Interaction modes between nanosized graphene flakes and liposomes: Adsorption,insertion and membrane fusion

BackgroundUnderstanding the effects of graphene-based nanomaterials on lipid membranes is critical to determine their environmental impact and their efficiency in the biomedical context. Graphene has been reported to favourably interact with biological and model lipid membranes.MethodsWe report on a systematic coarse-grained molecular dynamics study of the interaction modes of graphene nanometric flakes with POPC/cholesterol liposome membranes. We have simulated graphene layers with a variety of sizes and oxidation degrees, and we have analyzed the trajectories, the interaction modes, and the energetics of the observed phenomena.ResultsThree interaction modes are reported. Graphene can be transiently adsorbed onto the liposome membrane and/or inserted in its hydrophobic region. Inserted nanosheets prefer a perpendicular orientation, and tilt in order to maximize the contact with phospholipid tails while avoiding the contact with cholesterol molecules. When placed between two liposomes, graphene facilitates their fusion in a single vesicle.ConclusionsGraphene can be temporary adsorbed on the liposome before insertion. Bilayer curvature has an influence on the orientation of inserted graphene particles. Cholesterol molecules are depleted from the surrounding of graphene particles. Graphene layers may catalyse membrane fusion by bypassing the energy barrier required in stalk formation.General significanceNanometric graphene layers can be adsorbed/inserted in lipid-based membranes in different manners and affect the cholesterol distribution in the membrane, implying important consequences on the structure and functionality of biological cell membranes, and on the bioaccumulation of graphene in living organisms. The graphene-mediated mechanism opens new possibilities for vesicle fusion in the experimental context.     

How does protein architecture facilitate the transduction of ATP chemical-bond energy into mechanical work? The cases of nitrogenase and ATP binding-cassette proteins

Transduction of adenosine triphosphate (ATP) chemical-bond energy into work to drive large-scale conformational changes is common in proteins. Two specific examples of ATP-utilizing proteins are the nitrogenase iron protein and the ATP binding-cassette transporter protein, BtuCD. Nitrogenase catalyzes biological nitrogen fixation whereas BtuCD transports vitamin B(12) across membranes. Both proteins drive their reactions with ATP. To interpret how the mechanical force generated by ATP binding and hydrolysis is propagated in these proteins, a coarse-grained elastic network model is employed. The analysis shows that subunits of the proteins move against each other in a concerted manner. The lowest-frequency modes of the nitrogenase iron protein and of the ATP binding-cassette transporter BtuCD protein are found to link the functionally critical domains, and these modes are suggested to be responsible for (at least the initial stages) large-scale ATP-coupled conformational changes.     

Internal forces, tension and energy density in tethered cellular membranes

We analyze tethered cellular membranes by considering the membrane resultants, tension and densities of two modes of energy, bending and adhesion. These characteristics are determined based on a computational (finite-difference) analysis of membrane shape. We analyze the relative contribution and distribution of the membrane characteristics in four typical zones of the membrane surface. Using an axisymmetric model, we found that the meridional and circumferential components of the resultant are different near the tether body and they converge to the value of membrane tension farther from the tether. At the beginning of the area of membrane detachment from the cytoskeleton, the density of bending energy is on the same order of magnitude as membrane tension (resultant). Away from the tether, the bending energy density quickly decreases and becomes of the same order as that of the adhesion energy in the membrane-cytoskeleton attachment area. In that area, both modes of energy are significantly smaller than the membrane tension. We also consider the effect of the membrane bending modulus on the distribution of the membrane characteristics. An increase in the bending modulus results in changing the length and position on the membrane surface of zone 1 characterized by significant evolution of the resultant components. It also results in shortening zone 2 that covers the rest of the area of membrane detachment. The obtained results can help in a better interpretation of the measurements of membrane mechanical properties as well as in analyses of proteins and channels in curved membranes.     

What can we learn about amphiphile-membrane interaction from model lipid membranes?

Surface-active amphiphiles find applications in a wide range of areas of industry such as agrochemicals, personal care, and pharmaceuticals. In many of these applications, interaction with cell membranes is a key factor for achieving their purpose. How do amphiphiles interact with lipid membranes? What are their bases for membrane specificity? Which biophysical properties of membranes are susceptible to modulation by amphiphilic membrane-effectors? What aspects of this interaction are important for performing their function? In our work on membrane biophysics over the years, questions like these have arisen and we now share some of our findings and discuss them in this review. This topic was approached focusing on the membrane properties and their alterations rather than on the amphiphile structure requirements for their interaction. Here, we do not aim to provide a comprehensive list of the modes of action of amphiphiles of biological interest but to help in understanding them.     

