Yukihiro Tamba

Magainin 2-Induced Pore Formation in the Lipid Membranes Depends on Its Concentration in the Membrane Interface

Antimicrobial peptide magainin 2 forms pores in lipid membranes to induce leakage of internal contents of cells, which is a main cause of its bactericidal activity. However, the conditions and the mechanism of its pore formation remain unclear. In this report, to reveal the effect of the surface charge density of membranes on magainin 2-induced pore formation, we investigated the interaction of magainin 2 with giant unilamellar vesicles (GUVs) composed of a mixture of electrically neutral dioleoylphosphatidylcholine (DOPC) and negatively charged dioleoylphosphatidylglycerol (DOPG) in various ratios, using the single GUV method. We found that magainin 2 induced pores in the membranes of all kinds of single GUVs. For GUVs with the same charge density, the rate of the pore formation increased with magainin 2 concentration. The magainin 2 concentrations in a buffer required to induce the same rate of the pore formation greatly increased with a decrease in the surface charge density; e.g., the magainin 2 concentrations required for the pore formation in 30% DOPG/70% DOPC-GUVs were 50 times higher than those in 60% DOPG/40% DOPC-GUVs. However, after we converted the magainin 2 concentration in the buffer into that in the membrane interface, Xbmag, we found that Xbmag mainly determines the rate of the pore formation in various GUVs. These data support our model of two-state transition from the binding state to the pore state of the GUV for magainin 2-induced pore formation.

Kinetic Pathway of Antimicrobial Peptide Magainin 2-Induced Pore Formation in Lipid Membranes

The pore formation in lipid membranes induced by the antimicrobial peptide magainin 2 is considered to be the main cause for its bactericidal activity. To reveal the mechanism of the pore formation, it is important to elucidate the kinetic pathway of magainin 2-induced pore formation in lipid membranes. In this report, to examine the change in pore size over time during pore formation which can monitor its kinetic pathway, we investigated the rate of the leakage of various sized fluorescent probes through the magainin 2-induced pores in single giant unilamellar vesicles (GUVs) of 50% dioleoylphosphatidylglycerol (DOPG)/50% dioleoylphosphatidylcholine (DOPC) membrane. Magainin 2- induced leakage of Texas-Red dextran 10,000, Texas-Red dextran 3000, and Alexa-Fluor trypsin inhibitor occurred in two stages; a transient rapid leakage in the initial stage followed by a stage of slow leakage. In contrast, magainin 2 induced a transient, but very small (10-20%), leakage of fluorescent probes of a larger size such as Texas-Red dextran 40,000 and FITC-BSA. These results indicate that magainin 2 molecules initially induce a large, transient pore in lipid membranes following which the radius of the pore decreases to a stable smaller size. We estimated the radius of these pores, which increases with an increase in magainin 2 concentration. On the basis of these data, we propose a hypothesis on the mechanism of magainin 2-induced pore formation.

A membrane filtering method for the purification of giant unilamellar vesicles

The use of giant unilamellar vesicles (GUVs) for investigating the properties of biomembranes is advantageous compared to the use of small-sized vesicles such as large unilamellar vesicles (LUVs). Experimental methods using GUVs, such as the single GUV method, would benefit if there was a methodology for obtaining a large population of similar-sized GUVs composed of oil-free membranes. We here describe a new membrane filtering method for purifying GUVs prepared by the natural swelling method and demonstrate that, following purification of GUVs composed of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes suspended in a buffer, similar-sized GUVs with diameters of 10-30 μm are obtained. Moreover, this method enabled GUVs to be separated from water-soluble fluorescent probes and LUVs. These results suggest that the membrane filtering method can be applied to GUVs prepared by other methods to purify larger-sized GUVs from smaller GUVs, LUVs, and various water-soluble substances such as proteins and fluorescent probes. This method can also be used for concentration of dilute GUV suspensions.

