Pore Formation In Lipid Membranes Research Articles

Energetics of toroidal pore formation in lipid membranes using molecular dynamics simulations

Energetics of toroidal pore formation in lipid membranes using molecular dynamics simulations

The Possibility of Pore Formation in Lipid Membranes by Several Molecules of Amphipathic Peptides

The Possibility of Pore Formation in Lipid Membranes by Several Molecules of Amphipathic Peptides

Beyond structural models for the mode of action: How natural antimicrobial peptides affect lipid transport

Hypothesis: Most textbook models for antimicrobial peptides (AMP) mode of action are focused on structural effects and pore formation in lipid membranes, while these deformations have been shown to require high concentrations of peptide bound to the membrane. Even insertion of low amounts of peptides in the membrane is hypothesized to affect the transmembrane transport of lipids, which may play a key role in the peptide effect on membranes.Experiments: Here we combine state-of-the-art small angle X-ray/neutron scattering (SAXS/SANS) techniques to systematically study the effect of a broad selection of natural AMPs on lipid membranes. Our approach enables us to relate the structural interactions, effects on lipid exchange processes, and thermodynamic parameters, directly in the same model system.Findings: The studied peptides, indolicidin, aurein 1.2, magainin II, cecropin A and LL-37 all cause a general acceleration of essential lipid transport processes, without necessarily altering the overall structure of the lipid membranes or creating organized pore-like structures. We observe rapid scrambling of the lipid composition associated with enhanced lipid transport which may trigger lethal signaling processes and enhance ion transport. The reported membrane effects provide a plausible canonical mechanism of AMP-membrane interaction and can reconcile many of the previously observed effects of AMPs on bacterial membranes.

Effect of Lipid Structure and Material Properties on the Membrane Stability to Pore Formation

Biological membranes are natural barriers that preserve the integrity of the cell and its organelles. Even temporary damage of the cell membrane can break its normal homeostasis that will start the recovery mechanisms, apoptosis and inflammatory reactions. Formation of pores, through defects, in the lipid bilayer of biological membranes can lead to serious disruption of the normal lifecycle of the cells and the development of pathologies.A two-pronged approach was used to study the energetics of pore formation in lipid membranes assembled by isoprenoid lipids (e.g., DPhPC and Di-O-PhPC). The first prong studied the line tension of DPhPC, Di-O-PhPC, DOPC, and POPC using MD simulations and experiments with an electrical breakdown technique. Resistance to pore formation can be characterized by pore edge line tension, which describes the energy barrier height that should be overcome to irreversibly expand the pore, i.e. to rupture the membrane. A method of measuring pore line tension in MD, determining the lateral tension of the lipid membrane with the static pore, and experiments revealed that branched lipid significantly increased the pore energy and membrane resistance to pore formation. Additionally, work on GUVs and MD showed that low-weight solvent significantly lowers pore edge energy. The second prong used MD to quantify other key material properties that can affect pore formation (i.e., the compressibility modulus, bending modulus, per area free energy change with respect to curvature, and the intrinsic curvature). DPhPC's bending modulus is about half that of DPPC, but DPhPC's compressibility modulus is about two times larger than DPPC. Finally, DPhPC's effect on monolayer spontaneous curvature is similar to that of a PE lipid. Thus both structural peculiarities of lipid tails and solvents strongly influence membrane stability.

Free energy analysis of membrane pore formation process in the presence of multiple melittin peptides

Understanding the molecular mechanism underlying pore formation in lipid membranes by antimicrobial peptides is of great importance in biological sciences as well as in drug design applications. Melittin has been widely studied as a pore forming peptide, though the molecular mechanism for pore formation is still illusive. We examined the free energy barrier for the creation of a pore in lipid membranes with and without multiple melittin peptides. It was found that six melittin peptides significantly stabilized a pore, though a small barrier (a few kBT) for the formation still existed. With five melittin peptides or fewer, the pore formation barrier was much higher, though the established pore was in a local energy minimum. Although seven melittins effectively reduced the free energy barrier, a single melittin peptide left the pore after a long time MD simulation probably because of the overcrowded environment around the bilayer pore. Thus, it is highly selective for the number of melittin peptides to stabilize the membrane pore, as was also suggested by the line tension evaluations. The free energy cost required to insert a single melittin into the membrane is too high to explain the one-by-one insertion mechanism for pore formation, which also supports the collective melittin mechanism for pore formation.