The mechanism of Hsp70 chaperones: (entropic) pulling the models together

Hsp70s are conserved molecular chaperones that can prevent protein aggregation, actively unfold, solubilize aggregates, pull translocating proteins across membranes and remodel native proteins complexes. Disparate mechanisms have been proposed for the various modes of Hsp70 action: passive prevention of aggregation by kinetic partitioning, peptide-bond isomerase, Brownian ratcheting or active power-stroke pulling. Recently, we put forward a unifying mechanism named 'entropic pulling', which proposed that Hsp70 uses the energy of ATP hydrolysis to recruit a force of entropic origin to locally unfold aggregates or pull proteins across membranes. The entropic pulling mechanism reproduces the expected phenomenology that inspired the other disparate mechanisms and is, moreover, simple.     

The elastic deformability of closed multilayered membranes is the same as that of a bilayer membrane

The elastic behavior of closed multilayered membranes is analyzed with the assumption that the constituent layers are in close contact but are unconnected in the sense that they are free to slide by one another. The system exhibits three independent elastic deformation modes for any number of the constituent layers equal to or larger than two. These are the area expansivity of the membrane neutral surface, and the local and non-local membrane bending. The corresponding elastic moduli are expressed in terms of the elastic moduli of the constituent layers, their areas, and distances between their neutral surfaces. Closed multilayered membranes only differ from a closed bilayer membrane in that for any of their shapes some of the constituent layers are expanded and some compressed.     

Modelling and simulations of multi-component lipid membranes and open membranes via diffuse interface approaches

Diffuse interface (phase field) models are developed for multi-component vesicle membranes with different lipid compositions and membranes with free boundary. These models are used to simulate the deformation of membranes under the elastic bending energy and the line tension energy with prescribed volume and surface area constraints. By comparing our numerical simulations with recent biological experiments, it is demonstrated that the diffuse interface models can effectively capture the rich phenomena associated with the multi-component vesicle transformation and thus offering great functionality in their simulation and modelling.     

Membrane insertion and lateral mobility of synthetic amphiphilic signal peptides in lipid model membranes

Amphiphilic signal sequences with the potential to form alpha-helices with a polar, charged face and an apolar face are common in proteins which are imported into mitochondria, in the PTS permeases of bacteria, and in bacterial rhodopsins. Synthetic peptides of such sequences partition into the surface region of lipid membranes where they can adopt different secondary structures. A finely controlled balance of electrostatic and hydrophobic interactions determines the 'affinity' of amphiphilic signal peptides for lipid membranes, as well as the structure, orientation and depth of penetration of these peptides in lipid bilayer membranes. The ability of an individual peptide to associate with lipid bilayer membranes in several different modes is, most likely, a general feature of amphiphilic signal peptides and is reflected in several common physical properties of their amino acid sequences.     

Thermodynamics of Micelle Formation and Membrane Fusion Modulate Antimicrobial Lipopeptide Activity

Antimicrobial lipopeptides (AMLPs) are antimicrobial drug candidates that preferentially target microbial membranes. One class of AMLPs, composed of cationic tetrapeptides attached to an acyl chain, have minimal inhibitory concentrations in the micromolar range against a range of bacteria and fungi. Previously, we used coarse-grained molecular dynamics simulations and free energy methods to study the thermodynamics of their interaction with membranes in their monomeric state. Here, we extended the study to the biologically relevant micellar state, using, to our knowledge, a novel reaction coordinate based on hydrophobic contacts. Using umbrella sampling along this reaction coordinate, we identified the critical transition states when micelles insert into membranes. The results indicate that the binding of these AMLP micelles to membranes is thermodynamically favorable, but in contrast to the monomeric case, there are significant free energy barriers. The height of these free energy barriers depends on the membrane composition, suggesting that the AMLPs’ ability to selectively target bacterial membranes may be as much kinetic as thermodynamic. This mechanism highlights the importance of considering oligomeric state in solution as criterion when optimizing peptides or lipopeptides as antibiotic leads.     

