Conformation and dynamics of melittin bound to magnetically oriented lipid bilayers by solid-state (31)P and (13)C NMR spectroscopy

The conformation and dynamics of melittin bound to the dimyristoylphosphatidylcholine (DMPC) bilayer and the magnetic orientation in the lipid bilayer systems were investigated by solid-state (31)P and (13)C NMR spectroscopy. Using (31)P NMR, it was found that melittin-lipid bilayers form magnetically oriented elongated vesicles with the long axis parallel to the magnetic field above the liquid crystalline-gel phase transition temperature (T(m) = 24 degrees C). The conformation, orientation, and dynamics of melittin bound to the membrane were further determined by using this magnetically oriented lipid bilayer system. For this purpose, the (13)C NMR spectra of site-specifically (13)C-labeled melittin bound to the membrane in the static, fast magic angle spinning (MAS) and slow MAS conditions were measured. Subsequently, we analyzed the (13)C chemical shift tensors of carbonyl carbons in the peptide backbone under the conditions where they form an alpha-helix and reorient rapidly about the average helical axis. Finally, it was found that melittin adopts a transmembrane alpha-helix whose average axis is parallel to the bilayer normal. The kink angle between the N- and C-terminal helical rods of melittin in the lipid bilayer is approximately 140 degrees or approximately 160 degrees, which is larger than the value of 120 degrees determined by x-ray diffraction studies. Pore formation was clearly observed below the T(m) in the initial stage of lysis by microscope. This is considered to be caused by the association of melittin molecules in the lipid bilayer.     

A molecular dynamics study of the bee venom melittin in aqueous solution, in methanol, and inserted in a phospholipid bilayer

The structural properties of melittin, a small amphipathic peptide found in the bee venom, are investigated in three different environments by molecular dynamics simulation. Long simulations have been performed for monomeric melittin solvated in water, in methanol, and shorter ones for melittin inserted in a dimyristoylphosphatidylcholine bilayer. The resulting trajectories were analysed in terms of structural properties of the peptide and compared to the available NMR data. While in water and methanol solution melittin is observed to partly unfold, the peptide retains its structure when embedded in a lipid bilayer. The latter simulation shows good agreement with the experimentally derived ³J-coupling constants. Generally, it appears that higher the stability of the helical conformation of melittin, lower is the dielectric permittivity of the environment. In addition, peptide-lipid interactions were investigated showing that the C-terminus of the peptide provides an anchor to the lipid bilayer by forming hydrogen bonds with the lipid head groups.     

Analysis of antimicrobial peptide interactions with hybrid bilayer membrane systems using surface plasmon resonance

The lipid binding behaviour of the antimicrobial peptides magainin 1, melittin and the C-terminally truncated analogue of melittin (21Q) was studied with a hybrid bilayer membrane system using surface plasmon resonance. In particular, the hydrophobic association chip was used which is composed of long chain alkanethiol molecules upon which liposomes adsorb spontaneously to create a hybrid bilayer membrane surface. Multiple sets of sensorgrams with different peptide concentrations were generated. Linearisation analysis and curve fitting using numerical integration analysis were performed to derive estimates for the association (k(a)) and dissociation (k(d)) rate constants. The results demonstrated that magainin 1 preferentially interacted with negatively charged dimyristoyl-L-alpha-phosphatidyl-DL-glycerol (DMPG), while melittin interacted with both zwitterionic dimyristoyl-L-alpha-phosphatidylcholine and anionic DMPG. In contrast, the C-terminally truncated melittin analogue, 21Q, exhibited lower binding affinity for both lipids, showing that the positively charged C-terminus of melittin greatly influences its membrane binding properties. Furthermore the results also demonstrated that these antimicrobial peptides bind to the lipids initially via electrostatic interactions which then enhances the subsequent hydrophobic binding. The biosensor results were correlated with the conformation of the peptides determined by circular dichroism analysis, which indicated that high alpha-helicity was associated with high binding affinity. Overall, the results demonstrated that biosensor technology provides a new experimental approach to the study of peptide-membrane interactions through the rapid determination of the binding affinity of bioactive peptides for phospholipids.     

