Shape transformation of lipid vesicles induced by diffusing macromolecules

The attachment of macromolecules to the surface of a lipid vesicle may cause its deformations such as budding or creation of cylindrical protrusions. Diffusion of the macromolecules in the membranes may cause its shape transformations. The process of shrinking the protrusions due to diffusion of the macromolecules is investigated. It is assumed that macromolecules modify locally the spontaneous curvature and bending rigidity of the lipid membrane. Both spontaneous curvature and bending rigidities depend on the concentration of membrane components. It has been shown that cylindrical protrusions are created when the macromolecules which induce large spontaneous curvature are accumulated at a piece of the vesicle surface. It has been observed that here the elastic constants influence very little the evolution of the vesicle shape caused by diffusing macromolecules and the most important is the value the spontaneous curvature imposed by the macromolecules.     

Rationalizing the membrane interactions of cationic amphipathic antimicrobial peptides by their molecular shape

Biophysical and structural studies of cationic amphipathic antimicrobial peptides have revealed new mechanistic details concerning their membrane interactions. In interfacial environments the peptides adopt amphipathic conformations and the resulting distribution of polar, charged and hydrophobic residues allows them to partition into the bilayer interface. For several helical peptides it was found that their long axis is oriented parallel to the membrane surface, an arrangement which results in considerable perturbations in the packing of the lipid bilayer. Within the molecular shape concept the peptides act as wedge-like structures which impose positive curvature strain on the membrane. As a consequence a wide variety of morphologies are observed of peptide–lipid mixtures which strongly depend on the detailed peptide sequence, the membrane lipid composition, buffer, temperature and other environmental parameters. Therefore, the peptide–lipid systems are best described by phase diagrams, similar to the ones of detergent–lipid mixtures, encompassing on the one extreme regions where the peptide stabilizes the bilayer and on the other extreme regions where membrane lysis occurs. The effects of peptide sequence, membrane penetration depth, lipid composition and membrane surface charge density on membrane-association, -morphology and the resulting phase boundaries are discussed.     

Coupling of bending and stretching deformations in vesicle membranes

Biomimetic membranes are fluid and can undergo two different elastic deformations, bending and stretching. The bending of a membrane is primarily governed by two elastic parameters: its spontaneous (or preferred) curvature m and its bending rigidity κ. These two parameters define an intrinsic tension scale, the spontaneous tension 2 κm². Membrane stretching and compression, on the other hand, are determined by the mechanical tension acting within the membrane. For vesicle membranes, the two elastic deformations are coupled via the enclosed vesicle volume even in the absence of mechanical forces as shown here by minimizing the combined bending and stretching energy with respect to membrane area for fixed vesicle volume. As a consequence, the mechanical tension within a vesicle membrane depends on the spontaneous curvature and on the bending rigidity. This interdependence, which is difficult to grasp intuitively, is then illustrated for a variety of simple vesicle shapes. Depending on the vesicle morphology, the magnitude of the mechanical tension can be comparable to or can be much smaller than the spontaneous tension.     

Fusogenic tilted peptides induce nanoscale holes in supported phosphatidylcholine bilayers

Tilted peptides are known to insert in lipid bilayers with an oblique orientation, thereby destabilizing membranes and facilitating membrane fusion processes. Here, we report the first direct visualization of the interaction of tilted peptides with lipid membranes using in situ atomic force microscopy (AFM) imaging. Phase-separated supported dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers were prepared by fusion of small unilamellar vesicles and imaged in buffer solution, in the absence and in the presence of the simian immunodeficiency virus (SIV) peptide. The SIV peptide was shown to induce the rapid appearance of nanometer scale bilayer holes within the DPPC gel domains, while keeping the domain shape unaltered. We attribute this behavior to a local weakening and destabilization of the DPPC domains due to the oblique insertion of the peptide molecules. These results were directly correlated with the fusogenic activity of the peptide as determined using fluorescently labeled DOPC/DPPC liposomes. By contrast, the nontilted ApoE peptide did not promote liposome fusion and did not induce bilayer holes but caused slight erosion of the DPPC domains. In conclusion, this work provides the first direct evidence for the production of stable, well-defined nanoholes in lipid bilayer domains by the SIV peptide, a behavior that we have shown to be specifically related to the tilted character of the peptide. A molecular mechanism underlying spontaneous insertion of the SIV peptide within lipid bilayers and the subsequent removal of bilayer patches is proposed, and its relevance to membrane fusion processes is discussed.     

