Charged Lipid Bilayers Research Articles

Turning a negative into a positive.

Negatively charged lipid bilayers enhance the interaction between a chemokine and an atypical chemokine receptor.

Resolving the interactions between hydrophilic CdTe quantum dots and positively charged membranes at the nanoscale

The use of quantum dot nanoparticles (QDs) in bio-applications has gained quite some interest and requires a deep understanding of their interactions with model cell membranes. This involves assessing the extent of nanoparticle disruption of the membrane and how it depends on both nanoparticle and membrane physicochemical properties. Surface charge plays an important role in nanoparticle adsorption, which is primarily driven by electrostatic interactions; yet, once adsorbed, most reported works overlook the subsequent spatial nanoparticle insertion and location within the membrane. There is therefore a need for studies to assess the mutual role of membrane and nanoparticle charge into membrane structure and stability at the nanoscale, with a view to better design and control the functionality of these nanomaterials. In this work, we have resolved the extent of the interactions between hydrophilic, negatively charged CdTe QDs and positively charged lipid bilayers. A multiscale combination of surface-sensitive techniques enabled probing how surface charge mediates QD adsorption and membrane reorganization. Increasing membrane surface charge results into a larger adsorption of oppositely charged QDs, concomitantly inducing structural changes. Hydration of the membrane hydrophobic parts by QDs goes deeper into the inner leaflet with increasing membrane charge, resulting in supported lipid bilayers with decreased nanomechanical stability.

Human HSP70-escort protein 1 (hHep1) interacts with negatively charged lipid bilayers and cell membranes.

Human Hsp70-escort protein 1 (hHep1) is a cochaperone that assists in the function and stability of mitochondrial HSPA9. Similar to HSPA9, hHep1 is located outside the mitochondria and can interact with liposomes. In this study, we further investigated the structural and thermodynamic behavior of interactions between hHep1 and negatively charged liposomes, as well as interactions with cellular membranes. Our results showed that hHep1 interacts peripherally with liposomes formed by phosphatidylserine and cardiolipin and remains partially structured, exhibiting similar affinities for both. In addition, after being added to the cell membrane, recombinant hHep1 was incorporated by cells in a dose-dependent manner. Interestingly, the association of HSPA9 with hHep1 improved the incorporation of these proteins into the lipid bilayer. These results demonstrated that hHep1 can interact with lipids also present in the plasma membrane, indicating roles for this cochaperone outside of mitochondria.

Anomalous Diffusion of Polyelectrolyte Segments on Supported Charged Lipid Bilayers.

This work provides mesoscale models for the anomalous diffusion of a polymer chain on a heterogeneous surface with rearranging randomly distributed adsorption sites. Both the "bead-spring" model and oxDNA model were simulated on supported lipid bilayer membranes with various molar fractions of charged lipids, using Brownian dynamics method. Our simulation results demonstrate that "bead-spring" chains exhibit sub-diffusion on charged lipid bilayers which agrees with previous experimental observations for short-time dynamics of DNA segments on membranes. In addition, the non-Gaussian diffusive behaviors of DNA segments have not been observed in our simulations. However, a simulated 17 base pairs double stranded DNA, using oxDNA model, performs normal diffusion on supported cationic lipid bilayers. Due to the number of positively charged lipids attracted by short DNA is small, the energy landscape that the short DNA experiences during diffusion is not as heterogeneous as that experienced by long DNA chains, which results in normal diffusion rather than sub-diffusion for short DNA.

Coarse-Grained Molecular Dynamics of pH-Sensitive Lipids.

pH-sensitive lipids represent a class of lipids that can be protonated and destabilized in acidic environments, as they become positively charged in response to low-pH conditions. They can be incorporated into lipidic nanoparticles such as liposomes, which are able to change their properties and allow specific drug delivery at the acidic conditions encountered in some pathological microenvironments. In this work, we used coarse-grained molecular-dynamic simulations to study the stability of neutral and charged lipid bilayers containing POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and various kinds of ISUCA ((F)2-(imidazol-1-yl)succinic acid)-derived lipids, which can act as pH-sensitive molecules. In order to explore such systems, we used a MARTINI-derived forcefield, previously parameterized using all-atom simulation results. We calculated the average area per lipid, the second-rank order parameter and the lipid diffusion coefficient of both lipid bilayers made of pure components and mixtures of lipids in different proportions, under neutral or acidic conditions. The results show that the use of ISUCA-derived lipids disturbs the lipid bilayer structure, with the effect being particularly marked under acidic conditions. Although more-in depth studies on these systems must be carried out, these initial results are encouraging and the lipids designed in this research could be a good basis for developing new pH-sensitive liposomes.