Molecular dynamics simulation of melittin in a dimyristoylphosphatidylcholine bilayer membrane.

Molecular dynamics trajectories of melittin in an explicit dimyristoyl phosphatidylcholine (DMPC) bilayer are generated to study the details of lipid-protein interactions at the microscopic level. Melittin, a small amphipathic peptide found in bee venom, is known to have a pronounced effect on the lysis of membranes. The peptide is initially set parallel to the membrane-solution interfacial region in an alpha-helical conformation with unprotonated N-terminus. Solid-state nuclear magnetic resonance (NMR) and polarized attenuated total internal reflectance Fourier transform infrared (PATIR-FTIR) properties of melittin are calculated from the trajectory to characterize the orientation of the peptide relative to the bilayer. The residue Lys7 located in the hydrophobic moiety of the helix and residues Lys23, Arg24, Gln25, and Gln26 at the C-terminus hydrophilic form hydrogen bonds with water molecules and with the ester carbonyl groups of the lipids, suggesting their important contribution to the stability of the helix in the bilayer. Lipid acyl chains are closely packed around melittin, contributing to the stable association with the membrane. Calculated density profiles and order parameters of the lipid acyl chains averaged over the molecular dynamics trajectory indicate that melittin has effects on both layers of the membrane. The presence of melittin in the upper layer causes a local thinning of the bilayer that favors the penetration of water through the lower layer. The energetic factors involved in the association of melittin at the membrane surface are characterized using an implicit mean-field model in which the membrane and the surrounding solvent are represented as structureless continuum dielectric material. The results obtained by solving the Poisson-Bolztmann equation numerically are in qualitative agreement with the detailed dynamics. The influence of the protonation state of the N-terminus of melittin is examined. After 600 ps, the N-terminus of melittin is protonated and the trajectory is continued for 400 ps, which leads to an important penetration of water molecules into the bilayer. These observations provide insights into how melittin interacts with membranes and the mechanism by which it enhances their lysis.     

Modulation of melittin-induced lysis by surface charge density of membranes.

Phosphorus NMR spectroscopy was used to characterize the importance of electrostatic interactions in the lytic activity of melittin, a cationic peptide. The micellization induced by melittin has been characterized for several lipid mixtures composed of saturated phosphatidylcholine (PC) and a limited amount of charged lipid. For these systems, the thermal polymorphism is similar to the one observed for pure PC: small comicelles are stable in the gel phase and extended bilayers are formed in the liquid crystalline phase. Vesicle surface charge density influences strongly the micellization. Our results show that the presence of negatively charged lipids (phospholipid or unprotonated fatty acid) reduces the proportion of lysed vesicles. Conversely, the presence of positively charged lipids leads to a promotion of the lytic activity of the peptide. The modulation of the lytic effect is proposed to originate from the electrostatic interactions between the peptide and the bilayer surface. Attractive interactions anchor the peptide at the surface and, as a consequence, inhibit its lytic activity. Conversely, repulsive interactions favor the redistribution of melittin into the bilayer, causing enhanced lysis. A quantitative analysis of the interaction between melittin and negatively charged bilayers suggests that electroneutrality is reached at the surface, before micellization. The surface charge density of the lipid layer appears to be a determining factor for the lipid/peptide stoichiometry of the comicelles; a decrease in the lipid/peptide stoichiometry in the presence of negatively charged lipids appears to be a general consequence of the higher affinity of melittin for these membranes.     

Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes

The binding of bee venom melittin to negatively charged unilamellar vesicles and planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was studied with circular dichroism and deuterium NMR spectroscopy. The melittin binding isotherm was measured for small unilamellar vesicles containing 10 or 20 mol % POPG. Due to electrostatic attraction, binding of the positively charged melittin was much enhanced as compared to the binding to neutral lipid vesicles. However, after correction for electrostatic effects by means of the Gouy-Chapman theory, all melittin binding isotherms could be described by a partition Kp = (4.5 +/- 0.6) x 10(4) M-1. It was estimated that about 50% of the total melittin surface was embedded in a hydrophobic environment. The melittin partition constant for small unilamellar vesicles was by a factor of 20 larger than that of planar bilayers and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium NMR studies were performed with coarse lipid dispersions. Binding of melittin to POPC/POPG (80/20 mol/mol) membranes caused systematic changes in the conformation of the phosphocholine and phosphoglycerol head groups which were ascribed to the influence of electrostatic charge on the choline dipole. While the negative charge of phosphatidylglycerol moved the N+ end of the choline -P-N+ dipole toward the bilayer interior, the binding of melittin reversed this effect and rotated the N+ end toward the aqueous phase. No specific melittin-POPG complexes could be detected. The phosphoglycerol head group was less affected by melittin binding than its choline counterpart.     

Penetratin-membrane association: W48/R52/W56 shield the peptide from the aqueous phase

Using molecular dynamics simulations, we studied the mode of association of the cell-penetrating peptide penetratin with both a neutral and a charged bilayer. The results show that the initial peptide-lipid association is a fast process driven by electrostatic interactions. The homogeneous distribution of positively charged residues along the axis of the helical peptide, and especially residues K46, R53, and K57, contribute to the association of the peptide with lipids. The bilayer enhances the stability of the penetratin helix. Oriented parallel to the lipid-water interface, the subsequent insertion of the peptide through the bilayer headgroups is significantly slower. The presence of negatively charged lipids considerably enhances peptide binding. Lateral side-chain motion creates an opening for the helix into the hydrophobic core of the membrane. The peptide aromatic residues form a pi-stacking cluster through W48/R52/W56 and F49/R53, protecting the peptide from the water phase. Interaction with the penetratin peptide has only limited effect on the overall membrane structure, as it affects mainly the conformation of the lipids which interact directly with the peptide. Charge matching locally increases the concentration of negatively charged lipids, lateral lipid diffusion locally decreases. Lipid disorder increases, through decreased order parameters of the lipids interacting with the penetratin side chains. Penetratin molecules at the membrane surface do not seem to aggregate.     

The interaction of bee melittin with lipid bilayer membranes

The influence of melittin and the related 8-26 peptide on the stability and electrical properties of bilayer lipid membranes is reported. Melittin, unlike the 8-26 peptide, has a dramatic influence on lipid membranes, causing rupture at dilute concentrations. The circular dichroism of melittin demonstrated that under physiological conditions, in water, melittin is in extended conformation, which is enhanced in aqueous ethanol. However in 'membrane-like' conditions it is essentially alpha-helical. Secondary structure predictions were used to locate possible alpha-helical nucleation centres and a model of melittin was built according to these predictions. It is postulated that melittin causes a wedge effect in membranes.     

Bilayer localization of membrane-active peptides studied in biomimetic vesicles by visible and fluorescence spectroscopies.

Depth of bilayer penetration and effects on lipid mobility conferred by the membrane-active peptides magainin, melittin, and a hydrophobic helical sequence KKA(LA)7KK (denoted KAL), were investigated by colorimetric and time-resolved fluorescence techniques in biomimetic phospholipid/poly(diacetylene) vesicles. The experiments demonstrated that the extent of bilayer permeation and peptide localization within the membrane was dependent upon the bilayer composition, and that distinct dynamic modifications were induced by each peptide within the head-group environment of the phospholipids. Solvent relaxation, fluorescence correlation spectroscopy and fluorescence quenching analyses, employing probes at different locations within the bilayer, showed that magainin and melittin inserted close to the glycerol residues in bilayers incorporating negatively charged phospholipids, but predominant association at the lipid-water interface occurred in bilayers containing zwitterionic phospholipids. The fluorescence and colorimetric analyses also exposed the different permeation properties and distinct dynamic influence of the peptides: magainin exhibited the most pronounced interfacial attachment onto the vesicles, melittin penetrated more into the bilayers, while the KAL peptide inserted deepest into the hydrophobic core of the lipid assemblies. The solvent relaxation results suggest that decreasing the lipid fluidity might be an important initial factor contributing to the membrane activity of antimicrobial peptides.     

