for the antibacterial activity of PMAP-23. Also, the fluorescence data proved that Trp(21) at the second alpha-helix is buried deep into the phospholipid in the membrane. Therefore, it implies that Trp(21) in the second alpha-helix at the C-terminus of PMAP-23 may play an important role on the interactions with the membrane and the flexible region including two proline residues may allow this alpha-helix to span the lipid bilayer.     

Implicit solvent model estimates of the stability of model structures of the alamethicin channel

Alamethicin is a hydrophobic helical peptide of 20 residues, which oligomerizes to form ion-conducting channels in membranes. The behavior of an intact alamethicin channel in POPC bilayers was recently studied, using 2 ns molecular dynamics (MD) simulations of a model hexameric channel. These simulations produced numerous conformations of the channel. In the present study, we used 11 of these channel conformations and carried out continuum-solvent model calculations, similar to those used for the monomers in our previous studies, to investigate the energetics of the channel inside the lipid bilayer. Our results suggest that, out of the 11 channel conformations produced by the MD simulations, only four are stable inside the lipid bilayer, with water-to-membrane free energies of transfer ranging from ~–6 to ~–10 kcal/mol. Analysis of the results suggests two causes for the apparent instability of the remainder of the structures inside the lipid bilayer, both resulting from the desolvation of channel polar groups (i.e. their transfer from the aqueous phase into the bilayer). The first is specific, uncompensated backbone hydrogen bonds, which exist in the region of the channel exposed to the hydrocarbon of the lipid bilayer. The second is exposure of intra-pore water molecules to the surrounding lipid. Thus, the association of these structures with the membrane involves a large electrostatic desolvation free-energy penalty. The apparent conflict between continuum-solvent and MD calculations, and its significance for the interpretation of membrane proteins simulations, are discussed.     

Regulation of channel function due to physical energetic coupling with a lipid bilayer

Regulation of membrane protein functions due to hydrophobic coupling with a lipid bilayer has been investigated. An energy formula describing interactions between lipid bilayer and integral ion channels with different structures, which is based on the screened Coulomb interaction approximation, has been developed. Here the interaction energy is represented as being due to charge-based interactions between channel and lipid bilayer. The hydrophobic bilayer thickness channel length mismatch is found to induce channel destabilization exponentially while negative lipid curvature linearly. Experimental parameters related to channel dynamics are consistent with theoretical predictions. To measure comparable energy parameters directly in the system and to elucidate the mechanism at an atomistic level we performed molecular dynamics (MD) simulations of the ion channel forming peptide–lipid complexes. MD simulations indicate that peptides and lipids experience electrostatic and van der Waals interactions for short period of time when found within each other’s proximity. The energies from these two interactions are found to be similar to the energies derived theoretically using the screened Coulomb and the van der Waals interactions between peptides (in ion channel) and lipids (in lipid bilayer) due to mainly their charge properties. The results of in silico MD studies taken together with experimental observable parameters and theoretical energetic predictions suggest that the peptides induce ion channels inside lipid membranes due to peptide–lipid physical interactions. This study provides a new insight helping better understand of the underlying mechanisms of membrane protein functions in cell membrane leading to important biological implications.     

Effect of membrane tension on the electric field and dipole potential of lipid bilayer membrane

The dipole potential of lipid bilayer membrane controls the difference in permeability of the membrane to oppositely charged ions. We have combined molecular dynamics (MD) simulations and experimental studies to determine changes in electric field and electrostatic potential of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer in response to applied membrane tension. MD simulations based on CHARMM36 force field showed that electrostatic potential of DOPC bilayer decreases by ~45mV in the physiologically relevant range of membrane tension values (0 to 15dyn/cm). The electrostatic field exhibits a peak (~0.8×10(9)V/m) near the water/lipid interface which shifts by 0.9? towards the bilayer center at 15dyn/cm. Maximum membrane tension of 15dyn/cm caused 6.4% increase in area per lipid, 4.7% decrease in bilayer thickness and 1.4% increase in the volume of the bilayer. Dipole-potential sensitive fluorescent probes were used to detect membrane tension induced changes in DOPC vesicles exposed to osmotic stress. Experiments confirmed that dipole potential of DOPC bilayer decreases at higher membrane tensions. These results are suggestive of a potentially new mechanosensing mechanism by which mechanically induced structural changes in the lipid bilayer membrane could modulate the function of membrane proteins by altering electrostatic interactions and energetics of protein conformational states.     