Exploring peptide membrane interaction using surface plasmon resonance: differentiation between pore formation versus membrane disruption by lytic peptides

Lytic peptides comprise a large group of membrane-active peptides used in the defensive and offensive systems of all organisms. Differentiating between their modes of interaction with membranes is crucial for understanding how these peptides select their target cells. Here we utilized SPR to study the interaction between lytic peptides and lipid bilayers (L1 sensor chip). Using studies also on hybrid monolayers (HPA sensor chip) revealed that SPR is a powerful tool for obtaining a real-time monitoring of the steps involved in the mode of action of membrane-active peptides, some of which previously could not be detected directly by other techniques and reported here for the first time. We investigated the mode of action of peptides that represent two major families: (i) the bee venom, melittin, as a model of a non-cell-selective peptide that forms transmembrane pores and (ii) magainin and a diastereomer of melittin (four amino acids were replaced by their D enantiomers), as models of bacteria-selective non-pore-forming peptides. Fitting the SPR data to different interaction models allows differentiating between two major steps: membrane binding and membrane insertion. Melittin binds to PC/cholesterol approximately 450-fold better than its diastereomer and magainin, mainly because it is inserted into the inner leaflet (2/3 of the binding energy), whereas the other two are not. In contrast, there is only a slight difference in the binding of all the peptides to negatively charged PE/PG mono- and bilayer membranes (in the first and second steps), indicating that the inner leaflet contributes only slightly to their binding to PE/PG bilayers. Furthermore, the 100-fold stronger binding of the cell-selective peptides to PE/PG as compared with PC/cholesterol resulted only from electrostatic attraction to the negatively charged headgroups of the outer leaflet. These results clearly differentiate between the two general mechanisms: pore formation by melittin only in zwitterionic membranes and a detergent-like effect (carpet mechanism) for all the peptides in negatively charged membranes, in agreement with their biological function.     

Is lipid bilayer binding a common property of inhibitor cysteine knot ion-channel blockers?

Recent studies of several ICK ion-channel blockers suggest that lipid bilayer interactions play a prominent role in their actions. Structural similarities led to the hypothesis that bilayer interactions are important for the entire ICK family. We have tested this hypothesis by performing direct measurements of the free energy of bilayer partitioning (DeltaG) of several peptide blockers using our novel quenching-enhanced fluorescence titration protocol. We show that various ICK peptides demonstrate markedly different modes of interaction with large unilamellar lipid vesicles. The mechanosensitive channel blocker, GsMTx4, and its active diastereomeric analog, D-GsMTx4, bind strongly to both anionic and zwitterionic membranes. One potassium channel gating modifier, rHpTx2gs, interacts negligibly with both types of vesicles at physiological pH, whereas another, SGTx1, interacts only with anionic lipids. The slope of DeltaG dependence on surface potential is very shallow for both GsMTx4 and D-GsMTx4, indicating complex interplay of their hydrophobic and electrostatic interactions with lipid. In contrast, a cell-volume regulator, GsMTx1, and SGTx1 exhibit a very steep DeltaG dependence on surface potential, resulting in a strong binding only for membranes rich in anionic lipids. The high variability of 5 kcal/mole in observed DeltaG shows that bilayer partitioning is not a universal property of the ICK peptides interacting with ion channels.     

Energetics and dynamics of SNAREpin folding across lipid bilayers

Membrane fusion occurs when SNAREpins fold up between lipid bilayers. How much energy is generated during SNAREpin folding and how this energy is coupled to the fusion of apposing membranes is unknown. We have used a surface forces apparatus to determine the energetics and dynamics of SNAREpin formation and characterize the different intermediate structures sampled by cognate SNAREs in the course of their assembly. The interaction energy-versus-distance profiles of assembling SNAREpins reveal that SNARE motifs begin to interact when the membranes are 8 nm apart. Even after very close approach of the bilayers (approximately 2-4 nm), the SNAREpins remain partly unstructured in their membrane-proximal region. The energy stabilizing a single SNAREpin in this configuration (35 k(B)T) corresponds closely with the energy needed to fuse outer but not inner leaflets (hemifusion) of pure lipid bilayers (40-50 k(B)T).     

Functional motions of influenza virus hemagglutinin: a structure-based analytical approach.

Influenza virus hemagglutinin (HA), a homotrimeric integral membrane glycoprotein essential for viral infection, is engaged in two biological functions: recognition of target cells' receptor proteins and fusion of viral and endosomal membranes, both requiring substantial conformational flexibility from the part of the glycoprotein. The different modes of collective motions underlying the functional mobility/adaptability of the protein are determined in the present study using an extension of the Gaussian network model (GNM) to treat concerted anisotropic motions. We determine the molecular mechanisms that may underlie HA function, along with the structural regions or residues whose mutations are expected to impede function. Good agreement between theoretically predicted fluctuations of individual residues and corresponding x-ray crystallographic temperature factors is found, which lends support to the GNM elucidation of the conformational dynamics of HA by focusing upon a subset of dominant modes. The lowest frequency mode indicates a global torsion of the HA trimer about its longitudinal axis, accompanied by a substantial mobility at the viral membrane connection. This mode is proposed to constitute the dominant molecular mechanism for the translocation and aggregation of HAs, and for the opening and dilation of the fusion pore. The second and third collective modes indicate a global bending, allowing for a large lateral surface exposure, which is likely to facilitate the close association of the viral and endosomal membranes before pore opening. The analysis of kinetically hot residues, in contrast, reveals a localization of energy centered around the HA2 residue Asp112, which apparently triggers the solvent exposure of the fusion peptide.     