Pro-inflammatory protein S100A9 alters membrane organization by dispersing ordered domains

Pro-inflammatory, calcium-binding protein S100A9 is localized in the cytoplasm of many cells and regulates several intracellular and extracellular processes. S100A9 is involved in neuroinflammation associated with the pathogenesis of Alzheimer's disease (AD). The number of studies on the impact of S100A9 in co-aggregation processes with amyloid-like proteins is increasing. However, there is still a lack of data on how this protein interacts with lipid membranes. We employed atomic force microscopy (AFM), dynamic light scattering (DLS), and fluorescence measurements (Laurdan and Thioflavin-T) to study the interaction between protein and the membrane surface. We used lipid vesicles in bulk and planar tethered lipid bilayers as biomimetic membrane models. We demonstrated that the protein accumulates on negatively charged lipid bilayers but with no further loss of the bilayer's integrity. The most important result is that the initial adsorption and accumulation of apo-form of S100A9 on the lipid membrane surface is lipid phase-sensitive. The breaking down of raft-like and disappearance of gel-like domains indicate that protein incorporates into the hydrophobic part of the lipid bilayer. We observed the most noticeable loss of integrity in lipid bilayers constructed from a lipid mixture (brain total lipid extract). Understanding the function and interactions of these proteins in cellular environments might expand the development of new diagnostic and therapeutic approaches for AD or other related diseases.

Single-Molecule Trapping and Measurement in a Nanostructured Lipid Bilayer System.

The repulsive electrostatic force between a biomolecule and a like-charged surface can be geometrically tailored to create spatial traps for charged molecules in solution. Using a parallel-plate system composed of silicon dioxide surfaces, we recently demonstrated single-molecule trapping and high precision molecular charge measurements in a nanostructured free energy landscape. Here we show that surfaces coated with charged lipid bilayers provide a system with tunable surface properties for molecular electrometry experiments. Working with molecular species whose effective charge and geometry are well-defined, we demonstrate the ability to quantitatively probe the electrical charge density of a supported lipid bilayer. Our findings indicate that the fraction of charged lipids in nanoslit lipid bilayers can be significantly different from that in the precursor lipid mixtures used to generate them. We also explore the temporal stability of bilayer properties in nanofluidic systems. Beyond their relevance in molecular measurement, such experimental systems offer the opportunity to examine lipid bilayer formation and wetting dynamics on nanostructured surfaces.

Investigations into the membrane activity of arenicin antimicrobial peptide AA139

Arenicin-3 is an amphipathic β-hairpin antimicrobial peptide that is produced by the lugworm Arenicola marina. In this study, we have investigated the mechanism of action of arenicin-3 and an optimized synthetic analogue, AA139, by studying their effects on lipid bilayer model membranes and Escherichia coli bacterial cells. The results show that simple amino acid changes can lead to subtle variations in their interaction with membranes and therefore alter their pre-clinical potency, selectivity and toxicity. While the mechanism of action of arenicin-3 is primarily dependent on universal membrane permeabilization, our data suggest that the analogue AA139 relies on more specific binding and insertion properties to elicit its improved antibacterial activity and lower toxicity, as exemplified by greater selectivity between lipid composition when inserting into model membranes i.e. the N-terminus of AA139 seems to insert deeper into lipid bilayers than arenicin-3 does, with a clear distinction between zwitterionic and negatively charged lipid bilayer vesicles, and AA139 demonstrates a cytoplasmic permeabilization dose response profile that is consistent with its greater antibacterial potency against E. coli cells compared to arenicin-3.