The influence of anisotropic membrane inclusions on curvature elastic properties of lipid membranes

A membrane inclusion can be defined as a complex of protein or peptide and the surrounding significantly distorted lipids. We suggest a theoretical model that allows for the estimation of the influence of membrane inclusions on the curvature elastic properties of lipid membranes. Our treatment includes anisotropic inclusions whose energetics depends on their in-plane orientation within the membrane. On the basis of continuum elasticity theory, we calculate the inclusion-membrane interaction energy that reflects the protein or peptide-induced short-ranged elastic deformation of a bent lipid layer. A numerical estimate of the corresponding interaction constants indicates the ability of inclusions to sense membrane bending and to accumulate at regions of favorable curvature, matching the effective shape of the inclusions. Strongly anisotropic inclusions interact favorably with lipid layers that adopt saddlelike curvature; such structures may be stabilized energetically. We explore this possibility for the case of vesicle budding where we consider a shape sequence of closed, axisymmetric vesicles that form a (saddle-curvature adopting) membrane neck. It appears that not only isotropic but also strongly anisotropic inclusions can significantly contribute to the budding energetics, a finding that we discuss in terms of recent experiments.     

Spontaneous vesiculation

The thermodynamics of vesicle formation was analyzed by using the elastic bending energy approach. Several different possibilities of spontaneous vesiculation, due to soft bilayers, non-zero spontaneous curvature and Gaussian curvature, respectively, were presented and discussed. Intermediate structures in the closed vesicle–disklike mixed micelle phase transition could be either cup-like particles or open bilayers partially rolled into lipid tubules.     

Coacervation of poly-electrolytes in the presence of lipid bilayers: mutual alteration of structure and morphology

Many intrinsically disordered peptides have been shown to undergo liquid–liquid phase separation and form complex coacervates, which play various regulatory roles in the cell. Recent experimental studies found that such phase separation processes may also occur at the lipid membrane surface and help organize biomolecules during signaling events; in some cases, phase separation of proteins at the membrane surface was also observed to lead to significant remodeling of the membrane morphology. The molecular mechanisms that govern the interactions between complex coacervates and lipid membranes and the impacts of such interactions on their structure and morphology, however, remain unclear. Here we study the coacervation of poly-glutamate (E₃₀) and poly-lysine (K₃₀) in the presence of lipid bilayers of different compositions. We carry out explicit-solvent coarse-grained molecular dynamics simulations by using the MARTINI (v3.0) force-field. We find that more than 20% anionic lipids are required for the coacervate to form stable contact with the bilayer. Upon wetting, the coacervate induces negative curvature to the bilayer and facilitates local lipid demixing, without any peptide insertion. The magnitude of negative curvature, extent of lipid demixing, and asphericity of the coacervate increase with the concentration of anionic lipids. Overall, we observe a decrease in the number of contacts among the polyelectrolytes as the droplet spreads over the bilayer. Therefore, unlike previous suggestions, interactions among polyelectrolytes do not constitute a driving force for the membrane bending upon wetting by the coacervate. Rather, analysis of interaction energy components suggests that bending of the membrane is favored by enhanced interactions between polyelectrolytes with lipids as well as with counterions. Kinetic studies reveal that, at the studied polyelectrolyte concentrations, the coacervate formation precedes bilayer wetting.

Intrinsically disordered polyelectrolytes undergoing liquid–liquid phase separation to form complex coacervates on a membrane, which profoundly alters the membrane morphology.     

Retinoid chromophores as probes of membrane lipid order

There is a great need for development of independent methods to study the structure and function of membrane-associated proteins and peptides. Polarized light spectroscopy (linear dichroism, LD) using shear-aligned lipid vesicles as model membranes has emerged as a promising tool for the characterization of the binding geometry of membrane-bound biomolecules. Here we explore the potential of retinoic acid, retinol, and retinal to function as probes of the macroscopic alignment of shear-deformed 100 nm liposomes. The retinoids display negative LD, proving their preferred alignment perpendicular to the membrane surface. The magnitude of the LD indicates the order retinoic acid > retinol > retinal regarding the degree of orientation in all tested lipid vesicle types. It is concluded that mainly nonspecific electrostatic interactions govern the apparent orientation of the retinoids within the bilayer. We propose a simple model for how the effective orientation may be related to the polarity of the end groups of the retinoid probes, their insertion depths, and their angular distribution of configurations around the membrane normal. Further, we provide evidence that the retinoids can sense subtle structural differences due to variations in membrane composition and we explore the pH sensitivity of retinoic acid, which manifests in variations in absorption maximum wavelength in membranes of varying surface charge. Based on LD measurements on cholesterol-containing liposomes, the influence of membrane constituents on bending rigidity and vesicle deformation is considered in relation to the macroscopic alignment, as well as to lipid chain order on the microscopic scale.     