Numerical studies of the membrane fluorescent dyes dynamics in ground and excited states

Fluorescence methods are widely used in studies of biological and model membranes. The dynamics of membrane fluorescent markers in their ground and excited electronic states and correlations with their molecular surrounding within the fully hydrated phospholipid bilayer are still not well understood. In the present work, Quantum Mechanical (QM) calculations and Molecular Dynamics (MD) simulations are used to characterize location and interactions of two membrane polarity probes (Prodan; 6-propionyl-2-dimethylaminonaphthalene and its derivative Laurdan; 2-dimethylamino-6-lauroylnaphthalene) with the dioleoylphosphatidylcholine (DOPC) lipid bilayer model. MD simulations with fluorophores in ground and excited states are found to be a useful tool to analyze the fluorescent dye dynamics and their immediate vicinity. The results of QM calculations and MD simulations are in excellent agreement with available experimental data. The calculation shows that the two amphiphilic dyes initially placed in bulk water diffuse within 10 ns towards their final location in the lipid bilayer. Analysis of solvent relaxation process in the aqueous phase occurs on the picoseconds timescale whereas it takes nanoseconds at the lipid/water interface. Four different relaxation time constants, corresponding to different relaxation processes, where observed when the dyes were embedded into the membrane.     

Coarse-grained simulations of the membrane-active antimicrobial Peptide maculatin 1.1

Maculatin 1.1 (M1.1) is a membrane-active antimicrobial peptide (AMP) from an Australian tree frog that forms a kinked amphipathic α-helix in the presence of a lipid bilayer or bilayer-mimetic environment. To help elucidate its mechanism of membrane-lytic activity, we performed a total of ∼8 μs of coarse-grained molecular dynamics (CG-MD) simulations of M1.1 in the presence of zwitterionic phospholipid membranes. Several systems were simulated in which the peptide/lipid ratio was varied. At a low peptide/lipid ratio, M1.1 adopted a kinked, membrane-interfacial location, consistent with experiment. At higher peptide/lipid ratios, we observed spontaneous, cooperative membrane insertion of M1.1 peptide aggregates. The minimum size for formation of a transmembrane (TM) aggregate was just four peptides. The absence of a simple and well-defined central channel, along with the exclusion of lipid headgroups from the aggregates, suggests that a pore-like model is an unlikely explanation for the mechanism of membrane lysis by M1.1. We also performed an extended 1.25 μs simulation of the permeabilization of a complete liposome by multiple peptides. Consistent with the simpler bilayer simulations, formation of monomeric interfacial peptides and TM peptide clusters was observed. In contrast, major structural changes were observed in the vesicle membrane, implicating induced membrane curvature in the mechanism of active antimicrobial peptide lysis. This contrasted with the behavior of the nonpore-forming model peptide WALP23, which inserted into the vesicle to form extended clusters of TM α-helices with relatively little perturbation of bilayer properties.     

Molecular dynamics methods to predict peptide locations in membranes: LAH4 as a stringent test case

Determining the structure of membrane-active peptides inside lipid bilayers is essential to understand their mechanism of action. Molecular dynamics simulations can easily provide atomistic details, but need experimental validation. We assessed the reliability of self-assembling (or “minimum-bias”) and potential of mean force (PMF) approaches, using all-atom (AA) and coarse-grained (CG) force-fields. The LAH4 peptide was selected as a stringent test case, since it is known to attain different orientations depending on the protonation state of its four histidine residues.In all simulations the histidine side-chains inserted in the membrane when neutral, while they interacted with phospholipid headgroups in their charged state. This led to transmembrane orientations for neutral-His LAH4 in all minimum-bias AA simulations and in most CG trajectories. By contrast, the charged-His peptide stabilized membrane defects in AA simulations, whereas it was located at the membrane surface in some CG trajectories, and interacted with both lipid leaflets in others. This behavior is consistent with the higher antimicrobial activity and membrane-permeabilizing behavior of the charged-His LAH4. In addition, good agreement with solid-state NMR orientational data was observed in AA simulations.PMF calculations correctly predicted a higher membrane affinity for the neutral-His peptide. Interestingly, the structures and relative populations of PMF local free-energy minima corresponded to those determined in the less computationally demanding minimum-bias simulations.These data provide an indication about the possible membrane-perturbation mechanism of the charged-His LAH4 peptide: by interacting with lipid headgroups of both leaflets through its cationic side-chains, it could favor membrane defects and facilitate translocation across the bilayer.     

Role of calcium in reactive oxygen species-induced apoptosis in Candida albicans: an antifungal mechanism of antimicrobial peptide,PMAP-23

