Effect of lipid headgroup composition on the interaction between melittin and lipid bilayers

Journal of Colloid and Interface Science 311 (2007) 59–69 www.elsevier.com/locate/jcis

Effect of lipid headgroup composition on the interaction between melittin and lipid bilayers Adam A. Strömstedt a,∗ , Per Wessman b , Lovisa Ringstad a , Katarina Edwards b , Martin Malmsten a a Department of Pharmacy, Uppsala University, P.O. Box 580, SE-751 23 Uppsala, Sweden b Department of Physical and Analytical Chemistry, Uppsala University, P.O. Box 579, SE-751 23 Uppsala, Sweden

Received 13 November 2006; accepted 25 February 2007 Available online 2 March 2007

Abstract The effect of the lipid polar headgroup on melittin-phospholipid interaction was investigated by cryo-TEM, fluorescence spectroscopy, ellipsometry, circular dichroism, electrophoresis and photon correlation spectroscopy. In particular, focus was placed on the effect of the lipid polar headgroup on peptide adsorption to, and penetration into, the lipid bilayer, as well as on resulting colloidal stability effects for large unilamellar liposomes. The effect of phospholipid headgroup properties on melittin-bilayer interaction was addressed by comparing liposomes containing phosphatidylcholine, -acid, and -inositol at varying ionic strength. Increasing the bilayer negative charge leads to an increased liposome tolerance toward melittin which is due to an electrostatic arrest of melittin at the membrane interface. Balancing the electrostatic attraction between the melittin positive charges and the phospholipid negative charges through a hydration repulsion, caused by inositol, reduced this surface arrest and increased liposome susceptibility to the disruptive actions of melittin. Furthermore, melittin was demonstrated to induce liposome structural destabilization on a colloidal scale which coincided with leakage induction for both anionic and zwitterionic systems. The latter findings thus clearly show that coalescence, aggregation, and fragmentation contribute to melittin-induced liposome leakage, and that detailed molecular analyses of melittin pore formation are incomplete without considering also these colloidal aspects. © 2007 Elsevier Inc. All rights reserved. Keywords: Adsorption; Bilayer; Circular dichroism; Ellipsometry; Fluorescence; Liposome; Melittin

1. Introduction Melittin is a well characterized peptide composing 50% of the dry weight of the venom of the European honey bee, Apis mellifera. It is a cationic, 26 amino acid long, peptide with the sequence NH2 -GIGAVLKVLTTGLPALISWIKRKRQQCONH2 . The net charge of melittin is +5 to +6 at neutral pH with all charges but one located near the C-terminal [1]. The peptide predominantly displays a random coil conformation in aqueous solution below the dimerisation concentration [2] whereas it adopts a predominantly α-helical structure when bound to lipid membranes [3]. Several models have been proposed for the leakage induction mechanism of melittin. Both liposome fusion and micellization has been suggested under certain conditions [4–6] and there is also studies supporting * Corresponding author. Fax: +46 18 4714377.

E-mail address: [email protected] (A.A. Strömstedt). 0021-9797/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2007.02.070

pore formation [7–11], although the detailed nature of the pore is still under debate, e.g. regarding the formation of toroidal [11,12] or barrel-stave model pores [13]. Although melittin has been found to bind to lipid membranes in monomeric form [14] it may self-associate at higher concentration, and multimerisation may play a role also for membrane defect formation under some conditions [15,16]. Numerous aspects of melittin interaction with liposomes have been investigated, including the effects of length [17] and saturation [18] of the aliphatic chain of the phospholipid, effects of cholesterol and other sterols [19], effects of varying lipid packing parameters [12], and the presence of large polar headgroups, such as PEG [20]. Primarily, studies on headgroup effect on bilayer-melittin interaction have focused on choline (PC) [3,5,15], ethanolamine (PE) [4,21,22], serine (PS) [5,23], phospatidic acid (PA) [24], and glycerol (PG) [12,25]. It is by now well-established that anionic lipid headgroups confer higher resistance for liposomes against melittin-induced