Intramembrane serine protease catalysis in different physical environments

The cleavage of proteins inside cell membranes initiates pathological pathways and is a subject of a major interest. Thus, the nature of the coupling between the physical environment and the function of such proteins has recently attracted significant experimental and theoretical effort. However, it is difficult to determine the nature of this coupling in a unique way by experimental and theoretical studies without separating the chemical and the environmental factors. This work presents calculations of the activation barriers of the intramembrane rhomboid protease in neutral and charged lipid bilayers as well as in detergent micelle, trying to explore the environmental effect.

Effect of Environmental Factors on the Catalytic Activity of Intramembrane Serine Protease.

The cleavage of protein inside cell membranes regulates pathological pathways and is a subject of major interest. Thus, the nature of the coupling between the physical environment and the function of such proteins has recently attracted significant experimental and theoretical efforts. However, it is difficult to determine the nature of this coupling uniquely by experimental and theoretical studies unless one can separate the chemical and the environmental factors. This work describes calculations of the activation barriers of the intramembrane rhomboid protease in neutral and charged lipid bilayers and in detergent micelle, trying to explore the environmental effect. The calculations of the chemical barrier are done using the empirical valence bond (EVB) method. Additionally, the renormalization method captures the energetics and dynamical effects of the conformational change. The simulations indicate that the physical environment around the rhomboid protease is not a major factor in changing the chemical catalysis and that the conformational and substrate dynamics do not exhibit long-time coupling. General issues about the action of membrane-embedded enzymes are also considered.

Interaction of a short antimicrobial peptide on charged lipid bilayer: A case study on aurein 1.2 peptide

Aurein 1.2 (aurein) is a short but active α-helical antimicrobial peptide discovered in Australian tree frogs (Litoria aurea). It shows inhibition on a broad spectrum of bacteria and cancer cells. With well-defined helicity, amphipathicity, and cationic charges, it readily binds to membranes and causes membrane change and disruption. This study provides details on how aurein interacts with charged lipid membranes by using neutron membrane diffraction (NMD) and neutron spin echo (NSE) spectroscopy on complex peptide-membrane systems. NMD provides higher resolution lipid bilayer structures than solution scattering. NMD revealed the peptide is mostly associated in the lipid headgroup region. Even at moderately high concentrations (e.g., peptide:lipid ratio of 1:30), aurein is located at the acyl chain-headgroup region without deep penetration into the hydrophobic acyl chain. However, it does reduce the elasticity of the membrane at that concentration, which was corroborated by the NSE results. Furthermore, NSE shows that aurein first softens the membrane, like many other α-helical peptides at low concentration, but then makes the membrane much more rigid, even without membrane pore formation. Combining our previous studies, the evidence shows that aurein at relatively low concentrations still modifies lipid distribution significantly and can cause membrane thinning and lateral segregation of charged lipids. At the same time, the membrane's mechanical properties are modified with much slower lipid diffusion. This suggests that aurein can attack the microbial membrane without the need to form membrane pores or disintegrate membranes; instead, it promotes the formation of domains at low concentration.

Binding free energy calculation of human beta defensin 3 with negatively charged lipid bilayer using free energy perturbation method

Human β defensin type 3 (hBD-3) is a cationic peptide having strong antimicrobial activities even at high salt concentrations. The conserved sequence is believed to contribute to its unique antibacterial activities. To design novel drugs based on hBD-3, predicting the binding free energy contribution of each residue on hBD-3 with bacterial membrane is important. Firstly, the stable binding structure of hBD-3 dimer in analog form bound on POPG lipid bilayer was predicted using NAMD simulations, which was confirmed by RMSD, buried surface area, hydrogen bonds, distance map, and insertion depth map calculations. Then, free energy perturbation (FEP) method was applied to calculate the binding free energy of each residue by mutating it into Alanine. It was found that the positively charged residues on the tail region of hBD-3 contribute significantly to its binding with membrane. The result emphasized the importance of electrostatic interactions to hBD-3's binding with bacterial membrane.

Formation of Giant Lipid Vesicle Containing Dual Functions Facilitates Outer Membrane Phospholipase.