Structural properties of signal peptides and their membrane insertion

Structural properties of the amino acid sequences from 22 signal peptides have been analyzed and compared with peptides known to interact with biological membranes and liposomes, melittin, a lytic peptide of bee venom, and the non-polar C-terminal segment of cytochrome b5. All these peptides evidence a double amphipatic structure with an hydrophobic core of 9 to 24 amino acid residues and two charged polar ends. They all exhibit a high potential for making alpha-helix and, to a lesser degree, extended or beta-sheet conformation with low or negative potentials for making reverse turns or aperiodic conformation. A model of spontaneous insertion of these peptides into the lipid bilayer without specific surface receptor protein is proposed, where the two polar ends interact with each polar face of the lipid bilayer and the hydrophobic core inserts into the non-hydrogen bonding environment of the fatty acid side chains. This insertion could be the molecular trigger for ribophorin assembly around the signal peptide and subsequent attachment to the ribosome prior to the transfer of the polypeptide chain through the endoplasmic reticulum membrane.     

Structural and thermodynamic analyses of the interaction between melittin and lipopolysaccharide

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is the very first site of interactions with the antimicrobial peptides. In this work, we have determined a solution conformation of melittin, a well-known membrane active amphiphilic peptide from honey bee venom, by transferred nuclear Overhauser effect (Tr-NOE) spectroscopy in its bound state with lipopolysaccharide. The LPS bound conformation of melittin is characterized by a helical structure restricted only to the C-terminus region (residues A15-R24) of the molecule. Saturation transfer difference (STD) NMR studies reveal that several C-terminal residues of melittin including Trp19 are in close proximity with LPS. Isothermal titration calorimetry (ITC) data demonstrates that melittin binding to LPS or lipid A is an endothermic process. The interaction between melittin and lipid A is further characterized by an equilibrium association constant (Ka) of 2.85 x 10(6) M(-1) and a stoichiometry of 0.80, melittin/lipid A. The estimated free energy of binding (delta G0), -8.8 kcal mol(-1), obtained from ITC experiments correlates well with a partial helical structure of melittin in complex with LPS. Moreover, a synthetic peptide fragment, residues L13-Q26 or mel-C, derived from the C-terminus of melittin has been found to contain comparable outer membrane permeabilizing activity against Escherichia coli cells. Intrinsic tryptophan fluorescence experiments of melittin and mel-C demonstrate very similar emission maxima and quenching in presence of LPS micelles. The Red Edge Excitation Shift (REES) studies of tryptophan residue indicate that both peptides are located in very similar environment in complex with LPS. Collectively, these results suggest that a helical conformation of melittin, at its C-terminus, could be an important element in recognition of LPS in the outer membrane.     

Insertion and pore formation driven by adsorption of proteins onto lipid bilayer membrane-water interfaces

We describe the binding of proteins to lipid bilayers in the case for which binding can occur either by adsorption to the lipid bilayer membrane-water interface or by direct insertion into the bilayer itself. We examine in particular the case when the insertion and pore formation are driven by the adsorption process using scaled particle theory. The adsorbed proteins form a two-dimensional "surface gas" at the lipid bilayer membrane-water interface that exerts a lateral pressure on the lipid bilayer membrane. Under conditions of strong intrinsic binding and a high degree of interfacial converge, this pressure can become high enough to overcome the energy barrier for protein insertion. Under these conditions, a subtle equilibrium exists between the adsorbed and inserted proteins. We propose that this provides a control mechanism for reversible insertion and pore formation of proteins such as melittin and magainin. Next, we discuss experimental data for the binding isotherms of cytochrome c to charged lipid membranes in the light of our theory and predict that cytochrome c inserts into charged lipid bilayers at low ionic strength. This prediction is supported by titration calorimetry results that are reported here. We were furthermore able to describe the observed binding isotherms of the pore-forming peptides endotoxin (alpha 5-helix) and of pardaxin to zwitterionic vesicles from our theory by assuming adsorption/insertion equilibrium.     