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leakage compared to zwitterionic ones for the few cases investigated (PC/PE/PG, PC/PS, and PC/PA/PG) [5,8,12,26]. So far, investigations have focused on the effect of melittin on anionic versus zwitterionic headgroups and there has not been any studies on broader ranges of headgroup combinations to any appreciable extent, nor on variations in electrostatic screening. Furthermore, detailed adsorption experiments of melittin at supported lipid bilayers and cryo-TEM experiments on the effect of melittin on liposome structure and stability have not been performed until now. In the present investigation we therefore address these issues through comparison between phosphatidylcholine, -acid, and -inositol headgroups. The latter two are anionic headgroups, identical except for an inositol moiety outside the phosphate charge orientating itself perpendicular to the bilayer to maximize its hydration [27]. Since these anionic lipids form liposomes with comparable electrostatic potential, the main difference between the anionic systems is the presence of the additional hydration force in the case of inositol, which is expected to balance the attractive melittin-headgroup interaction and to affect peptide adsorption to, and incorporation into, the anionic lipid bilayer, thus also affecting liposome defect formation. 2. Experimental 2.1. Materials The lipids used, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sodium 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), and phosphatidylinositol (soy, sodium salt) were all obtained from Avanti Polar Lipids (Alabaster, AL), and of 99+% purity. Cholesterol (99+% purity) was from Sigma–Aldrich (Steinheim, Germany). The quenchers, 5-doxyl stearate and 16-doxyl stearate, were from Aldrich Chemical Company (Milwaukee, WI), while poly-L-lysine (Mw = 170 kDa) and n-dodecyl-βD-maltoside (DDM) (98+% purity) were both from Sigma– Aldrich (St. Louis, MO). The fluorescent marker 5(6)-carboxyfluorescein (99+% purity) was from Acros Organics (Geel, Belgium). Other chemicals used were of analytical grade. Water used was from a Millipore Milli-Q Plus 185 ultra-pure water system. All measurements were performed at pH 7.4 in phosphate-buffered saline (PBS) buffer (0.01 M sodium phosphate buffer with 0.15 M NaCl) or the same buffer without NaCl (PB). Lyophilized and HPLC purified (lipase free) European honey bee venom (melittin) of 97+% purity was obtained from Sigma–Aldrich (St. Louis, MO). The same melittin batch (115H8255) was used for all experiments. The peptide was dissolved in either PBS or PB. In order to remove any residual peptide aggregates, melittin solutions were filtered through a 0.22 µm Millex-GV4 filter (Millipore) prior to storage (small aliquots in dark at −20 ◦ C).

Fig. 1. Binding isotherm describing the partitioning process of melittin into liposomes of either DOPC/cholesterol (60/40 mol/mol) (PC) or PI/DOPC/cholesterol (30/30/40 mol/mol) (PI). Reff is the peptide to lipid molar ratio in the membrane, while [P ]aq is the molar concentration of free peptide in the buffer. For DOPA/DOPC/cholesterol (30/30/40 mol/mol) liposomes, virtually all melittin is adsorbed, rendering a [P ]aq ≈ 0 and those results are therefore not included in the graph. The insert shows the blue shift of the melittin tryptophan residue with 5 µM melittin free in PBS (solid line) or bound to PC bilayers (dashed line). The curves have been normalized at 390 nm and the Y -axis is in arbitrary units.

used as bilayer mounting surface. These were cleaned at 80 ◦ C for 5 min in water solutions of first 3.6% NH4 OH and 4.3% H2 O2 followed by 4.6% HCl and 4.3% H2 O2 . The resulting surfaces displayed an advancing contact angle of less than 10◦ . The slides were then kept in 95% ethanol. Prior to use, the surfaces were cleaned by gas plasma discharges with a Harrick Plasma Cleaner PDC-32G (Harrick Plasma, Ithaca, NY) at 18 W in 0.2 Torr, for 5 min. 2.3. Liposome preparation Dry lipid films were formed on flask walls by dissolving phospholipid(s) and cholesterol in chloroform followed by evaporation under nitrogen gasflow and subsequently also in vacuum over night at room temperature. PBS or PB was then added together with 100 mM of 5(6)-carboxyfluorescein, and the lipids resuspended. The suspensions were then freeze-thawn by alternate placement of the suspension-containing vial in liquid nitrogen and in a 62 ◦ C water bath, in each case followed by vortexing, for eight cycles. Liposome polydispersity was then reduced by extrusion through polycarbonate filters with 100 nm pores using a LipoFast miniextruder (Avestin, Ottawa, Canada). Nonencapsulated carboxyfluorescein was removed by filtration through two subsequent Sephadex G-25 Medium PD-10 columns (Amersham Biosciences, Uppsala, Sweden). They were then refrigerated at 4 ◦ C in glass vials between experiments. 2.4. Partition studies

2.2. Surfaces Polished silica slides, prepared by thermal oxidation to an oxide layer thickness of 30 nm (Okmetic, Espoo, Finland), were

In order to estimate the partitioning of melittin between liposomes and bulk, steady state fluorescence measurements were performed with a SPEX Fluorolog 1650 0.22-m double spec-

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trometer (SPEX Industries, Edison, NJ). The excitation wavelength was set to 280 nm and emission spectra were acquired between 320 and 390 nm. During each experiment aliquots of a 10 mM liposome suspension (prepared without carboxyfluorescein) were added to an aqueous melittin solution and an emission spectrum was acquired after each addition. The emission spectrum of the single tryptophan residue in melittin shifts toward lower wavelengths upon peptide partitioning into lipid membranes (insert, Fig. 1). This blue shift, which arises due to the less polar environment experienced by the tryptophan residue in the lipid membrane, was quantified by taking the ratio of the fluorescence intensities, corrected for the inner filter effects, at 325 and 355 nm, respectively. The degree of blue shift was used to determine the fraction of melittin that had partitioned into the lipid membranes, α, which followed as a function of the lipid to peptide molar mixing ratio, Ri . The fraction of membrane associated melittin and the mixing ratio was then used to construct binding isotherms describing the partition process. The isotherms were fitted to the following expression [28]: Kp Reff α/Ri = = L, (1 − α)[P ]tot [P ]aq γp

