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J. Biol. Chem., Vol. 279, Issue 53, 55042-55050, December 31, 2004

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Dissection of Antibacterial and Toxic Activity of Melittin

A LEUCINE ZIPPER MOTIF PLAYS A CRUCIAL ROLE IN DETERMINING ITS HEMOLYTIC ACTIVITY BUT NOT ANTIBACTERIAL ACTIVITY^*

Neeta Asthana, Sharada Prasad Yadav¶, and Jimut Kanti Ghosh||

From the Molecular and Structural Biology Division, Central Drug Research Institute, Lucknow 226 001, India

Received for publication, August 4, 2004 , and in revised form, September 29, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Melittin, a naturally occurring antimicrobial peptide, exhibits strong lytic activity against both eukaryotic and prokaryotic cells. Despite a tremendous amount of work done, very little is known about the amino acid sequence, which regulates its toxic activity. With the goal of understanding the basis of toxic activity and poor cell selectivity in melittin, a leucine zipper motif has been identified. To evaluate the possible structural and functional roles of this motif, melittin and its two analogs, after substituting the heptadic leucine by alanine, were synthesized and characterized. Functional studies indicated that alanine substitution in the leucine zipper motif resulted in a drastic reduction of the hemolytic activity of melittin. However, interestingly, both the designed analogs exhibited antibacterial activity comparable to melittin. Mutations caused a significant decrease in the membrane permeability of melittin in zwitterionic but not in negatively charged lipid vesicles. Although both the analogs exhibited similar secondary structures in the presence of negatively charged lipid vesicles as melittin, they failed to adopt a significant helical structure in the presence of zwitterionic lipid vesicles. Results suggest that the substitution of heptadic leucine by alanine impaired the assembly of melittin in an aqueous environment and its localization only in zwitterionic but not in negatively charged membrane. Altogether, the results suggest the identification of a structural element in melittin, which probably plays a prominent role in regulating its toxicity but not antibacterial activity. The results indicate that cell selectivity in some antimicrobial peptides can probably be introduced by modulating their assembly in an aqueous environment.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Melittin, the major component of European bee venom from Apis mellifera, is a well studied peptide, which is known for its strong cytolytic and antimicrobial activities (1, 2). It belongs to the family of cytolytic peptides whose members are believed to be the key components of defense and offense mechanisms of all organisms (3, 4).

A considerable number of structure-function studies have been carried out to understand the molecular mechanism of the hemolytic and antimicrobial activities of melittin. The replacement of the first 20 amino acids by another helix-forming sequence did not alter the antimicrobial and hemolytic activity of melittin (5). However, the deletion of Leu-6, Lys-7, Val-8, Leu-9, Leu-13, Leu-16, Ile-17, Trp-19, and Ile-20 dramatically reduced the hemolytic activity of melittin and to a lesser extent the antimicrobial activity also (6). The crucial roles of both termini of melittin in its functional properties have been demonstrated by the design of several analogs with deletion of N-(7, 8) and C-terminal (9) sequences.

Several efforts have been made to design analogs of melittin with decreased hemolytic but similar antimicrobial activity. For example, the diastereoisomers of melittin having D-amino acid analogs (10) at a few positions, a hybrid peptide consisting of cecropin A and melittin sequences (11), and an analog designed from the C-terminal 15-residue fragment (12) exhibited similar antibacterial activity to melittin but much less hemolytic activity. The crystal structure of melittin has been solved, which indicated a tetrameric helix-bend-helix structure (13) with two helical segments, positioned between amino acid residues 1–10 and 13–26 and the bend region within residues 11–12.

Biophysical properties of melittin such as self-association and phospholipid membrane-interaction have been studied extensively to understand the mode of action of melittin (14–17). Although in water melittin exists as monomer with mostly random coil conformation, an increase in peptide concentration or the addition of salt results in the transformation of monomer to tetramer with a pronounced helical structure (18–20). Melittin has been used as a model for studying the general features of lipid interaction of membrane proteins. Membrane-interaction studies suggest that melittin forms transmembrane pores in zwitterionic lipid bilayers by a barrel-stave mechanism (21–24) and in negatively charged lipid vesicles it acts like a detergent through a carpet mechanism (24, 25).