Membrane association and selectivity of the antimicrobial peptide NK‐2: a molecular dynamics simulation study

In an effort to better understand the initial mechanism of selectivity and membrane association of the synthetic antimicrobial peptide NK‐2, we have applied molecular dynamics simulation techniques to elucidate the interaction of the peptide with the membrane interfaces. A homogeneous dipalmitoylphosphatidylglycerol (DPPG) and a homogeneous dipalmitoylphosphatidylethanolamine (DPPE) bilayers were taken as model systems for the cytoplasmic bacterial and human erythrocyte membranes, respectively. The results of our simulations on DPPG and DPPE model membranes in the gel phase show that the binding of the peptide, which is considerably stronger for the negatively charged DPPG lipid bilayer than for the zwitterionic DPPE, is mostly governed by electrostatic interactions between negatively charged residues in the membrane and positively charged residues in the peptide. In addition, a characteristic distribution of positively charged residues along the helix facilitates a peptide orientation parallel to the membrane interface. Once the peptides reside close to the membrane surface of DPPG with the more hydrophobic side chains embedded into the membrane interface, the peptide initially disturbs the respective bilayer integrity by a decrease of the order parameter of lipid acyl chain close to the head group region, and by a slightly decrease in bilayer thickness. We found that the peptide retains a high content of helical structure on the zwitterionic membrane‐water interface, while the loss of α‐helicity is observed within a peptide adsorbed onto negatively charged lipid membranes. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Phase separations induced by melittin in negatively-charged phospholipid bilayers as detected by fluorescence polarization and differential scanning calorimetry

Interactions between melittin and a variety of negatively-charged lipid bilayers have been investigated by intrinsic fluorescence, fluorescence polarization of 1,6-diphenylhexatriene and differential scanning calorimetry. (1) Intrinsic fluorescence of the single tryptophan residue of melittin shows that binding of this peptide to negatively-charged phospholipids is directly related to the surface charge density, but is unaffected by the physical state of lipids, fluid or gel, single-shell vesicles or unsonicated dispersions. (2) Changes in the thermotropic properties of negatively-charged lipids upon melittin binding allow to differentiate two groups of lipids: (i) A progressive disappearance of the transition, without any shift in temperature, is observed with monoacid C₁₄ lipids such as dimyristoylphosphatidylglycerol and -serine (group 1). (ii) With a second group of lipids (group 2), a transition occurs even at melittin saturation, and two transitions are detected at intermediate melittin content, one corresponding to remaining unperturbed lipids, the other shifted downward by 10–20°C. This second group of lipids is constituted by monoacid C₁₆ lipids, dipalmitoylphosphatidylglycerol and -serine. Phosphatidic acids also enter this classification, but it is the net charge of the phosphate group which allows to discriminate: singly charged phosphatidic acids belong to group 2, whereas totally ionized ones behave like group 1 lipids, whatever the chain length. (3) It is concluded that melittin induces phase separations between unperturbed lipid regions which give a transition at the same temperature as pure lipid, and peptide rich domains in which the stoichiometry is 1 toxin per 8 phospholipids. The properties of such domains depend on the bilayer stability: in the case of C₁₆ aliphatic chains and singly charged polar heads, the lipid-peptide domains have a transition at a lower temperature than the pure lipid. With shorter C₁₄ chains or with two net charges by polar group, the bilayer structure is probably totally disrupted, and the new resulting phase can no longer lead to a cooperative transition.     

Molecular details of melittin-induced lysis of phospholipid membranes as revealed by deuterium and phosphorus NMR