(1)

where [P ]tot is the total concentration of melittin, Reff the actual or effective peptide to total lipid ratio in the membrane, [P ]aq the actual concentration of free melittin in the buffer, Kp the partition coefficient (M−1 ), and γPL an activity coefficient introduced in order to account for electrostatic melittin-melittin interactions in the membrane. According to the Gouy–Chapman approach, where the melittin molecules are treated as point charges, the activity coefficient can be described by   γpL = exp 2zp sinh−1 (zp bReff ) . (2) Here zp is the effective number of charges of melittin in the membrane and b is a parameter that is determined by the ionic strength in the buffer [29]. Once Kp and zp are known for a specific lipid composition, Reff for any chosen Ri can be calculated by using an iterative process involving the expression below in combination with Eq. (2): Reff =

Kp [P ]tot [P ]L = L , [L] γp + Kp [L]

(3)

where [P ]L is the concentration of peptides adsorbed to the liposomes and [L] is the concentration of lipids. 2.5. Ellipsometry Melittin adsorption to supported lipid bilayers was studied by in situ null ellipsometry using an Optrel Multiskop ellipsometer (Optrel, Germany) with a 100 mW argon laser at 532 nm, and an angle of incidence of 67.66◦ . Measurements were carried out at 37 ◦ C in a 5 ml cuvette under stirring (300 rpm). The adsorption was followed by monitoring adsorption induced changes in the amplitude and phase of light reflected on the adsorbing surface. From this, the refractive index (n) and layer thickness (d) of the adsorbed layer were determined. With these parameters the adsorbed amount (Γ ) was

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calculated according to de Feijter et al. [30], using a refractive index increment (dn/dc) of 0.154 cm3 /g [31,32]. Corrections were routinely made to compensate for any change in bulk refractive index caused by changes in temperature or excess electrolyte concentration. Zwitterionic bilayers were deposited by coadsorption from a mixed micellar solution containing DOPC/cholesterol (60/40 mol/mol) and DDM as described in detail previously [31]. In short, mixed micellar solutions were prepared by dissolving dried lipid films of DOPC and cholesterol in a 19 mM DDM water solution. The resulting micellar solution, containing 97.3 mol% DDM, 1.6 mol% DOPC, and 1.1 mol% cholesterol, was then added to the cuvette and the deposition monitored. After the lipid adsorption had stabilised, any deposited DDM was removed by rinsing with Milli-Q water (5 ml/min). By repeating this procedure and gradually lowering the concentrations of the micellar solution, stable, densely packed bilayers are formed, with a defect density of less than 10% [31]. With anionic lipid mixtures the mixed micelle approach resulted in sub-bilayer and patchy adsorption. Therefore liposome adsorption was used for generation of supported anionic lipid layers. DOPA/DOPC/cholesterol (30/30/40 mol/mol) (PA) or PI/DOPC/cholesterol (30/30/40 mol/mol) (PI) liposomes to be deposited were prepared as described above but without carboxyfluorescein. To reduce adsorption of peptide directly to the silica substrate through any defects in the supported bilayer, polylysine was preadsorbed (from a 50 ppm aqueous solution). Nonadsorbed polylysine was removed by rinsing with water at 5 ml/min for 20 min. The resulting adsorbed amount of polylysin was 0.045 ± 0.010 mg/m2 . Liposomes were then added (20 µM in PBS), again followed by rinsing with buffer (5 ml/min for 15 min) when the liposome adsorption had stabilized. In the PA containing systems, the final layer formed had similar properties to that formed by the zwitterionic lipid system, and suggest that a layer fairly close to a complete bilayer is formed, while the bilayer formed in the case of PI was inferred to contain some patchiness. After lipid bilayer formation, melittin was added cumulatively to concentrations of 0.025, 0.1, and 0.4 µM and finally to 1.6 µM in the cuvette, and the adsorption monitored. All measurements were done in at least duplicate. 2.6. Liposome leakage assay Liposome integrity was followed by monitoring carboxyfluorescein efflux to the liposome-external lower concentration environment, resulting in loss of self-quenching and thus in an increased fluorescence signal [33]. This fluorescence increase was measured with a SPEX Fluorolog-2 spectrofluorometer (SPEX Industries, Edison, NJ) using an excitation wavelength of 492 nm and an emission wavelength of 515 nm (their respective maxima). Measurements were conducted at a fixed lipid concentration of 20 µM and with five to seven different melittin concentrations within the leakage interval, i.e., the peptide to lipid interval corresponding to 0–100% leakage, ranging from 3.1 nM to 1.6 µM at 37 ◦ C and under constant stirring. In each experiment, an initial signal acquisition for 10 min