Pore formation in lipid membrane II: Energy landscape under external stress

Lipid membranes are extremely stable envelopes allowing cells to survive in various environments and to maintain desired internal composition. Membrane permeation through formation of transversal pores requires substantial external stress. Practically, pores are usually formed by application of lateral tension or transmembrane voltage. Using the same approach as was used for obtaining continuous trajectory of pore formation in the stress-less membrane in the previous article, we now consider the process of pore formation under the external stress. The waiting time to pore formation proved a non-monotonous function of the lateral tension, dropping from infinity at zero tension to a minimum at the tension of several millinewtons per meter. Transmembrane voltage, on the contrary, caused the waiting time to decrease monotonously. Analysis of pore formation trajectories for several lipid species with different spontaneous curvatures and elastic moduli under various external conditions provided instrumental insights into the mechanisms underlying some experimentally observed phenomena.

Pore formation in lipid membrane I: Continuous reversible trajectory from intact bilayer through hydrophobic defect to transversal pore

Lipid membranes serve as effective barriers allowing cells to maintain internal composition differing from that of extracellular medium. Membrane permeation, both natural and artificial, can take place via appearance of transversal pores. The rearrangements of lipids leading to pore formation in the intact membrane are not yet understood in details. We applied continuum elasticity theory to obtain continuous trajectory of pore formation and closure, and analyzed molecular dynamics trajectories of pre-formed pore reseal. We hypothesized that a transversal pore is preceded by a hydrophobic defect: intermediate structure spanning through the membrane, the side walls of which are partially aligned by lipid tails. This prediction was confirmed by our molecular dynamics simulations. Conversion of the hydrophobic defect into the hydrophilic pore required surmounting some energy barrier. A metastable state was found for the hydrophilic pore at the radius of a few nanometers. The dependence of the energy on radius was approximately quadratic for hydrophobic defect and small hydrophilic pore, while for large radii it depended on the radius linearly. The pore energy related to its perimeter, line tension, thus depends of the pore radius. Calculated values of the line tension for large pores were in quantitative agreement with available experimental data.

Probing a Continuous Polar Defect: A Reaction Coordinate for Pore Formation in Lipid Membranes.

Various biophysical processes involve the formation of aqueous pores over lipid membranes, including processes of membrane fusion, antimicrobial peptide activity, lipid flip-flop, and membrane permeation. Reliable and efficient free-energy calculations of pore formation using molecular dynamics simulations remained challenging due to the lack of good reaction coordinates (RCs) for pore formation. We present a new RC for pore formation that probes the formation and rupture of a continuous polar defect over the membrane. Potential of mean force (PMF) calculations along the new RC rapidly converge and exhibit no hysteresis between pore-opening and pore-closing pathways, in contrast to calculations based on previous RCs. We show that restraints along the new RC may restrain the system tightly to the transition state of pore formation, rationalizing the absence of hysteresis. We observe that the PMF of pore formation in a tension-free membrane of dimyristoylphosphatidylcholine (DMPC) reveals a free-energy barrier for pore nucleation, confirming a long-hypothesized metastable prepore state. We test the influence of the lipid force field, the cutoff distance used for Lennard-Jones interactions, and the lateral membrane size on the free energies of pore formation. In contrast to PMF calculations based on previous RCs, we find that such parameters have a relatively small influence on the free energies of pore nucleation. However, the metastability of the open pore in DMPC may depend on such parameters. The RC has been implemented into an extension of the GROMACS simulation software. The new RC allows for reliable and computationally efficient free-energy calculations of pore formation in lipid membranes.

Mechanics of water pore formation in lipid membrane under electric field

Transmembrane water pores are crucial for substance transport through cell membranes via membrane fusion, such as in neural communication. However, the molecular mechanism of water pore formation is not clear. In this study, we apply all-atom molecular dynamics and bias-exchange metadynamics simulations to study the process of water pore formation under an electric field. We show that water molecules can enter a membrane under an electric field and form a water pore of a few nanometers in diameter. These water molecules disturb the interactions between lipid head groups and the ordered arrangement of lipids. Following the movement of water molecules, the lipid head groups are rotated and driven into the hydrophobic region of the membrane. The reorientated lipid head groups inside the membrane form a hydrophilic surface of the water pore. This study reveals the atomic details of how an electric field influences the movement of water molecules and lipid head groups, resulting in water pore formation.