Molecular view of the role of fusion peptides in promoting positive membrane curvature

Fusion peptides are moderately hydrophobic segments of viral and nonviral membrane fusion proteins that enable these proteins to fuse two closely apposed biological membranes. In vitro assays furthermore show that even isolated fusion peptides alone can support membrane fusion in model systems. In addition, the fusion peptides have a distinct effect on the phase diagram of lipid mixtures. Here, we present molecular dynamics simulations investigating the effect of a particular fusion peptide, the influenza hemagglutinin fusion peptide and some of its mutants, on the lipid phase diagram. We detect a systematic shift toward phases with more positive mean curvature in the presence of the peptides, as well as an occurrence of bicontinuous cubic phases, which indicates a stabilization of Gaussian curvature. The wild-type fusion peptide has a stronger effect on the phase behavior as compared to the mutants, which we relate to its boomerang shape. Our results point to a different role of fusion peptides than hitherto assumed, the stabilization of pores rather than stalks along the fusion pathway.     

A Monte Carlo study of fluctuating polymer-grafted membranes

Using Monte Carlo simulations of an off-lattice model, we study the elastic properties of polymer-grafted membranes. Our results are found to be in good agreement with those predicted by the classical path approximation of the self-consistent field theory and scaling theory based on de Gennes' blob picture. In particular, we found that when the membrane is grafted on both sides by brushes with same molecular weight N and grafting density sigma, the excess bending modulus induced by the polymers scales as N3 sigmaalpha where alpha is consistent with 7/3, as predicted by the self-consistent field theory, and 5/2, as predicted by the scaling theory. When the polymers are grafted to one side of the membrane only, the membrane bends away from the polymers with a spontaneous curvature with a scaling that is consistent with both scaling and self-consistent field theories. When the thickness of the brush exceeds the membrane's spontaneous radius of curvature, the bending modulus approaches a constant which is of the same order as the bending modulus of the bare membrane.     

Loosening of Lipid Packing Promotes Oligoarginine Entry into Cells

Despite extensive use of arginine‐rich cell‐penetrating peptides (CPPs)—including octaarginine (R8)—as intracellular delivery vectors, mechanisms for their internalization are still under debate. Lipid packing in live cell membranes was characterized using a polarity‐sensitive dye (di‐4‐ANEPPDHQ), and evaluated in terms of generalized polarization. Treatment with membrane curvature‐inducing peptides led to significant loosening of the lipid packing, resulting in an enhanced R8 penetration. Pyrenebutyrate (PyB) is known to facilitate R8 membrane translocation by working as a hydrophobic counteranion. Interestingly, PyB also actively induced membrane curvature and perturbed lipid packing. R8 is known to directly cross cell membranes at elevated concentrations. The sites of R8 influx were found to have looser lipid packing than surrounding areas. Lipid packing loosening is proposed as a key factor that governs the membrane translocation of CPPs.     

Electrostatic properties of membrane lipids coupled to metarhodopsin II formation in visual transduction

Changes in lipid composition have recently been shown to exert appreciable influences on the activities of membrane-bound proteins and peptides. We tested the hypothesis that the conformational states of rhodopsin linked to visual signal transduction are related to biophysical properties of the membrane lipid bilayer. For bovine rhodopsin, the meta I-meta II conformational transition was studied in egg phosphatidylcholine (PC) recombinants versus the native rod outer segment (ROS) membranes by means of flash photolysis. Formation of metarhodopsin II was observed by the change in absorbance at 478 nm after a single actinic flash was delivered to the sample. The meta I/meta II ratio was investigated as a function of both temperature and pH. The data clearly demonstrated thermodynamic reversibility of the transition for both the egg PC recombinants and the native ROS membranes. A significant shift of the apparent pK(a) for the acid-base equilibrium to lower values was evident in the egg PC recombinant, with little meta II produced under physiological conditions. Calculations of the membrane surface pH using a Poisson-Boltzmann model suggested the free energies of the meta I and meta II states were significantly affected by electrostatic properties of the bilayer lipids. In the ROS membranes, phosphatidylserine (PS) is needed for full formation of meta II, in combination with phosphatidylethanolamine (PE) and polyunsaturated docosahexaenoic acid (DHA; 22:6omega3) chains. We propose that the PS surface potential leads to an accumulation of hydronium ions, H(3)O(+), in the electrical double layer, which drive the reaction together with the large negative spontaneous curvature (H(0)) conferred by PE plus DHA chains. The elastic stress/strain of the bilayer arises from an interplay of the approximately zero H(0) from PS and the negative H(0) due to the PE headgroups and polyunsaturated chains. The lipid influences are further explained in terms of matching of the bilayer spontaneous curvature to the curvature at the lipid/rhodopsin interface, as formulated by the Helfrich bending energy. These new findings guide current ideas as to how bilayer properties govern the conformational energetics of integral membrane proteins. Moreover, they yield knowledge of how membrane lipid-protein interactions involving acidic phospholipids such as PS and neutral polyunsaturated DHA chains are implicated in key biological functions such as vision.     