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was performed to reveal any spontaneous leakage. Throughout, spontaneous leakage under the timescale of the experiment was found to be less than 2%. The effect of melittin addition to the liposome dispersions was then monitored for 45 min, well after the leakage plateau had been reached. To establish the maximum leakage signal, Triton X100 (Sigma–Aldrich, Steinheim, Germany) was added to a concentration of 0.05 vol% and then measurements continued for another 5 min. Control measurements at 20 ◦ C instead of 37 ◦ C were performed with no observed difference in leakage potential. 2.7. Zeta potential The zeta potential of the liposomes in the presence and absence of melittin was measured using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). Measurements were performed at 20 ◦ C using disposable capillary zetacells. Salt free buffer (PB) was used in order to avoid problems with electrode oxidation and deteriorating measurement reproducibility at high electrolyte concentration. The buffer was also degassed in order to minimize bubble formation in the cuvette. Samples were measured after 45 min of incubation and each experiment was performed in triplicate. The lipid concentration used in the measurements was 40 µM, while melittin concentration was varied from 25 nM to 3.2 µM. Using this approach, the zeta potential in the absence of melittin was found to be, −40 and −51 mV for the 30 mol% anionic PA and PI liposomes, respectively, while the PC liposomes had a zeta potential of −9 mV. The minor negative potential found for PC is in agreement with previous investigations [34,35], and has been speculated as being due to the finite separation between the positive and negative charges in the zwitterionic group affecting the counterion distributions, and resulting in a lower pKa for the phoshate group [36]. However, also minor hydrolysis of the phospholipid, yielding lysophosphatidylcholine and fatty acid, as well as surface enrichment of polarizable anions [37], may contribute to the observed effect. 2.8. Circular dichroism The secondary structure of melittin, either free in buffer or together with liposomes, was analyzed by circular dichroism using a JASCO J810 spectropolarimeter (JASCO Corporation, Easton, MD). The measurements were performed at 20 ◦ C in a 2 mm quartz cuvette. The peptide secondary structure was investigated in the range 200–260 nm at a scan rate of 50 nm/min with 30 accumulations, for samples containing 2 µM melittin and/or 800 µM lipid. The helix content of melittin was quantified by comparing the degree of depolarisation at 222 nm [38] to 225 nm [39] with reference spectra for samples containing 100% α-helix and 100% random coil. As reference, samples of 0.133 mM monomeric poly-L-lysine was used, in 0.1 M NaOH or in 0.1 M HCl, respectively [39]. To avoid any instrumental baseline drift between measurements, the background value (detected at 250–260 nm, where no peptide signal is present) was subtracted for each individual sample measurement. Signals from non peptide components

were also corrected for. Measurements were performed in triplicate. 2.9. Tryptophan quenching Tryptophan quenching was monitored for liposomes in which a portion of the anionic phospholipids (or zwitterionic were no anionic phospholipids were used) was replaced by either of two different doxyl-stearate to a total of 10 mol% of the lipids. The two types of doxyl-stearate had the doxyl group linked to either the 5th (D5) or 16th (D16) carbon, counted from the stearate carboxyl group. Since melittin contain only one tryptophan residue, and the doxyl group quenches the tryptophan only when in close proximity, the magnitude of the fluorescence quenching gives an indication on the extent of membrane penetration of the tryptophan indole sidechain. The tryptophan fluorescence was monitored by a SPEX Fluorolog2 spectrofluorometer in a quartz cuvette at a concentration of 2 µM melittin and 800 µM lipid at 37 ◦ C with excitation and emission wavelength set at 289 and 355 nm, respectively. Measurements were done in triplicate. 2.10. Cryo-TEM The cryogenic transmission electron microscopy (cryoTEM) investigations were performed with a Zeiss EM 902A transmission electron microscope (Carl Zeiss NTS, Oberkochen, Germany), operating at 80 kV and in zero loss brightfield mode. Digital images were recorded under low dose conditions with a BioVision Pro-SM Slow Scan CCD camera (Proscan GmbH, Scheuring, Germany) and analysis software (Soft Imaging System GmbH, Münster, Germany). In order to visualize as many details as possible, an underfocus of 1–2 µm was used to enhance the image contrast. The method for sample preparation is summarized here but more thoroughly described elsewhere [40]. Samples were equilibrated at 25 ◦ C and at close to 100% atmospheric humidity within a climate chamber. A small drop (∼1 µl) of sample was deposited on a copper grid covered with a perforated polymer film and with thin evaporated carbon layers on both sides. Excess liquid was thereafter removed by means of blotting with a filter paper, leaving a thin film of the solution on the grid. Immediately after blotting, the sample was vitrified in liquid ethane, held just above its freezing point. Samples were kept below −165 ◦ C in a protected atmosphere during transfer and examination. 2.11. Light scattering The hydrodynamic diameter of the liposomes were studied by photon correlation spectroscopy at a scattering angle of 173◦ using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) at 20 ◦ C using disposable plastic cuvettes. Samples were measured 45 min after melittin administration and each experiment was performed in triplicate. Lipid concentration was fixed at 40 µM and the melittin concentration was varied from 25 nM to 3.2 µM.

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Fig. 2. Adsorption isotherms of melittin on bilayers of either DOPC/cholesterol (60/40 mol/mol) (PC), DOPA/DOPC/cholesterol (30/30/40 mol/mol) (PA) or PI/DOPC/cholesterol (30/30/40 mol/mol) (PI). Adsorption was measured for PBS (ionic strength 0.174), and with PI also for PB (ionic strength 0.024). The error bars represent SD from duplicate or triplicate measurements.