Experimental Estimation of Membrane Tension Induced by Osmotic Pressure

Osmotic pressure (Π) induces the stretching of plasma membranes of cells or lipid membranes of vesicles, which plays various roles in physiological functions. However, there have been no experimental estimations of the membrane tension of vesicles upon exposure to Π. In this report, we estimated experimentally the lateral tension of the membranes of giant unilamellar vesicles (GUVs) when they were transferred into a hypotonic solution. First, we investigated the effect of Π on the rate constant, kp, of constant-tension (σex)-induced rupture of dioleoylphosphatidylcholine (DOPC)-GUVs using the method developed by us recently. We obtained the σex dependence of kp in GUVs under Π and by comparing this result with that in the absence of Π, we estimated the tension of the membrane due to Π at the swelling equilibrium, σosmeq. Next, we measured the volume change of DOPC-GUVs under small Π. The experimentally obtained values of σosmeq and the volume change agreed with their theoretical values within the limits of the experimental errors. Finally, we investigated the characteristics of the Π-induced pore formation in GUVs. The σosmeq corresponding to the threshold Π at which pore formation is induced is similar to the threshold tension of the σex-induced rupture. The time course of the radius change of GUVs in the Π-induced pore formation depends on the total membrane tension, σt; for small σt, the radius increased with time to an equilibrium one, which remained constant for a long time until pore formation, but for large σt, the radius increased with time and pore formation occurred before the swelling equilibrium was reached. Based on these results, we discussed the σosmeq and the Π-induced pore formation in lipid membranes.

Simulations of Pore Formation in Lipid Membranes: Reaction Coordinates, Convergence, Hysteresis, and Finite-Size Effects.

Transmembrane pores play an important role in various biophysical processes such as membrane permeation, membrane fusion, and antimicrobial peptide activity. In principal, all-atom molecular dynamics (MD) simulations provide an accurate model of pore formation in lipid membranes. However, the free energy landscape of transmembrane pore formation remains poorly understood, partly because potential of mean force (PMF) calculations of pore formation strongly depend on the choice of the reaction coordinate. In this study, we used umbrella sampling to compute PMFs for pore formation using three different reaction coordinates, namely, (i) a coordinate that steers the lipids in the lateral direction away from the pore center, (ii) the distance of a single lipid phosphate group from the membrane center, and (iii) the average water density inside a membrane-spanning cylinder. Our results show that while the three reaction coordinates efficiently form pores in membranes, they suffer from strong hysteresis between pore-opening and pore-closing simulations, suggesting that they do not restrain the systems close to the transition state for pore formation. The two reaction coordinates that act via restraining the lipids lead to more pronounced hysteresis compared with the coordinate acting on the water molecules. By comparing PMFs computed from membranes with different numbers of lipids, we observed significant artifacts from the periodic boundary conditions in small simulation systems. Further analysis suggests that the formation and disruption of a continuous hydrogen-bonding network across the membrane corresponds to the transition state for pore formation. Our study provides molecular insights into the critical steps of transmembrane pore formation, and it may guide the development of efficient reaction coordinates for pore formation.

Communication: Activation energy of tension-induced pore formation in lipid membranes.

Tension plays a vital role in pore formation in biomembranes, but the mechanism of pore formation remains unclear. We investigated the temperature dependence of the rate constant of constant tension (σ)-induced pore formation in giant unilamellar vesicles of lipid membranes using an experimental method we developed. By analyzing this result, we determined the activation energy (Ua) of tension-induced pore formation as a function of tension. A constant (U0) that does not depend on tension was found to contribute significantly to Ua. Analysis of the activation energy clearly indicated that the dependence of Ua on σ in the classical theory is correct, but that the classical theory of pore formation is not entirely correct due to the presence of U0. We can reasonably consider that U0 is a nucleation free energy to form a hydrophilic pre-pore from a hydrophobic pre-pore or a region with lower lateral lipid density. After obtaining U0, the evolution of a pre-pore follows a classical theory. Our data provide valuable information that help explain the mechanism of tension-induced pore formation in biomembranes and lipid membranes.

Electrostatic interaction effects on tension-induced pore formation in lipid membranes.

We investigated the effects of electrostatic interactions on the rate constant (k(p)) for tension-induced pore formation in lipid membranes of giant unilamellar vesicles under constant applied tension. A decrease in salt concentration in solution as well as an increase in surface charge density of the membranes increased k(p). These data indicate that k(p) increases as the extent of electrostatic interaction increases. We developed a theory on the effect of the electrostatic interactions on the free energy profile of the membrane containing a prepore and also on the values of k(p); this theory explains the experimental results and fits the experimental data reasonably well in the presence of weak electrostatic interactions. Based on these results, we conclude that a decrease in the free energy barrier of the prepore state due to electrostatic interactions is the main factor causing an increase in k(p).

Effects of dimethyl sulfoxide on lipid membrane electroporation.

Pores can be generated in lipid membranes by the application of an external electric field or by the addition of particular chemicals such as dimethyl sulfoxide (DMSO). Molecular dynamics (MD) has been shown to be a useful tool for unveiling many aspects of pore formation in lipid membranes in both situations. By means of MD simulations, we address the formation of electropores in cholesterol-containing lipid bilayers under the influence of DMSO. We show how a combination of physical and chemical mechanisms leads to more favorable conditions for generating membrane pores and, in particular, how the addition of DMSO to the medium significantly reduces the minimum electric field required to electroporate a lipid membrane. The strong alteration of membrane transversal properties and the energetic stabilization of the hydrophobic pore stage by DMSO provide the physicochemical mechanisms that explain this effect.