Solid-state deuterium and phosphorus-31 nuclear magnetic resonance (2H and 31P NMR) studies of deuterium-enriched phosphatidylcholine [( 3',3'-2H2]DPPC, [sn-2-2H31]DPPC) and ditetradecylphosphatidylglycerol (DMPG-diether), as water dispersions, were undertaken to investigate the action of melittin on zwitterionic and negatively charged membrane phospholipids. When the lipid-to-protein ratio (Ri) is greater than or equal to 20, the 2H and 31P NMR spectral features indicate that the system is constituted by large bilayer structures of several thousand angstrom curvature radius, at T greater than Tc (Tc, temperature of "gel-to-liquid crystal" phase transition of pure lipid dispersions). At T approximately Tc, a detailed analysis of the lipid chain ordering shows that melittin induces a slight disordering of the "plateau" positions concomitantly with a substantial ordering of positions near the bilayer center. At T much greater than Tc, an apparent general chain disordering is observed. These findings suggest that melittin is in contact with the acyl chain segments and that its position within the bilayer may depend on the temperature. On a cooling down below Tc, for Ri greater than 20, two-phase spectra are observed, i.e., narrow single resonances superimposed on gel-type phosphorus and deuterium powder patterns. These narrow resonances are characteristic of small structures (vesicles, micelles, ... of a few hundred angstrom curvature radius) undergoing fast isotropic reorientation, which averages to zero both the quadrupolar and chemical shift anisotropy interactions. On an increase of the temperature above Tc, the NMR spectra indicate that the system returns reversibly to large bilayer structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Design and characterization of anchoring amphiphilic peptides and their interactions with lipid vesicles.

In an effort to develop a polymer/peptide assembly for the immobilization of lipid vesicles, we have made and characterized four water-soluble amphiphilic peptides designed to associate spontaneously and strongly with lipid vesicles without causing significant leakage from anchored vesicles. These peptides have a primary amphiphilic structure with the following sequences: AAAAAAAAAAAAWKKKKKK, AALLLAAAAAAAAAAAAAAAAAAAWKKKKKK, and KKAALLLAAAAAAAAAAAAAAAAAAAWKKKKKK and its reversed homologue KKKKKKWAAAAA AAAAAAAAAAAAAALLLAAKK. Two of the four peptides have their hydrophobic segments capped at both termini with basic residues to stabilize the transmembrane orientation and to increase the affinity for negatively charged vesicles. We have studied the secondary structure and the membrane affinity of the peptides as well as the effect of the different peptides on the membrane permeability. The influence of the hydrophobic length and the role of lysine residues were clearly established. First, a hydrophobic segment of 24 amino acids, corresponding approximately to the thickness of a lipid bilayer, improves considerably the affinity to zwitterionic lipids compared to the shorter one of 12 amino acids. The shorter peptide has a low membrane affinity since it may not be long enough to adopt a stable conformation. Second, the presence of lysine residues is essential since the binding is dominated by electrostatic interactions, as illustrated by the enhanced binding with anionic lipids. The charges at both ends, however, prevent the peptide from inserting spontaneously in the bilayer since it would involve the translocation of a charged end through the apolar core of the bilayer. The direction of the amino acid sequence of the peptide has no significant influence on its behavior. None of these peptides perturbs membrane permeability even at an incubation lipid to peptide molar ratio of 0.5. Among the four peptides, AALLLAAAAAAAAAAAAAAAAAAAWKKKKKK is identified as the most suitable anchor for the immobilization of lipid vesicles.     

Influence of the arrangement and secondary structure of melittin peptides on the formation and stability of toroidal pores

Melittin interactions with lipid bilayers and melittin formed pores are extensively studied to understand the mechanism of the toroidal pore formation. Early experimental studies suggested that melittin peptide molecules are anchored by their positively charged residues located next to the C-terminus to only one leaflet of the lipid bilayer (asymmetric arrangement). However, the recent non-linear spectroscopic experiment suggests a symmetric arrangement of the peptides with the C-terminus of the peptides anchored to both bilayers. Therefore, we present here a computational study that compares the effect of symmetric and asymmetric arrangements of melittin peptides in the toroidal pore formation. We also investigate the role of the peptide secondary structure during the pore formation. Two sets of the symmetric and asymmetric pores are prepared, one with a helical peptide from the crystal structure and the other set with a less helical peptide. We observe a stable toroidal pore being formed only in the system with a symmetric arrangement of the less helical peptides. Based on the simulation results we propose that the symmetric arrangement of the peptides might be more favorable than the asymmetric arrangement, and that the helical secondary structure is not a prerequisite for the formation of the toroidal pore.     