3. Results and discussion 3.1. Melittin partitioning For technical reasons the lipid concentrations used in the present study varied, dependent on the method used, in a span between 20 and 2000 µM. Knowledge about the melittin partition behaviour was therefore needed in order to correlate results obtained with different techniques. This information was, furthermore, of importance for the comparison of data obtained in membrane systems with different lipid composition. To obtain an estimate of the partition coefficient and the effective number of charges of the membrane associated peptides, Kp and zp , we used a spectroscopic method based on the measurement of changes in melittin intrinsic fluorescence (see above for details). The determined Kp and zp values were then used to calculate Reff , the effective peptide to lipid (P/L) molar ratio in the membrane, at the relevant lipid concentrations. For the DOPC/cholesterol (60/40 mol/mol) system, Kp and zp were determined to be 2.6 × 104 M−1 and 5.5, respectively (Fig. 1). As expected, the peptide-membrane interactions were significantly increased upon inclusion of the anionic PA and PI lipids. Accordingly, for membranes supplemented with 30 mol% PI the measurements indicated Kp and zp values of 9.8 × 104 M−1 and 1.1, respectively. For membranes supplemented with 30 mol% PA the spectroscopic investigations in fact suggested that virtually all melittin was membrane associated at lipid concentrations corresponding to 20 µM or more. Thus, for the PA-based systems Reff may be taken as 1/Ri . Further information on the adsorption of melittin on bilayers of the compositions corresponding to DOPC/cholesterol (60/40 mol/mol) (PC), DOPA/DOPC/cholesterol (30/30/40 mol/mol) (PA) and PI/DOPC/cholesterol (30/30/40 mol/mol) (PI) liposome systems was observed by ellipsometry on supported bilayers (Fig. 2). In agreement with the partitioning results (Fig. 1) the adsorption of melittin at the zwitterionic PC bilayer is significantly lower than that at the ionic PA and PI bilayers.

Fig. 3. Peptide to lipid (P/L) molar ratio generating 50% leakage of trapped carboxyfluorescein for liposomes with different mole fractions of anionic phospholipids ranging from entirely zwitterionic to completely anionic. All liposomes contained 40% cholesterol which is included in the lipid ratio. The error bars are the deviations from linearity from 2 to 99% leakage out of 5 to 7 measurements.

This is also expected based on the strong electrostatic interaction between the positively charged melittin and the anionic PA and PI (−40 and −51 mV, respectively, as shown in Fig. 5). 3.2. Effects of melittin on liposome leakage It is by now rather well established that negative charged liposomes are more resistant toward melittin-induced leakage than zwitterionic liposomes [5,26]. Our results on liposome leakage from DOPA/DOPC/cholesterol and PI/DOPC/cholesterol liposomes of different phospholipids composition (Fig. 3) support these findings and show that both anionic lipid systems hinder melittin-induced liposome leakage. However, the P/L ratio required to induce 50% leakage is significantly higher for PA-containing liposomes than for PI-containing ones of the same molar ratio of anionic lipid (and comparable zeta poten-

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For all liposomes, the negative electrostatic potential decreases with increasing levels of the positively charged melittin added to the liposomes (Fig. 5). While charge reversal is observed at high melittin concentrations in the case of PC, no such charge reversal is observed for PA- and PI-containing liposomes. It should also be noted that the effective reduction in liposome zeta potential caused by melittin addition is not strongly correlated in itself with leakage induction (Fig. 5, cf. arrows corresponding to 50% leakage for each liposome type). 3.3. Bilayer induced changes in melittin secondary structure

Fig. 4. Melittin-induced liposome leakage for DOPC/cholesterol (60/40 mol/mol) (PC), DOPA/DOPC/cholesterol (30/30/40 mol/mol) and PI/DOPC/ cholesterol (30/30/40 mol/mol) (PI) at varying molar ratios of adsorbed peptide to lipid (Reff ) in PBS. Reff is equivalent to total peptide to lipid molar ratio for PA.

Fig. 5. The zeta potential of liposomes containing either DOPC/cholesterol (60/40 mol/mol) (PC), DOPA/DOPC/cholesterol (30/30/40 mol/mol) (PA) or PI/DOPC/cholesterol (30/30/40 mol/mol) (PI) in the presence of melittin at various peptide to lipid (P/L) molar ratios. Arrows show the P/L ratio required to generate 50% leakage for PC, PA, and PI. The error bars represent SD from triplicate measurements.

tial, Fig. 5). The difference between the two types of anionic liposomes increases with increasing molar fraction of the anionic phospholipids. Independent of liposome composition, the leakage is generated within the first minute following melittin addition. By combining the results from the partitioning study with the leakage measurements, we could show the actual adsorbed peptide to lipid molar ratio for each respective leakage induction level (Fig. 4). This approach augments the already substantial difference in the membrane disruptive potency for melittin in each system. The data clearly show that although the absorbed amounts are in the order of PC < PI < PA, the leakage inducing effect per adsorbed melittin molecule follows the opposite trend, i.e., PA < PI < PC.