Ca2+ ion transport through channels formed by α-hemolysin analyzed using a microwell array on a Si substrate

For the functional analysis of ion channel activity, an artificial lipid bilayer suspended over microwells was formed that ruptured giant unilamellar vesicles on a Si substrate. Ca(2+) ion indicators (fluo-4) were confined in the microwells by sealing the microwells with a lipid bilayer. An overhang formed at the microwells prevented the lipid membrane from falling into them and allowed the stable confinement of the fluorescent probes. The transport of Ca(2+) ions through the channels formed by α-hemolysin inserted in a lipid membrane was analyzed by employing the fluorescence intensity change of fluo-4 in the microwells. The microwell volume was very small (1-100 fl), so a highly sensitive monitor could be realized. The detection limit is several tens of ions/s/μm(2), and this is much smaller than the ion current in a standard electrophysiological measurement. Smaller microwells will make it possible to mimic a local ion concentration change in the cells, although the signal to noise ratio must be further improved for the functional analysis of a single channel. We demonstrated that a microwell array with confined fluorescent probes sealed by a lipid bilayer could constitute a basic component of a highly sensitive biosensor array that works with functional membrane proteins. This array will allow us to realize high throughput and parallel testing devices.

Design and facile synthesis of neoglycolipids as lactosylceramide mimetics and their transformation into glycoliposomes.

Neoglycolipids composed of disaccharide glycoside and phospholipid were designed and prepared as mimetics of lactosylceramide. The lactosyl- and N-acetyllactosaminyl-phospholipids (Lac-DPPA and LacNAc-DPPA) were enzymatically synthesized from lactose and LacNAc respectively by cellulase-mediated condensation with 1,6-hexanediol, followed by conjugation of the resulting glycosides and dipalmitoylphosphatidyl choline (DPPC) mediated by Streptomyces phospholipase D. Alternatively, allyl β-lactoside was ozonolyzed to give an aldehyde, which was condensed with dipalmytoyl phosphatidyl ethanolamine to afford a second type of glycolipid (Lac-DPPE). NMR spectroscopy indicated that the neoglycolipids behave differently in different solvent systems. X-ray diffraction clearly showed that multilamellar vesicles (MLVs) of Lac-DPPE and Lac-DPPA-MLV are in the bilayer gel phase at 20 °C, whereas those of Lac-DPPE-MLV were in the lamellar liquid-crystalline phase at 50 °C. Differential scanning calorimetry showed that Lac-DPPE-MLV had complex thermotropic behavior depending on the incubation conditions. After a long incubation at 10 °C, endothermic transitions are observed at 39.6, 42.3 °C, and 42.9 °C. These neoglycolipids have the ability to trap calcein, a chelating derivative of fluorescein, in MLVs and showed specific binding to lectin in plate assays using fluorescently labeled compounds.

Confinement of Fluorescent Probes in Microwells on Si Substrates by Sealing with Lipid Bilayers

We investigated the optimum architecture for confining fluorescent probes in microwells on a Si substrate by covering it with a lipid bilayer. We modified the structure of the wells to prevent the lipid membrane from falling into them, and the overhang shape at the aperture improved the probability of confinement. The fluorescence intensity from the calcein confined in the wells remained unchanged for one hour or more, indicating that the probes remain stably in the wells without flowing out. An artificial cell sealed with the suspended membrane is a promising tool for the functional analysis of membrane proteins.

Visualization of Single Membrane Protein Structure in Stretched Lipid Bilayer Suspended over Nanowells

In this study, we observed the topology of a single protein in a stretched lipid bilayer (membrane) suspended over a nanoscale well using a fast-scanning atomic force microscope (AFM). The membrane was located stably enough on the well to prevent the leakage of a liquid placed in the well, and it allowed us to observe membrane stretching using an AFM. We successfully observed the gradual stretching of the suspended membrane. We also observed single bacteriorhodopsin proteins in the stretched membrane, and found that they maintained their trimeric structure, but that the distances between the trimers increased.

Laser Light and X-Ray Diffraction Studies of Oriented Isotactic Polypropylene (iPP) Prepared by Temperature Slope Crystallization

Isotactic polypropylene (iPP) film was melt-crystallized in a temperature gradient. The iPP film showed well oriented α- and β-crystalline textures along the gradient. The crystalline structure, phase transition boundary and lamellar twisting were examined by X-ray diffraction and laser light diffraction (LLD). On the α-β boundary, LLD shows a sharp streak perpendicular to the boundary, where the a-axis of the β-crystal is oriented perpendicular to the temperature gradient. Apart from the boundary, the a-axis of the β-crystal becomes parallel to the gradient. The β-crystal shows lamellar twisting with a pitch of 200 μm at room temperature. When heated the β-crystal, the lamellar distance of 295Å at room temperature decreases to 285Å at 80–100°C and then increases to more than 300Å above 120°C. During the heating, the value of the twist period increases from 200 to 210 μm at 90–100°C, and then to above 224 μm at 140°C. The increase of the twist period is related to the increasing crystalline thickness of the β-lamellae.