Entry of Cell-Penetrating Peptide Transportan 10 into a Single Vesicle by Translocating Across Lipid Membrane and Its Induced Pores

The cell-penetrating peptide, transportan 10 (TP10), can translocate across the plasma membrane of living cells and thus can be used for the intracellular delivery of biological cargo such as proteins. However, the mechanisms underlying its translocation and the delivery of large cargo remain unclear. In this report we investigated the entry of TP10 into a single giant unilamellar vesicle (GUV) and the TP10-induced leakage of fluorescent probes using the single GUV method. GUVs of 20% dioleoylphosphatidylglycerol (DOPG)/80% dioleoylphosphatidylcholine (DOPC) were prepared, and they contained a water-soluble fluorescent dye, Alexa Fluor 647 hydrazide (AF647), and smaller vesicles composed of 20% DOPG/80% DOPC. The interaction of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) with these loaded GUVs was investigated using confocal microscopy. The fluorescence intensity of the GUV membrane increased with time to a saturated value, then the fluorescence intensity due to the membranes of the smaller vesicles inside the GUV increased prior to leakage of AF647. This result indicates that CF-TP10 entered the GUV from the outside by translocating across the lipid membrane before CF-TP10-induced pore formation. The rate constant of TP10-induced pore formation in lipid membranes increased with an increase in TP10 concentration. Large molecules such as Texas Red Dextran 40,000, and vesicles with a diameter of 1-2 μm, permeated through the TP10-induced pores or local rupture in the lipid membrane. These results provide the first direct experimental evidence that TP10 can deliver large cargo through lipid membranes, without the need for special transport mechanisms such as those found in cells.

3P220 張力が誘起する脂質膜中のポア形成に対する静電相互作用の効果の理論(生体膜・人工膜 : ダイナミクス,ポスター,第52回日本生物物理学会年会(2014年度))

3P220 張力が誘起する脂質膜中のポア形成に対する静電相互作用の効果の理論(生体膜・人工膜 : ダイナミクス,ポスター,第52回日本生物物理学会年会(2014年度))

The single GUV method for revealing the functions of antimicrobial, pore-forming toxin, and cell-penetrating peptides or proteins.

We recently developed the single giant unilamellar vesicle (GUV) method for investigating the functions and dynamics of biomembranes. The single GUV method can provide detailed information on the elementary processes of physiological phenomena in biomembranes, such as their rate constants. Here we describe the process of pore formation induced by the antimicrobial peptide (AMP), magainin 2, and the pore-forming toxin (PFT), lysenin, as revealed by the single GUV method. We obtained the rate constants of several elementary steps, such as peptide/protein-induced pore formation in lipid membranes and the membrane permeation of fluorescent probes through the pores. Information on the entry of the cell-penetrating peptide (CPP), transportan 10 (TP10), into a single GUV and its induced pore formation in lipid membranes was also obtained. We compare the single GUV method with other methods for investigating the interaction of peptides/proteins with lipid membranes (i.e., the large unilamellar vesicle (LUV) suspension method, the GUV suspension method, and single channel recording), and discuss the pros and cons of the single GUV method. On the basis of these data, we discuss the advantages of the single GUV method.

3P217 細胞透過ペプチドであるトランスポータン10 のベシクル内への進入と脂質膜中のポア形成に対する張力の効果(生体膜・人工膜 : ダイナミクス,ポスター,第52回日本生物物理学会年会(2014年度))

3P217 細胞透過ペプチドであるトランスポータン10 のベシクル内への進入と脂質膜中のポア形成に対する張力の効果(生体膜・人工膜 : ダイナミクス,ポスター,第52回日本生物物理学会年会(2014年度))

3P218 張力が誘起する脂質膜中のポア形成に対する静電相互作用の効果(生体膜・人工膜 : ダイナミクス,ポスター,第52回日本生物物理学会年会(2014年度))

3P218 張力が誘起する脂質膜中のポア形成に対する静電相互作用の効果(生体膜・人工膜 : ダイナミクス,ポスター,第52回日本生物物理学会年会(2014年度))

Rate Constant of Tension-Induced Pore Formation in Lipid Membranes

We investigated the effects of tension induced by micropipet aspiration on giant unilamellar vesicles (GUVs) composed of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC). We analyzed the time course of the fraction of intact GUVs among all of the GUVs under constant tension σ and obtained the rate constants of pore formation kp(σ). To determine kp, we developed an approach using the mean first passage time. The fitting of the theoretical curves of kp versus σ to the experimental data determined the line tension of a prepore, Γ. The value of Γ of a DOPG/DOPC bilayer was smaller than that of a DOPC bilayer.