Our circular dichroism measurements showed that for all systems investigated, melittin binding to liposomes induces a coil to helix transition. While the helix content of melittin in PBS buffer at the conditions investigated is 20 ± 1%, the helix content in the liposome systems is substantially higher, 77 ± 3%, 79 ± 1%, and 67 ± 2% for PC, PA, and PI liposomes, respectively. For PI liposomes in PB (no NaCl), the corresponding helix content of melittin is 64 ± 3%. It should be noted that these values largely represent melittin when bound to the liposomes, with little influence of melittin free in solution. This is inferred by increasing the liposome concentration a factor of two, and by reducing the melittin concentration by a factor of two, which both gave helix contents in good agreement with those reported above. Further, partition data support the assumption that virtually all melittin is membrane bound at the P/L ratio employed in the circular dichroism measurements. It should also be noted that these values of melittin helix content in buffer and when bound to zwitterionic and anionic liposomes agree well with those previously reported for related systems [3,41,42]. Overall, the differences observed between the anionic and the zwitterionic systems in terms of peptide adsorption and partitioning to the lipid membrane, and of resistance to structural changes and leakage induction, seems to be largely uncorrelated to the peptide secondary structure formed upon binding to the lipid bilayer. 3.4. Tryptophan depth of melittin bound to membranes Another factor which may, however, contribute to the differences observed in melittin action on anionic and zwitterionic liposomes is the extent of peptide penetration into the membrane interior. Indeed, quenching of the single melittin tryptophan residue by depth-specific doxyl quenching shows that melittin penetrates deeper into the bilayer in the case of PC liposome compared to the PA-containing system (Fig. 6). Thus, quenching is substantially higher in the D5 position for PC than for PA-containing liposomes. PI-containing liposomes represent a case in between PC and PA-containing liposomes, with intermediate quenching by the D5 doxyl stearate. Thus, in the case of PI, melittin does not penetrate into the bilayer to quite the same extent as for PC, but substantially more so than for PA. Decreasing the electrolyte concentration in the case of PI, however, yields quenching results more comparable to the PA case, i.e., with melittin penetrating the lipid bilayer to a lesser extent than observed in the high salt system. The D16-related

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Fig. 6. Tryptophan fluorescence of melittin measured when bound to liposomes containing position-specific doxyl-stearate. The doxyl quencher is positioned either shallow, at the 5th carbon (D5), or deeper, at the 16th carbon (D16). Results are shown for DOPC/cholesterol (60/40 mol/mol) (PC), DOPA/DOPC/cholesterol (30/30/40 mol/mol) (PA), and PI/DOPC/cholesterol (30/30/40 mol/mol) (PI), the latter either in PBS (ionic strength 0.174) or in PB (ionic strength 0.024). The error bars show standard deviations from triplicate experiments except for the “All” bars, where the standard deviation also includes the variation between the different liposome compositions. The peptide to lipid molar ratio is 1:400 for all samples.

quenching is similar for all systems and likely corresponds to the depth unspecificity generated from membrane fluidity, although it might be possible that it represent a small population of peptides that would cover the inside of pores or the edges of fragmenting membranes. In the absence of doxyl quencher, quenching is marginal, which indicates that the D16 quenching is due to the doxyl groups, rather than to any other residual quenching in the samples. In literature the penetration of the melittin tryptophan in anionic compared to zwitterionic membranes has been concluded to be either deeper [25,43,44] or shallower [5,45], depending on the method used. However, water penetration around membrane bound melittin has been shown to be higher for pure zwitterionic membranes than when mixed with anionic lipids [45]. This weakens the argument from water-based quenching and blue shift studies, which have generally portrayed a deeper penetration in cholesterol free anionic membranes. It should also be considered that there has not been a comparative study of penetration depth between anionic and zwitterionic membranes in the presence of cholesterol, prior to this study. Previous reports show a decrease of melittin tryptophan penetration in zwitterionic membranes when cholesterol is introduced [25,46]. This could also be the reason for the lower lytic activity for melittin in zwitterionic membranes when cholesterol is added [47], an effect that we can confirm (results not shown). However, as in the case of melittin in zwitterionic membranes, cholesterol increases water penetration in the headgroup region, although it reduces it in the interior of the membrane [48]. 3.5. Importance of headgroup electrostatics and hydratisation In analogy to the decreased melittin penetration into the lipid bilayer on decreasing the excess electrolyte concentra-

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Fig. 7. Leakage from liposomes containing PI/DOPC/cholesterol (30/30/40 mol/mol) (PI) at different peptide to lipid (P/L) molar ratios in PBS (ionic strength 0.174) or PB (ionic strength 0.024).

Fig. 8. Peptide to lipid (P/L) molar ratio generating 50% leakage of encapsulated carboxyfluorescein for liposomes of either DOPC/cholesterol (60/40 mol/mol) (PC), DOPA/DOPC/cholesterol (30/30/40 mol/mol) (PA), or PI/ DOPC/cholesterol (30/30/40 mol/mol) (PI), in PBS (ionic strength 0.174) or PB (ionic strength 0.024). The error bars are the deviations from linearity from 2 to 99% leakage out of at least 5 measurements.

tion in the case of PI-containing liposomes (see above), the P/L ratio needed to induce leakage is higher when melittin is incubated with PI liposomes in NaCl-free buffer (ionic strength 0.024) compared to buffer containing 150 mM NaCl (ionic strength 0.174) (Fig. 7). Consequently, also from a leakage perspective, decreasing the electrolyte concentration for the PI system renders it more similar to PA. Furthermore, PAcontaining liposomes display similar electrolyte concentration dependence, i.e., more melittin is required to cause liposome leakage at low electrolyte concentration (Fig. 8). Quantitatively, a P/L ratio 1.6 times higher is required to inflict 50% leakage for PA liposomes in the absence than in the presence of 150 mM NaCl, an electrolyte effect similar to the situation for PI. The peptide penetration results are therefore compatible with the liposome leakage results. Interestingly, the electrolyte effect seems to be reversed for PC liposomes, which display increased susceptibility to leakage induction in the presence of salt. Still, it has been reported that cationic peptides partition at a higher ratio to PC membranes when in solvent with higher NaCl concentrations [49].