Effect of Electrostatic Interactions on Phase Stability of Cubic Phases of Biomembranes

We investigated effect of electrostatic interactions due to surfacecharges on structures and stability of cubic phases of monoolein (MO)membrane using the small-angle X-ray scattering method. Firstly, wechanged the surface charge density of the membrane by usingdioleoylphosphatidic acid (DOPA). As increasing DOPA concentration in themembrane at 30 wt % lipid concentration, a Q224 to Q229 phasetransition occurred at 0.6 mol % DOPA, and at and above 25 mol %, DOPA/MOmembranes were in the Lα phase. NaCl in the bulk phase reduced theeffect of DOPA. These results indicate that as the electrostaticinteractions increase, the most stable phase changes as follows: Q224⇒ Q229 ⇒ Lα. The increase in DOPAconcentration reduced the absolute value of spontaneous curvature of themembrane, | H0 |. Secondly, we changed the surface charge of themembrane by adding a de novo designed peptide, which has netpositive charges and a binding site on the electrically neutral membraneinterface. The peptide-1 (WLFLLKKK) induced a Q224 to Q229phase transition in the MO membrane at low peptide concentration. As NaClconcentration increases, the MO/peptide-1 membrane changed from Q229to Q224 phase. The increase in peptide-1 concentration reduced |H0 |. Based on these results, the stability of the cubic phases and themechanism of phase transition between cubic phase and Lα phase arediscussed.

The size of the pore in lipid membranes induced by antimicrobial peptide magainin 2

Antimicrobial peptides found and isolated from a wide variety of organisms have an activity to kill bacteria. The target of these peptides is thought to be the lipid membrane region of the bacterial and fungal biomembranes. Using the single GUV (giant unilamellar vesicle) method, we have succeeded in revealing the elementary processes of the pore formation in lipid membranes induced by antimicrobial peptide, magainin 2. The statistical analysis of the pore formation in a GUV over many “single GUVs” enabled us to estimate the rate constant of the magainin 2-induced pore formation in lipid membranes. In this report, to reveal the size of the magainin 2-induced pores in lipid membranes, we investigated the interactions of magainin 2 with single GUVs containing various sizes of fluorescent probes. Under the conditions with no photobleaching of fluorescent probes, we investigated the interaction of magainin 2 with single GUVs of 50% dioleoylphosphatidylglycerol (DOPG)/ 50% dioleoylphosphatidylcholine(DOPC) membrane containing various sizes of fluorescent probes in 10 mM PIPES (pH 7.0), 150 mM NaCl (buffer A). Magainin 2 induced a transient (less than 10 s), but very small (10–20 %) leakage of Texas-Red dextran 40,000 (TRD-40k), Texas-Red dextran 70,000 (TRD-70k), and FITC-BSA, although the same concentrations of magainin 2 induced a complete leakage of calcein. In contrast, the magainin 2-induced leakage of Texas-Red dextran 10,000 (TRD-10k) and Texas-Red dextran 3,000 (TRD-3k) had two phases; the transient rapid leakage in the initial stage and the following slow leakage. These results indicate that in the initial stage of the magainin 2-induced pore formation, the size of the pore was large and then transformed into a small, steady size. The radius of the transient, large pore in the initial stage is larger than 6.4 nm (i.e., the Stokes-Einstein radius, RSE, of TRD-70k) and also that the radius of the small steady pore in the final stage is smaller than 3.5 nm (i.e., RSE of FITC-BSA). The amount of the leakage of TRD-10k in the initial stage increased with an increase in magainin 2 concentration. This result indicates that the radius of the large pore in the initial stage increased with an increase in magainin 2 concentration. We discuss these results from a point of view of the pore formation.