Giant lipid vesicles are used to study artificial cell models, as well as the encapsulation of biomolecules, and the reconstitution of membrane proteins on these vesicles. Recently, complex reactions in giant vesicles have been controlled by reconstituting numerous kinds of biomolecules. However, it is challenging to generate giant lipid vesicles containing a diverse set of proteins at concentrations sufficient to ensure proper functioning. Here, we describe an artificial cell model showing dual functions of small molecule transportation and small vesicle budding, using a dual functional membrane protein (transportation and phosphatase activity) called the outer membrane phospholipase (OmpLA). To the best of our knowledge, we have revealed for the first time the transportation of ions or small molecules through OmpLA on the charged lipid bilayer. The lipid composition controlled the orientation of OmpLA through proteinase K digestion. Finally, OmpLA enzyme activity of phospholipid hydrolysis caused the budding of small vesicles.

Molecular Dynamics Simulations of Human Beta-Defensin Type 3 Crossing Different Lipid Bilayers.

Human β defensin type 3 (hBD-3) is a small cationic cysteine-rich peptide. It has a broad spectrum of antimicrobial activities. However, at high concentrations, it also shows hemolytic activity by interrupting red blood cells. To understand the selectivity of hBD-3 disrupting cell membranes, investigating the capability of hBD-3 translocating through different membranes is important. Since hBD-3 in the analogue form in which all three pairs of disulfide bonds are broken has similar antibacterial activities to the wild-type, this project investigates the structure and dynamics of an hBD-3 analogue in monomer, dimer, and tetramer forms through both zwitterionic and negatively charged lipid bilayers using molecular dynamics (MD) simulations. One tetramer structure of hBD-3 was predicted by running all-atom MD simulations on hBD-3 in water at a high concentration, which was found to be stable in water during 400 ns all-atom simulations based on root-mean-squared deviation, root-mean-squared fluctuation, buried surface area, and binding interaction energy calculations. After that, hBD-3 in different forms was placed inside different membranes, and then steered MD simulation was conducted to pull the hBD-3 out of the membrane along the z-direction to generate different configurational windows to set up umbrella-sampling (US) simulations. Because extensive sampling is important to obtain accurate free energy barriers, coarse-grained US MD simulations were performed in each window. Based on the long-term simulation result, membrane thinning was found near hBD-3 in different lipid bilayers and in different hBD-3 oligomer systems. By calculating the root-mean-squared deviation of the z-coordinate of hBD-3 molecules, rotation of the oligomer inside the bilayer and stretching of the oligomer structure along the z-direction were observed. Although reorientation of lipid heads toward the hBD-3 tetramer was observed based on the density profile calculation, the order parameter calculation shows that hBD-3 disrupts 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) lipids more significantly and makes it less ordered than on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids. Calculating the free energy of hBD-3 through different lipid bilayers, it was found that generally hBD-3 encounters a lower energy barrier through negatively charged lipid membranes than the zwitterionic membrane. hBD-3 in different forms needs to overcome a lower energy barrier crossing the combined POPC+POPS bilayer through the POPS leaflet than through the POPC leaflet. Besides that, the potential of mean force result suggests that hBD-3 forms an oligomer translocating negatively charged lipid membranes at a low concentration. This study supplied new insight into the antibacterial mechanism of hBD-3 through different membranes.

Can di-4-ANEPPDHQ reveal the structural differences between nanodiscs and liposomes?