AFM: a nanotool in membrane biology

Cellular membranes are vital for life. They confine cells and cytosolic compartments and are involved in virtually every cellular process. Cellular membranes form cellular contacts and focal adhesions, anchor the cytoskeleton, generate energy gradients, transform energy, transduce signals, move cells, and actively form compartments to assemble different membrane proteins into functional entities. But how do cellular membranes perform these tasks? What do the machineries of cellular membranes look like, and how are they controlled and guided? Atomic force microscopy (AFM) allows the observation of biological surfaces in their native environment at a signal-to-noise ratio superior to that of any optical microscopic technique. With a spatial resolution approaching approximately 1 nm, AFM can identify the supramolecular assemblies, characteristic structure, and functional conformation of native membrane proteins. In recent years, AFM has evolved from imaging applications to a multifunctional "laboratory on a tip" that allows observation and manipulation of the machineries of cellular membranes. In the force spectroscopy mode, AFM detects interactions between two single cells at molecular resolution. Force spectroscopy can also be used to probe the local elasticity, chemical groups, and receptor sites of live cells. Other applications locate molecular interactions driving membrane protein folding, assembly, and their switching between functional states. It is also possible to examine the energy landscape of biomolecular reactions, as well as reaction pathways, associated lifetimes, and free energy. In this review, we provide a flavor of the fascinating opportunities offered by the use of AFM as a nanobiotechnological tool in modern membrane biology.     

Use of a real-time polymerase chain reaction thermocycler to study bacterial cell permeabilization by antimicrobial peptides

Antimicrobial peptides are good leads to develop new antibiotics, but knowledge of their mode of action is a prerequisite. Destruction of the microbial membranes through a detergent-like mechanism is one of these modes of action. This is usually studied by using a fluorescent nucleic acid stain such as SYTOX Green, which is impermeable to living cells. Using a simple protocol based on the use of a standard real-time thermocycler, we confirmed that the actions of the antimicrobial peptides LL-37 and magainin 2 on bacterial cells are different.     

The permeability of liposomes to nonelectrolytes. I. Activation energies for permeation.

The effect of temperature on the permeability of nonelectrolytes across liposomal membranes above and below their transition temperature has been studied by using an osmotic method. Below their transition temperature, liposomes are osmotically insensitive structures but, on addition of gramicidin A, the water permeability so increased that the permeability of solutes could be studied. The measured activation energies for permeation of a variety of nonelectrolytes has been found to increase when a) there is an increase in the capability of the solutes to form hydrogen bonds in water, b) the cholesterol concentration in the membranes increases and c) the membranes pass from a liquid-crystalline to a solid-crystalline state. The change in the activation energy for permeation per hydrogen bond is about 1.8 kcal/mole for all the different liposome systems investigated; the only solute tested that deviated from this correlation was urea, whose activation energy for permeation across a gramicidin-containing system was much lower than expected from its hydrogen-bonding capacity. This finding suggests that urea is permeating across the gramicidin pore. Although the literature contains only incomplete data relating the activation energies for permeation of nonelectrolytes across biological membranes to their hydrogen-bonding capacity, the available evidence suggests that there is a similar correlation to that found in liposomes. Thus, the average increase in the activation energy per hydrogen bond for permeation across ox red cell membranes (Jacobs, Glassman & Parpart, J. Cell. Comp. Physiol. 7:197, 1935) is 2.2 plus or minus 0.4 kcal/mole, a value that is similar to that obtained in liposomes. However, the activation energies for water and urea are - in such a system - very much lower than expected, suggesting that they, too, are permeating by some parallel route such as an aqueous pore.     

Effect of ionic polarizability on electrodiffusion in lipid bilayer membranes.