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Attractive electrostatic interactions between the C-terminal part of melittin and negatively charged lipid headgroups may require a more parallel orientation upon adsorption. This has been suggested as a possible mechanism behind the larger melittin resistance observed for anionic liposomes [8]. It has also been suggested that melittin-induced pore formation in the case of zwitterionic lipid membranes is triggered at a critical P/L ratio when the peptide changes its orientation from parallel toward perpendicular relative to the membrane normal [22]. However, the P/L ratio, where the change in orientation is proposed to occur for pure DOPC liposomes, is almost 12 times higher than what is required for 100% leakage in our studies on such liposomes (results not shown). It is possible that the perceived reorientation is a result of large scale structural effects (see below). Nevertheless, for the anionic membranes, the much higher melittin adsorption and leakage resilience, the results on shallower penetration and the peptides structural indifference toward headgroup composition, combined with the differences between PA- and PI-containing systems, as well as the effects of varying electrolyte concentrations, all point toward that the shallower, likely more parallel orientation of melittin, is a consequence of electrostatic interaction with the headgroups of the anionic lipids. 3.6. Structural effects caused by melittin In order to learn more about the possible structural effects of melittin on liposomes cryo-TEM was used for direct visualization. The investigation showed that there were considerable effects on the size and structure of the liposomes when treated with melittin. These changes could be seen at adsorbed concentrations within the leakage interval of the P/L ratio (Fig. 4). Fig. 9a shows PC liposomes prior to melittin addition. Spherical, mostly unilamellar liposomes in the size range 75–150 nm, some bilamellar and occasionally more elongated occur. CryoTEM showed a similar liposome structure also in samples composed of PI and PA (results not shown). No structural changes were revealed in PC samples upon addition of melittin in concentrations corresponding to a Reff of 4.4 × 10−4 and a leakage of 50%. The sample appearance changed markedly, however, when Reff was increased to 8.8 × 10−4 , corresponding to 70% leakage of the liposomes (Fig. 9b). The micrographs then displayed a population of very small liposomes with diameters in the range from 15 to 30 nm. In addition, larger liposomes, with diameters sometimes exceeding 300 nm, in the process of fusion or, alternatively, fission were frequently observed. Thus, membrane perturbations other than pore-like defects are likely to contribute to the leakage measured at melittin concentrations corresponding to Reff values of 8.8 × 10−4 , or higher. The cryo-TEM investigations revealed that melittin caused major structural changes also in samples containing PA- and PIsupplemented liposomes. In these cases, however, much larger membrane concentrations of peptide were needed to induce the structural transitions. Thus, no change in liposome structure was observed at Reff values corresponding to those where pure PC liposomes ruptured and fused (results not shown). Higher melittin concentrations triggered, however, important

alterations in the PA-based systems. At a Reff of 1.8 × 10−2 , corresponding to 100% leakage for PA liposomes, the micrographs displayed very small liposomes together with aggregated and fused structures (Fig. 9c). For PI-based systems severe structural alterations were revealed at a Reff of 4.8 × 10−3 (Fig. 9d). This is well below the Reff value of 6.9 × 10−3 needed for complete leakage of the PI liposomes. Interestingly, a new type of structure, not observed in the PC and PA systems, was exposed in the micrographs. These, electron dense, roughly spherical, particles of varying size were frequently detected in the PI-based system (black arrows in Fig. 9d). The compact structures often appeared attached to a liposomal bilayer but were also detected free in solution. Similar structures have earlier been observed in dispersed lipid systems and interpreted as representative of particles with reversed hexagonal phase structure [50]. The capability of melittin to induce reversed hexagonal phases in pure DOPA liposome systems have been seen, however, only at the high P/L ratio of 0.25 [24]. Finally, melittin adsorption generated rough contours on the large fused anionic liposomes (Figs. 9c and 9d) as compared to the smooth contours on the fused PC liposomes (Fig. 9b). This difference could simply be a result of the higher amount of adsorbed melittin on the anionic membranes, but it could also signify a difference in the manner of adsorption, the formation of rafts, or effects on membrane fluidity. Also from dynamic light scattering, evidence for melittininduced structural changes can be seen. The average liposome diameter increases gradually but marginally as the P/L ratio increases across the leakage interval (less than 1.2 times the initial diameter at 100% leakage). However, the volume size distribution curves before and after addition of melittin, of a concentration yielding 100% leakage, display a significant difference (Figs. 10a–10c). After melittin addition, the size distributions shifted to higher diameters for all three lipid systems, most noticeably so for PC were also a population of large aggregates were discovered (Fig. 10a). This is in agreement with the cryoTEM results that also showed large fused bodies especially in the PC system and predominantly in anionic systems the presence of a large number of budding or aggregating liposomes in complexes similar in size with the untreated liposomes (Fig. 9). The smaller aggregates present contribute to the still sizeable overlap with the untreated distribution. The higher effect on PC liposomes should be interpreted in the light of the much lower amount of adsorbed melittin required for total leakage and morphological transformation compared to the other two systems. Previously, melittin-induced liposome leakage has been claimed to precede large scale effects like liposome fusion or fission both in anionic and zwitterionic membranes [5]. This is one of the fundamental arguments for the presence and necessity of pore formation as a leakage inducer. Our results show that liposomes can undergo large scale morphological transformations, generated from both budding and fusion, already within the leakage interval. The liposomal population undergoes slight alterations in size distributions as soon as the P/L ratio reaches the leakage interval according to dynamic light scattering. Fusion seems less pronounced in anionic liposomes even at the same leakage levels as the zwitterionic ones. The