The potential-sensitive di-4-ANEPPDHQ dye is presently gaining popularity in structural studies of the lipid bilayer. Within the bilayer, dye environmental sensitivity originates from the excitation induced charge redistribution and is usually attributed to solvent relaxation. Here, di-4-ANEPPDHQ is utilized to compare the structure of neutral and negatively charged lipid bilayers between two model systems: the nanodiscs and the liposomes. Using the well-established approach of measuring solvatochromic shifts of the steady-state spectra to study the bilayer structural changes has proved insufficient in this case. By applying an in-depth analysis of time-resolved fluorescence decays and emission spectra, we distinguished and characterized two and three distinct emissive di-4-ANEPPDHQ species in the liposomes and the nanodiscs, respectively. These emissive species were ascribed to the dual emission of the dye rather than to solvent relaxation. An additional, long-lived component present in the nanodiscs was associated with a unique domain of high order, postulated recently. Our results reveal that the di-4-ANEPPDHQ steady-state fluorescence should be interpreted with caution. With the experimental approach presented here, the di-4-ANEPPDHQ sensitivity was improved. We confirmed that the bilayer structure is, indeed, altered in the nanodiscs. Moreover, molecular dynamic simulations showed a distribution of the probe in the nanodiscs plane, which is sensitive to lipid composition. In POPC nanodiscs, probe frequently interacts with MSP, while in POPC-POPG nanodiscs, such interactions are rare. We did not observe, however, any impact of those interactions on the probe fluorescence.

How arginine derivatives alter the stability of lipid membranes: dissecting the roles of side chains, backbone and termini

Arginine (R)-rich peptides constitute the most relevant class of cell-penetrating peptides and other membrane-active peptides that can translocate across the cell membrane or generate defects in lipid bilayers such as water-filled pores. The mode of action of R-rich peptides remains a topic of controversy, mainly because a quantitative and energetic understanding of arginine effects on membrane stability is lacking. Here, we explore the ability of several oligo-arginines R_n and of an arginine side chain mimic R_mathrm {Side} to induce pore formation in lipid bilayers employing MD simulations, free-energy calculations, breakthrough force spectroscopy and leakage assays. Our experiments reveal that R_mathrm {Side} but not R_n reduces the line tension of a membrane with anionic lipids. While R_n peptides form a layer on top of a partly negatively charged lipid bilayer, R_mathrm {Side} leads to its disintegration. Complementary, our simulations show R_mathrm {Side} causes membrane thinning and area per lipid increase beside lowering the pore nucleation free energy. Model polyarginine R_8 similarly promoted pore formation in simulations, but without overall bilayer destabilization. We conclude that while the guanidine moiety is intrinsically membrane-disruptive, poly-arginines favor pore formation in negatively charged membranes via a different mechanism. Pore formation by R-rich peptides seems to be counteracted by lipids with PC headgroups. We found that long R_n and R_mathrm {Side} but not short R_n reduce the free energy of nucleating a pore. In short R_n, the substantial effect of the charged termini prevent their membrane activity, rationalizing why only longer mathrm {R}_{n} are membrane-active.

Composition effects on photooxidative membrane destabilization by TiO2 nanoparticles

Membrane interactions and photooxidative membrane destabilization of titanium dioxide (TiO2) nanoparticles were investigated, focusing on the effects of membrane composition, notably phospholipid headgroup charge and presence of cholesterol. For this, we employed a battery of state-of-the-art methods for studies of bilayers formed by zwitterionic palmitoyloleoylphosphatidylcholine (POPC) containing also polyunsaturated palmitoylarachidonoylphosphocholine (PAPC), as well as its mixtures with anionic palmitoyloleoylphosphatidylglycerol (POPG) and cholesterol. It was found that the TiO2 nanoparticles display close to zero charge at pH 7.4, resulting in aggregation. At pH 3.4, in contrast, the 6 nm TiO2 nanoparticles are well dispersed due to a strongly positive ζ-potential. Mirroring this pH dependence, TiO2 nanoparticles were observed to bind to negatively charged lipid bilayers at pH 3.4, but much less so at pH 7.4. While nanoparticle binding has some destabilizing effect alone, illumination with ultraviolet (UV) light accentuates membrane destabilization, a result of oxidative stress caused by generated reactive oxygen species (ROS). Neutron reflectivity (NR), quartz crystal microbalance (QCM), and small-angle X-ray scattering (SAXS) results all demonstrate that membrane composition strongly influences membrane interactions and photooxidative destabilization of lipid bilayers. In particular, the presence of anionic POPG makes the bilayers more sensitive to oxidative destabilization, whereas a stabilizing effect was observed in the presence of cholesterol. Also, structural aspects of peroxidation were found to depend strongly on membrane composition, notably the presence of anionic phospholipids. The results show that membrane interactions and UV-induced ROS generation act in concert and need to be considered together to understand effects of lipid membrane composition on UV-triggered oxidative destabilization by TiO2 nanoparticles, e.g., in the context of oxidative damage of bacteria and cells.