Continuous stiffness mode nanoindentation response of poly(methyl methacrylate) surfaces

Indentation is a comparatively simple and virtually nondestructive method of determining mechanical properties of material surfaces by means of an indenter inducing a localized deformation. The paper present experimental results of the load-displacement curves, the hardness and the elastic modulus data, and associated analysis for poly(methyl methacrylate) (PMMA) surfaces as a function of contact displacement. The experimental results include continuous stiffness indentations performed using constant loading rate and constant displacement rate experiments. The continuous stiffness indentation involves continuous calculation of a material stiffness, and hence hardness and elastic modulus of surfaces, during discrete loading-unloading cycles, as in a conventional indentation routine, and in a comparatively smaller time constant. The dependence of the compliance curves, the hardness, the elastic modulus and the plasticity index upon the imposed penetration depth, the applied normal load and the deformation rate are described. Tip area and load frame calibrations for the continuous stiffness indentation are also reported. The paper includes practical considerations encountered during indentation of polymers specifically at low penetration depths. The experimental results show a peculiarly harder response of PMMA surfaces at the submicron (near to surface) layers.     

Effects of an amphipathic alpha-helical peptide on lateral pressure and water penetration in phosphatidylcholine and monoolein mixed membranes

The physicochemical properties of mixed membranes of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and a nonlamellar-forming lipid, 1-monoolein (MO), and the effects of an amphipathic alpha-helical peptide, 18A (DWLKAFYDKVAEKLKEAF), on the membranes were investigated by fluorescence measurements and 31P NMR. The intramolecular excimer formation of dipyrenylphosphatidylcholines showed that the increased lateral pressure near the bilayer center by MO is reduced by the lamellar-cubic phase transition at an MO mole fraction of 0.7, while the lateral pressure near the polar-apolar interface increases even through the phase transition. The fluorescence lifetime of 2-(9-anthroyloxy)stearic acid revealed that water penetration into the interface region increases with the MO fraction. The insertion of the 18A peptide into the membrane interface region decreased both the lateral pressure near the interface and water penetration, and shifted the lamellar-cubic phase transition to a higher MO fraction. This suggests that 18A induces a positive curvature strain and lowers the lateral pressure and water penetration. Furthermore, the increase in the MO fraction in POPC/MO LUV promoted partitioning of 18A to the membranes. This preferential binding to the MO-containing membranes is presumably ascribed to the propensity of 18A to reduce the membrane strain.     

In silico partitioning and transmembrane insertion of hydrophobic peptides under equilibrium conditions

Nascent transmembrane (TM) polypeptide segments are recognized and inserted into the lipid bilayer by the cellular translocon machinery. The recognition rules, described by a biological hydrophobicity scale, correlate strongly with physical hydrophobicity scales that describe the free energy of insertion of TM helices from water. However, the exact relationship between the physical and biological scales is unknown, because solubility problems limit our ability to measure experimentally the direct partitioning of hydrophobic peptides across lipid membranes. Here we use microsecond molecular dynamics (MD) simulations in which monomeric polyleucine segments of different lengths are allowed to partition spontaneously into and out of lipid bilayers. This approach directly reveals all states populated at equilibrium. For the hydrophobic peptides studied here, only surface-bound and transmembrane-inserted helices are found. The free energy of insertion is directly obtained from the relative occupancy of these states. A water-soluble state was not observed, consistent with the general insolubility of hydrophobic peptides. The approach further allows determination of the partitioning pathways and kinetics. Surprisingly, the transfer free energy appears to be independent of temperature, which implies that surface-to-bilayer peptide insertion is a zero-entropy process. We find that the partitioning free energy of the polyleucine segments correlates strongly with values from translocon experiments but reveals a systematic shift favoring shorter peptides, suggesting that translocon-to-bilayer partitioning is not equivalent but related to spontaneous surface-to-bilayer partitioning.     

