Hirotaka Ariyama

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.

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.

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.