Functionalized Graphene@Gold Nanostar/Lipid for Pancreatic Cancer Gene and Photothermal Synergistic Therapy under Photoacoustic/Photothermal Imaging Dual‐Modal Guidance

Nanomaterial-based pancreatic cancer treatment has received widespread attention and rapid development in the past few years. The major challenges include the poor combination of diagnosis and therapy, the difficulty in targeting therapy from the root and the unsatisfactory antitumor efficiency, which is accompanied by a great risk of relapse and metastasis. In this work, a positively charged lipid bilayer membrane is coated on reduced graphene oxide@gold nanostar (rGO@AuNS) for photoacoustic/photothermal dual-modal imaging-guided gene/photothermal synergistic therapy of pancreatic cancer. In addition, the cross-linking of folic acid on the surface of rGO@AuNS-lipid can specifically bind after recognizing folic acid receptors on the surface of cancer cells, and greatly improve the targeting ability of the nanomaterial and the performance of imaging diagnosis by receptor-mediated endocytosis. Moreover, the photothermal and gene (targeting G12V mutant K-Ras gene) synergistic therapy shows outstanding anticancer efficacy for pancreatic cancer tumor bearing mice, and it is noteworthy that the treatment groups have anti-liver metastasis of pancreatic cancer.

The Human LL-37(17-29) antimicrobial peptide reveals a functional supramolecular structure

Here, we demonstrate the self-assembly of the antimicrobial human LL-37 active core (residues 17–29) into a protein fibril of densely packed helices. The surface of the fibril encompasses alternating hydrophobic and positively charged zigzagged belts, which likely underlie interactions with and subsequent disruption of negatively charged lipid bilayers, such as bacterial membranes. LL-3717–29 correspondingly forms wide, ribbon-like, thermostable fibrils in solution, which co-localize with bacterial cells. Structure-guided mutagenesis analyses supports the role of self-assembly in antibacterial activity. LL-3717–29 resembles, in sequence and in the ability to form amphipathic helical fibrils, the bacterial cytotoxic PSMα3 peptide that assembles into cross-α amyloid fibrils. This argues helical, self-assembling, basic building blocks across kingdoms of life and points to potential structural mimicry mechanisms. The findings expose a protein fibril which performs a biological activity, and offer a scaffold for functional and durable biomaterials for a wide range of medical and technological applications.

Membrane Tension in Negatively Charged Lipid Bilayers in a Buffer under Osmotic Pressure.

Osmotic pressure (Π) induces membrane tension in cell membranes and the lipid bilayers of vesicles and plays an important role in the functions and physical properties of these membranes. We recently developed a method to determine quantitatively the membrane tension of giant unilamellar vesicles (GUVs) under Π and applied it to GUVs comprising electrically neutral dioleoylphosphatidylcholine (DOPC). Here, we examined the effect of Π on GUVs composed of DOPC and negatively charged dioleoylphosphatidylglycerol (DOPG) in a buffer containing a physiological concentration of ions. First, we examined the rate constant, kr, for constant tension (σex)-induced rupture of DOPG/DOPC (4/6)-GUVs under Π and obtained the dependence of kr on σex in GUVs for various values of Π. Comparing this dependence in the absence of Π provided values for membrane tension due to Π, σosm, which agree with the theoretical values within the experimental error. The values of σosm for DOPG/DOPC-GUVs were smaller than those for DOPC-GUVs under the same Π. Two factors, that is, the solute concentration in a GUV suspension and the elastic modulus of the GUV membrane, can reasonably explain this difference based on the theory of σosm. We also examined the effect of Π on the rate constant, kFF, for the transbilayer movement of lipid molecules in single GUVs. The values of kFF increased with increasing Π, indicating that kFF increased with σosm. This result supports the existence of prepores in stretched lipid bilayers. Based on these results, we discuss the membrane tension of DOPG/DOPC-GUVs under Π.