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Fig. 9. Representative cryo-TEM images of aggregates of the different lipid composition prior (a) and after (b–d) melittin addition. (a) DOPC/cholesterol (60/40 mol/mol) liposomes in the absence of melittin. (b) DOPC/cholesterol (60/40 mol/mol) aggregates with melittin added corresponding to a peptide concentration in the membrane, Reff , of 8.8 × 10−4 and causing 70% leakage (c) DOPA/DOPC/cholesterol (30/30/40 mol/mol) aggregates with melittin added corresponding to a Reff of 1.8 × 10−2 and 100% leakage. (d) PI/DOPC/cholesterol (30/30/40 mol/mol) aggregates with melittin added corresponding to a Reff of 4.8 × 10−3 and 70% leakage. Black arrows indicate electron dense structures, possibly reversed hexagonal phase. White arrows indicate ice crystals deposited on the sample after the vitrification. Scale bars indicate a size of 100 nm.

overall results from cryo-TEM and dynamic light scattering is that large scale structural transitions can not be excluded as responsible for at least part of the melittin-induced leakage in these systems of cholesterol supplemented phospholipid membranes. Coupling a hydrophilic compound on the anionic lipid headgroup does not necessarily confer increased protection for the membrane. In contrast, such a compound (in our case inositol) can be detrimental for the membrane integrity when treated with melittin. Fusion induced by divalent cations on anionic liposomes has previously been shown to be inhibited when introducing PI as a membrane constituent, even at lower membrane ratios than used in this study [51]. Still, our results show that melittin-induced fusion of PI liposomes occur at lower adsorbed amounts of melittin than if substituted for PA. Comparing PI with anionic PEG-lipids gives a taste of what complex effects interfacial molecules can have. Interfacial PEG (2–5 kDa) on anionic liposomes has been shown to promote the binding of melittin [20] while we have seen a slight reduction with inositol. Yet, while PEG seem to have a protective effect against melittin-induced leakage on anionic membranes [20], inositol has the opposite effect. It is clear that the smaller (180 Da) but more compact and rigid, inositol close to the membrane inter-

face compared to the long, flexible PEG has diametric effects on the lytic properties of melittin. Judging from the electrostatic and hydrophobic properties of melittin in its helical structure, it is possible that the inositol forces the C-terminal to protrude from the charged headgroups and away from the membrane normal, something that might not be reached by PEG that would constitute a lesser obstacle in the absolute vicinity of the headgroup charge. In the case of inositol, a protruding C-terminal of the melittin rigid helix could possibly mediate a deeper penetration of the N-terminal by lever action, and thus increase the lytical property. 4. Summary Melittin causes liposome structural destabilization through fusion, aggregation, and budding, which coincides with leakage induction. The melittin-induced morphological transitions are qualitatively similar for the phosphatidylcholine, -acid, and -inositol headgroups, but the headgroup properties strongly affect the amount of melittin needed to induce these transitions. Increasing the membrane negative charge leads to increased peptide adsorption but also to increased liposome tolerance, the latter due to melittin being arrested closer to the interface of

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Fig. 10. Size distribution of liposomes of either: (a) DOPC/cholesterol (60/40 mol/mol) (PC), (b) DOPA/DOPC/cholesterol (30/30/40 mol/mol) (PA), or (c) PI/ DOPC/cholesterol (30/30/40 mol/mol) (PI) without melittin or with melittin added to a concentration yielding 100% leakage.

the lipid bilayer. Balancing the electrostatic attraction between melittin and the phospholipid negative charges through a hydration repulsion caused by inositol reduced this surface arrest and increased liposome vulnerability toward melittin. This relaxation can be largely eliminated by reducing the excess electrolyte concentration, i.e., by shifting the interaction balance toward an increasing dominance of electrostatics and surface arrest. The findings demonstrate that properties of the lipid polar headgroup, other than simply anionic or zwitterionic, are important in these systems and given by a balance of interaction contributions. Our findings also demonstrate that any detailed molecular analysis of melittin pore formation in liposome systems must take colloidal effects into account. This is likely to be the case also for other types of membrane disruptive peptides, such as antibacterial peptides, given their chemical, structural and functional similarities. Acknowledgments To Ms. Lotta Wahlberg goes our deepest gratitude for her expert technical assistance. Professor Mats Almgren is gratefully acknowledged for discussions on fluorescence quenching and Professor Lennart Bergström for putting the Zetasizer to our disposal. This work was financed by the Swedish Research Council.

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