PMAP-23 (RIIDLLWRVRRPQKPKFVTVWVR-NH₂) is an antimicrobial peptide (AMP) derived from porcine myeloid. Membrane disruption is thought to underpin the anticandidal activity of PMAP-23. However, many AMPs act via mechanisms other than simple membrane permeabilisation. Here, we investigated the anticandidal mechanism of PMAP-23 at low concentrations. Membrane disruption and depolarisation and rapid K⁺ efflux were observed in Candida albicans cells treated with 5?µM PMAP-23. In contrast, 2.5?µM PMAP-23 caused membrane depolarisation and K⁺ efflux without membrane disruption. The lower PMAP-23 concentration increased cytosolic and mitochondrial Ca²⁺ levels. Disruption of Ca²⁺ homeostasis altered the NAD⁺/NADH ratio and resulted in reactive oxygen species (ROS) accumulation and glutathione oxidation. PMAP-23 treatment also stimulated apoptosis, as evidenced by metacaspase activation, DNA fragmentation, and phosphatidylserine externalisation. Pretreatment with the mitochondrial Ca²⁺ uptake inhibitor (ruthenium red) or ROS scavenger (N-acetylcysteine) attenuated these apoptotic events. Our results suggest that PMAP-23 induces apoptosis as antifungal mechanism, and mitochondrial Ca²⁺-induced ROS is major factor to trigger the apoptosis. Thus, the anticandidal activity of PMAP-23 is not based solely on disruption of biological membranes but also involves induction of apoptosis via mitochondrial Ca²⁺-dependent ROS. PMAP-23 mode of action sheds new light on the antifungal mechanism of antimicrobial peptides, supporting the role of Ca²⁺ and ROS in apoptosis regulation.     

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.     

Interaction of gabaergic ketones with model membranes: A molecular dynamics and experimental approach

γ-Aminobutyric-acid receptor (GABA_A-R), a membrane intrinsic protein, is activated by GABA and modulated by a wide variety of recognized drugs. GABA_A-R is also target for several insecticides which act by recognition of a non-competitive blocking site. Mentha oil is rich in several ketones with established activity against various insects/pests. Considering that mint ketones are highly lipophilic, their action mechanism could involve, at least in part, a non-specific receptor modulation by interacting with the surrounding lipids. In the present work, we studied in detail the effect on membranes of five cyclic ketones present in mint plants, with demonstrated insecticide and gabaergic activity. Particularly, we have explored their effect on the organization and dynamics of the membrane, by using Molecular Dynamics (MD) Simulation studies in a bilayer model of DPPC. We performed free diffusion MD and obtained spatially resolved free energy profiles of ketones partition into bilayers based on umbrella sampling. The most favored location of ketones in the membrane corresponded to the lower region of the carbonyl groups. Both hydrocarbon chains were slightly affected by the presence of ketones, presenting an ordering effect for the methylene groups closer to the carbonyl. MD simulations results were also contrasted with experimental data from fluorescence anisotropy studies which evaluate changes in membrane fluidity. In agreement, these assays indicated that the presence of ketones between lipid molecules induced an enhancement of the intermolecular interaction, increasing the molecular order throughout the bilayer thickness.     

Structural Plasticity in the Topology of the Membrane-Interacting Domain of HIV-1 gp41

We use a number of computational and experimental approaches to investigate the membrane topology of the membrane-interacting C-terminal domain of the HIV-1 gp41 fusion protein. Several putative transmembrane regions are identified using hydrophobicity analysis based on the Wimley-White scales, including the membrane-proximal external region (MPER). The MPER region is an important target for neutralizing anti-HIV monoclonal antibodies and is believed to have an interfacial topology in the membrane. To assess the possibility of a transmembrane topology of MPER, we examined the membrane interactions of a peptide corresponding to a 22-residue stretch of the MPER sequence (residues 662–683) using fluorescence spectroscopy and oriented circular dichroism. In addition to the previously reported interfacial location, we identify a stable transmembrane conformation of the peptide in synthetic lipid bilayers. All-atom molecular dynamics simulations of the MPER-derived peptide in a lipid bilayer demonstrate a stable helical structure with an average tilt of 24 degrees, with the five tryptophan residues sampling different environments inside the hydrocarbon core of the lipid bilayer, consistent with the observed spectral properties of intrinsic fluorescence. The degree of lipid bilayer penetration obtained by computer simulation was verified using depth-dependent fluorescence quenching of a selectively attached fluorescence probe. Overall, our data indicate that the MPER sequence can have at least two stable conformations in the lipid bilayer, interfacial and transmembrane, and suggest a possibility that external perturbations can switch the topology during physiological functioning.     

Interactions of the Anticancer Drug Tamoxifen with Lipid Membranes

Interactions of the hydrophobic anticancer drug tamoxifen (TAM) with lipid model membranes were studied using calcein-encapsulated vesicle leakage, attenuated total reflection Fourier transform infrared (FTIR) spectroscopy, small-angle neutron scattering (SANS), atomic force microscopy (AFM) based force spectroscopy, and all-atom molecular dynamics (MD) simulations. The addition of TAM enhances membrane permeability, inducing calcein to translocate from the interior to the exterior of lipid vesicles. A large decrease in the FTIR absorption band’s magnitude was observed in the hydrocarbon chain region, suggesting suppressed bond vibrational dynamics. Bilayer thickening was determined from SANS data. Force spectroscopy measurements indicate that the lipid bilayer area compressibility modulus K_A is increased by a large amount after the incorporation of TAM. MD simulations show that TAM decreases the lipid area and increases chain order parameters. Moreover, orientational and positional analyses show that TAM exhibits a highly dynamic conformation within the lipid bilayer. Our detailed experimental and computational studies of TAM interacting with model lipid membranes shed new light on membrane modulation by TAM.     