Melittin and the 8-26 fragment. Differences in ionophoric properties as measured by monolayer method

Melittin is a major (approximately 50%) protein component of bee venom. This peptide is an amphiphilic protein, because, while the amino acid residues 1-20 are predominantly hydrophobic (with the exception of Lys-7), residues 21-26 are hydrophilic. The binding properties to vesicles and lipid bilayers of melittin have provided much useful information regarding biological (hemolytic) activity (Habermann, E., 1972, Science [Wash. DC], 177:314-322). Recent studies have convincingly established that the melittin monolayer (at air-water interface) model membrane system allows one to analyze the various forces present in such structures. We present comparative monolayer studies of melittin and the peptide fragment 8-26 regarding the channel formation for the selective anion (Cl-) penetration in monolayers, analogous to melittin (tetramer) channel function in lipid bilayer. The differences in surface pressure and surface potential of monolayers between native melittin and the 8-26 fragment suggest that these may be ascribed to Lys-7.     

Cholesterol promotes the interaction of Alzheimer β-amyloid monomer with lipid bilayer

Cholesterol in the plasma membrane plays an important role in the pathogenesis of Alzheimer's disease, but the exact function of cholesterol in the regulation of amyloid-β (Aβ) generation, aggregation, and toxicity remains elusive. To gain insight into the bioactivity of cholesterol, we investigate the effect of cholesterol levels on the interaction of Aβ(1-42) monomer with the zwitterionic 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer containing different mole fractions of cholesterol from χ=0, 0.2, to 0.4 using all-atom molecular dynamics simulations. Simulation results show that an increased cholesterol level alters the structure, dynamics, and surface chemistry of the lipid bilayer, leading to increased bilayer thickness, hydrophobic chain order, surface hydrophobicity, and decreased lipid mobility. All these effects significantly promote the binding of Aβ to the lipid bilayer. Mechanistically, the adsorption of Aβ on the bilayer is a cooperative process. First, charged residues act as anchors to establish the initial binding of Aβ to phosphate headgroups of the bilayer driven by electrostatic interactions, which further facilitates hydrophobic residues to reside on the bilayer. Once hydrophobic residues especially from C-terminus are locked on the bilayer, the interactions among charged residues, lipid bilayer, and calcium ions are optimized to provide additional attractive forces to stabilize Aβ adsorbed on or inserted into the lipid bilayer. Inclusion of cholesterol makes this binding process more energetically favorable. Upon adsorption on the bilayer, Aβ appears to preferentially adopt α-helical or unstructured conformation. This work supports that cholesterol acts as a promoter for Aβ--membrane interactions, which would facilitate Aβ aggregation and membrane insertion.     

Site-specific deuterium order parameters and membrane-bound behavior of a peptide fragment from the intracellular domain of HIV-1 gp41.

The behavior of the cytolytic peptide fragment 828-848 (P828) from the carboxy-terminus of the envelope glycoprotein gp41 of HIV-1 in membranes was investigated by solid-state 2H NMR on P828 with the selectively deuterated isoleucines I3, I13, I16, and I20. The quadrupole splittings of the I3 side chain show significant sensitivity to the main phase-transition temperature of the lipid, consistent with partial penetration of the N-terminal peptide region into the hydrophobic core of the membrane. In contrast, the quadrupole splittings of I13, I16, and I20 are in agreement with a location of the C-terminal portion of the peptide near the lipid/water interface. The perturbation of the bilayer by the peptide was studied by 2H NMR on sn-1 chain deuterated 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine membranes. Peptide incorporation results in a significant reduction of lipid chain order toward the bilayer center, but only a modest reduction near the lipid glycerol. These observations suggest a penetration of the partially structured peptide backbone into the membrane/water interface region that reduces lateral packing density and decreases order in the hydrophobic core. In addition, the structure of the peptide was investigated free in water and bound to SDS micelles by high-resolution NMR. P828 is unstructured in water but exists in a flexible partially helical conformation when bound to negatively charged liposomes or micelles. The flexible helix covers the first 14 residues of the peptide, whereas the C-terminus of the peptide, where three of the six positively charged arginine residues are located, appears to be unstructured. The peptide-induced changes in lipid chain order profiles indicate that membrane curvature stress is the driving force for the cytolytic behavior of P828.