The interaction of a peptide with a scrambled hydrophobic/hydrophilic sequence (Pro-Asp-Ala-Asp-Ala-His-Ala-His-Ala-His-Ala-Ala-Ala-His-Gly) (PADH) with DPPC model membranes: a DSC study

Depending on their hydrophobicity, peptides can interact differently with lipid membranes inducing dramatic modifications into their host systems. In the present paper, the interaction of a synthetic peptide with a scrambled hydrophobic/hydrophilic sequence (Pro-Asp-Ala-Asp-Ala-His-Ala-His-Ala-His-Ala-Ala-Ala-His-Gly) (PADH) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) model membranes has been investigated by differential scanning calorimetry (DSC), adopting three different experimental approaches. In the first, the peptide is forced to be included into the hydrocarbon region of the lipid bilayer, by codissolving it with the lipid giving rise to mixed multilamellar vesicles–peptide systems; in the second, this system is passed through an extruder, thus producing large unilamellar vesicles–peptide systems; in the third, it is allowed to interact with the external surface of the membrane.

The whole of the DSC results obtained have shown that the incorporation of the peptide into the lipid bilayer by means of the first method induces a decrease in the enthalpy of the gel–liquid crystal transition of the membrane and a shift of the transition to the lower temperatures, thus resembling, in spite of its prevalently hydrophilic nature, the behavior of transbilayer hydrophobic peptides. The extrusion of these systems creates unilamellar vesicles free of peptides but of smaller size as evidenced by the decreased cooperativity of the transition. The peptide, added externally to the DPPC model membrane, has no effect on the phase behavior of the bilayer.

These findings suggest that the effect of the interaction of scrambled hydrophobic/hydrophilic peptides into lipid bilayers strongly affects the thermotropic behavior of the host membrane depending on the preparation method of the lipid/peptide systems. The whole of the results obtained in the present paper can be useful in approaching studies of bioactive peptides/lipids systems.     

Membrane interactions of host-defense peptides studied in model systems

Host-defense, antibiotic peptides are believed to generate their cytolytic effects by interacting with the membranes of bacterial cells. Direct analyses of peptide interactions with real cellular membranes are difficult, however, due to the high complexity of physiological membranes. This review summarizes experimental work aiming to understand peptide-membrane interactions and their relationships with the peptides' biological actions using specific model systems. Varied model assemblies have been constructed that generally aim to mimic the fundamental lipid bilayer organization of the membrane. The model systems we will describe include multilamellar and unilamellar vesicles, planar lipid bilayers, lipid monolayers and micelles, and colorimetric biomimetic membranes. The different artificial models have facilitated examination of specific biological or chemical parameters affecting peptide action, for example the effect of membrane lipid composition on peptide affinities and membrane penetration, the relationship between membrane fluidity and peptide interactions, the conformations of active peptides, and other factors. We evaluate the strengths and limitations of the various approaches, and point to future directions in the field.     

Lateral Organization of Host Heterogeneous Raft‐like Membranes Altered by the Myristoyl Modification of Tyrosine Kinase c‐Src

Membrane‐bound c‐Src non‐receptor tyrosine kinase, unlike other acyl‐modified lipid‐anchored proteins, anchors to the membrane by a myristoyl chain along with a polybasic residue stretch, which is shorter in chain length than its host membrane. The packing defect arising from this mismatched chain length of the host and the lipid anchor significantly affects the lateral organization of heterogeneous membranes. We reveal the mixing of phase domains and formation of novel nanoscale‐clusters upon membrane binding of the Myr‐Src (2–9) peptide. Fluorescence cross correlation spectroscopy was used to explore the nature of these clusters. We show that Myr‐Src (2–9) is able to oligomerize, and the peptide clusters are embedded in a lipid platform generated by lipid sorting. Further, using confocal fluorescence microscopy and FRET assays we show that localized charge enrichment and membrane curvature are able to shift the partition coefficient towards the more ordered lipid phase.     

Examination of membrane fusion by dissipative particle dynamics simulation and comparison with continuum elastic models

A dissipative particle dynamics model is applied to probe the lipidic membrane fusion. This model is verified by reproducing the lipid phase behavior. The classical stalk model has been visited and modified. The tilt deformation of the lipids and the noncircular shape of the stalk are supported. The stalk is shown to undergo asymmetric expansion to form the trans-monolayers contact (TMC). Unlike previous models, an energy barrier between the stalk and the TMC has been identified, implying that the TMC should be a metastable formation. This shows good agreement with the fusion experiments. Two typical elastic continuum models are compared with our result and possible modifications to the two elastic models are suggested. The effect of spontaneous curvature of lipid on selection of fusion pathway is also examined. It is observed that a bent stalk with pore or an inverted micellar intermediate will have more chance to occur than traditional stalk when the spontaneous curvature of the lipid becomes more negative.