The Structural Dynamics of the Flavivirus Fusion Peptide–Membrane Interaction

Membrane fusion is a crucial step in flavivirus infections and a potential target for antiviral strategies. Lipids and proteins play cooperative roles in the fusion process, which is triggered by the acidic pH inside the endosome. This acidic environment induces many changes in glycoprotein conformation and allows the action of a highly conserved hydrophobic sequence, the fusion peptide (FP). Despite the large volume of information available on the virus-triggered fusion process, little is known regarding the mechanisms behind flavivirus–cell membrane fusion. Here, we evaluated the contribution of a natural single amino acid difference on two flavivirus FPs, FLA_G (⁹⁸DRGWGNGCGLFGK¹¹⁰) and FLA_H (⁹⁸DRGWGNHCGLFGK¹¹⁰), and investigated the role of the charge of the target membrane on the fusion process. We used an in silico approach to simulate the interaction of the FPs with a lipid bilayer in a complementary way and used spectroscopic approaches to collect conformation information. We found that both peptides interact with neutral and anionic micelles, and molecular dynamics (MD) simulations showed the interaction of the FPs with the lipid bilayer. The participation of the indole ring of Trp appeared to be important for the anchoring of both peptides in the membrane model, as indicated by MD simulations and spectroscopic analyses. Mild differences between FLA_G and FLA_H were observed according to the pH and the charge of the target membrane model. The MD simulations of the membrane showed that both peptides adopted a bend structure, and an interaction between the aromatic residues was strongly suggested, which was also observed by circular dichroism in the presence of micelles. As the FPs of viral fusion proteins play a key role in the mechanism of viral fusion, understanding the interactions between peptides and membranes is crucial for medical science and biology and may contribute to the design of new antiviral drugs.     

Vstx1, a modifier of Kv channel gating, localizes to the interfacial region of lipid bilayers

VSTx1 is a tarantula venom toxin which binds to the archaebacterial voltage-gated potassium channel KvAP. VSTx1 is thought to access the voltage sensor domain of the channel via the lipid bilayer phase. In order to understand its mode of action and implications for the mechanism of channel activation, it is important to characterize the interactions of VSTx1 with lipid bilayers. Molecular dynamics (MD) simulations (for a total simulation time in excess of 0.2 micros) have been used to explore VSTx1 localization and interactions with zwitterionic (POPC) and with anionic (POPE/POPG) lipid bilayers. In particular, three series of MD simulations have been used to explore the net drift of VSTx1 relative to the center of a bilayer, starting from different locations of the toxin. The preferred location of the toxin is at the membrane/water interface. Although there are differences between POPC and POPE/POPG bilayers, in both cases the toxin forms favorable interactions at the interface, maximizing H-bonding to lipid headgroups and to water molecules while retaining interactions with the hydrophobic core of the bilayer. A 30 ns unrestrained simulation reveals dynamic partitioning of VSTx1 into the interface of a POPC bilayer. The preferential location of VSTx1 at the interface is discussed in the context of Kv channel gating models and provides support for a mode of action in which the toxin interacts with the Kv voltage sensor "paddle" formed by the S3 and S4 helices.     

2H-NMR study and molecular dynamics simulation of the location, alignment, and mobility of pyrene in POPC bilayers

The alignment of pyrene in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer was investigated using two different approaches, namely solid-state (2)H-NMR spectroscopy and molecular dynamics (MD) simulations. Quadrupolar splittings from (2)H-NMR spectra of deuterated pyrene-d(10) in an oriented lipid bilayer give information about the orientation of C-D bonds with respect to the membrane normal. From MD simulations, geometric information is accessible via trajectories. By defining molecular and bond order parameters, the data from MD trajectories and NMR spectra can be compared straightforwardly. To ensure that the results from both methods are comparable, parameters of the experimental and the simulation setup were chosen to be as similar as possible. From simulations, we saw that pyrene prefers a position inside the lipid membrane near the headgroups and has no tendency to diffuse from one monolayer of the membrane to the other. The results from simulation and NMR show that the normal of the molecular plane is aligned nearly perpendicular to the bilayer normal. The long axis of pyrene lies preferentially parallel to the bilayer normal within a range of +/-30 degrees . The results from the two different methods are remarkably consistent. The good agreement can be explained by the fact that the different kind of motions of a pyrene molecule are already averaged within a few nanoseconds, which is the timescale covered by the MD simulation.