Ion-carrier complexes and organic ions of similar size and shape have mobilities in lipid bilayer membranes which span several orders of magnitude. In this communication, an examination is made of the hypothesis that the basis for this unusually wide range of ionic mobilities is the potential energy barrier arising from image forces which selectively act on ions according to their polarizability. Using Poisson's equation to evaluate the electrostatic interaction between an ion and its surroundings, the potential energy barrier to ion transport due to image effects is computed, with the result that the potential energy barrier height depends strongly on ionic polarizability. Theoretical membrane potential energy profile calculations are used in conjunction with Nernst-Planck electrodiffusion equation to analyze the available mobility data for several ion-carrier complexes and lipid-soluble ions in lipid bilayer membranes. The variation among the mobilities of different ions is shown to be in agreement with theoretical predictions based on ionic polarizability and size. Furthermore, the important influence exerted by image forces on ion transport in lipid bilayer membranes compared to the frictional effect of membrane viscosity is established by contrasting available data on the activation energy of ionic conductivity with that for membrane fluidity.     

Different distribution of daunomycin in plasma membranes from drug-sensitive and drug-resistant P388 leukemia cells.

When the anthracycline daunomycin (DNM) is incorporated into isolated plasma membranes from P388 murine leukemia cells, the drug partitions between 'deep' and 'surface' membrane domains. Such domains have been characterized on the basis of: (1) fluorescence resonance energy transfer between 1,6-diphenylhexa-1,3,5-triene or 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene as energy donors, which are well known in their positioning within the membrane, and daunomycin as the energy acceptor, and (2) quenching of the fluorescence of the membrane-associated drug by the water-soluble quencher iodide. The distribution of DNM between the two plasma membrane domains is different depending on the cellular phenotype. Thus, in membranes from drug-sensitive cells, DNM is preferentially confined to 'surface' domains, while in membranes from drug-resistant cells, the drug distributes more homogeneously between 'surface' and 'deep' domains. Experiments using artificial lipid vesicles suggest that differences in the relative levels of certain lipids in the plasma membranes from drug-sensitive and drug-resistant cells, namely phosphatidylserine and cholesterol, are partly responsible for the observed differences in the distribution of DNM. Since drug-membrane interactions are important in anthracycline cytotoxicity, it is possible that our observations on a different membrane distribution of daunomycin, may be related to the different sensitivity to the drug exhibited by these cells.     

Thyroid hormones and their effects: a new perspective

The thyroid hormones are very hydrophobic and those that exhibit biological activity are 3',5',3,5-L-tetraiodothyronine (T4), 3',5,3-L-triiodothyronine (T3), 3',5',3-L-triiodothyronine (rT3) and 3,5',-L-diiothyronine (3,5-T2). At physiological pH, dissociation of the phenolic -OH group of these iodothyronines is an important determinant of their physical chemistry that impacts on their biological effects. When non-ionized these iodothyronines are strongly amphipathic. It is proposed that iodothyronines are normal constituents of biological membranes in vertebrates. In plasma of adult vertebrates, unbound T4 and T3 are regulated in the picomolar range whilst protein-bound T4 and T3 are maintained in the nanomolar range. The function of thyroid-hormone-binding plasma proteins is to ensure an even distrubtion throughout the body. Various iodothyronines are produced by three types of membrane-bound cellular deiodinase enzyme systems in vertebrates. The distribution of deiodinases varies between tissues and each has a distinct developmental profile. Thyroid hormones. (1) the nuclear receptor mode is especially important in the thyroid hormone axis that controls plasma and cellular levels of these hormones. (2) These hormones are strongly associated with membranes in tissues and normally rigidify these membranes. (3) They also affect the acyl composition of membrane bilayers and it is suggested that this is due to the cells responding to thyroid-hormone-induced membrane rigidificataion. Both their immediate effects on the physical state of membranes and the consequent changes in membrane composition result in several other thyroid hormone effects. Effects on metabolism may be due primarily to membrane acyl changes. There are other actions of thyroid hormones involving membrane receptors and influences on cellular interactions with the extracellulara matrix. The effects of thyroid hormones are reviewed and appear to b combinations of these various modes of action. During development, vertebrates show a surge in T4 and other thyroid hormones, as well as distinctive profiles in the appearance of the deiodinase enzymes and nuclear receptors. Evidence from the use of analogues supports multiple modes of action. Re-examination of data from th early 1960s supports a membrane action. Findings from receptor 'knockout' mice supports an important role for receptors in the development of the thyroid axis. These iodothyronines may be better thought of as 'vitamone'-like molecules than traditional hormonal messengers.     

The effect of rehydration on the thickness of erythrocyte membranes as observed by energy transfer

Erythrocyte membranes were embedded between two artificial chromophore layers which function as energy donor and acceptor, respectively. From the rate of energy transfer across the membranes their thickness was derived. In this manner the thickness of human erythrocyte membranes was found to increase from 65 Å in the air-dried state to approximately 100 Å upon rehydration in 7 mM phosphate buffer.