Despite all of the work done on melittin, it is not yet clear (i) why melittin has very low cell selectivity, i.e. why it exerts both high antibacterial and toxic activities, and (ii) is the same amino acid sequence responsible for both the hemolytic and antibacterial activities. Toward the identification of important structural and functional elements in melittin and to gain insight about the regulation of cell selectivity in an antimicrobial peptide in general, we have found a leucine zipper motif, located within the residues 6–20, which has not been reported earlier to the best of our knowledge. Because this motif is known to play important roles in the assembly of DNA-binding proteins (26, 27) or membrane-associated viral fusion proteins (28–30), it was of interest to look into the possible roles of this motif in the structural integrity and functional activity of melittin. For this purpose, two analogs of melittin were designed by selectively replacing heptadic leucine with single and double alanine and then synthesized. Hemolytic activity of single (MM-1) and double alanine mutants (MM-2) against human red blood cells was significantly less than that of melittin. Interestingly, MM-1 and MM-2 exhibited antibacterial activity comparable to melittin. Both of the designed analogs displayed contrasting membrane permeability in zwitterionic and negatively charged lipid vesicles. The results have been discussed in terms of the role of this motif in determining the hemolytic and antimicrobial activity of melittin.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Rink amide 4-methylbenzhydrylamine resin (loading capacity, 0.63 mmol/g) and all the N- Fmoc¹ and side-chain protected amino acids were purchased from Novabiochem, Switzerland. Coupling reagents for peptide synthesis such as 1-hydroxybenzotriazole, N,N'-diisopropylcarbodiimide, 1,1,3,3-tetramethyluronium tetrafluoroborate, and N,N'-diisopropylethylamine were purchased from Sigma. Dichloromethane, N,N'-dimethylformamide, and piperidine were of standard grades and procured from reputed local companies. Acetonitrile (HPLC grade) was procured from Merck, India, and trifluoroacetic acid was purchased from Sigma. Egg phosphatidylcholine (PC), egg phosphatidylglycerol (PG), and dimyristoyl phosphatidylethanolamine (PE) were procured from Northern Lipids Inc., and cholesterol (Chol) was purchased from Sigma. 3,3'-Dipropylthiadicarbocyanine iodide and 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD)-fluoride were purchased from Molecular Probes (Eugene, OR). The rest of the reagents were of analytical grade and procured locally; buffers were prepared in milli Q water.

Peptide Synthesis, Fluorescent Labeling, and Purification—All of the peptides were synthesized manually on solid phase. Stepwise solid phase syntheses were carried out on rink amide MBHA resin (0.15 mmol) utilizing the standard Fmoc chemistry, employing N,N'-diisopropylcarbodiimide/1-hydroxybenzotriazole or 1,1,3,3-tetramethyluronium tetrafluoroborate/1-hydroxybenzotriazole/N,N'-diisopropylethylamine coupling procedure (30–32) with a checking of the deprotection of the -amino group and the coupling of amino acids by usual procedures (33, 34). After the synthesis was over, each peptide was cleaved from the resin by a standard method as reported earlier (32) and precipitated with dry ether. Labeling at the N terminus of peptides with a fluorescent probe was achieved by a standard procedure (30, 32, 35, 36), and after sufficient labeling, the labeled peptides were cleaved from the resins and precipitated. All the peptides were purified by reverse phase HPLC on an analytical Vydac C18 column using a linear gradient of 0–80% acetonitrile for 45 min with a flow rate of 0.6 ml/min. Both acetonitrile and water contained 0.05% trifluoroacetic acid. The purified peptides were 95% homogeneous as shown by HPLC. Experimental molecular mass of the peptides, detected by electrospray-mass spectrometry analysis, corresponded to the desired values within 0.5 Da.

Preparation of Small Unilamellar Vesicles—Small unilamellar vesicles were prepared by a standard procedure (32, 35, 36) with required amounts of either of the PC/cholesterol (8:1 w/w), PC/PG/cholesterol (4:4:1 w/w), or PE/PG (7:3 w/w). Dry lipids were dissolved in CHCl₃/MeOH (2:1 v/v) in small glass vial. Solvents were evaporated under a stream of nitrogen resulting the formation of a thin film on the wall of a glass vessel. The thin film was resuspended in a buffer at a concentration of 8.2 mg/ml by vortex mixing. The lipid dispersions were then sonicated in a bath-type sonicator (Laboratory Supplies Company, New York) for 10–20 min until it became transparent. The lipid concentration was determined by phosphorus estimation (37).

Circular Dichroism Experiments—The circular dichroism (CD) spectra of peptides were recorded in phosphate-buffered saline (PBS, pH 7.4) and in the presence of phospholipid vesicles by utilizing a Jasco J-810 spectropolarimeter, calibrated routinely with 10-camphor sulfonic acid. The samples were scanned at room temperature (30 °C) in a capped quartz cuvette of 0.20-cm path length in the wavelength range of 250–195 nm. The fractional helicities were calculated with the help of mean residue ellipticity values at 222 nm by the following equation (38, 39),

(Eq. 1)

as already reported (30, 32), where []₂₂₂ was the experimentally observed mean residue ellipticity at 222 nm. The values for []¹⁰⁰₂₂₂ and []⁰₂₂₂ that correspond to 100 and 0% helix contents were considered to have mean residue ellipticity values of –32,000 and –2000, respectively, at 222 nm (39).

Membrane Permeability Assay, Dissipation of Diffusion Potential from the Small Unilamellar Lipid Vesicles—The ability of melittin and its analogs to destabilize the phospholipid bilayer was detected by the assay of peptide induced dissipation of diffusion potential across the lipid vesicles by employing a standard procedure (40, 41) as reported before (30, 32, 41). Lipid vesicles, prepared in K⁺ buffer (50 mM K₂SO₄, 25 mM HEPES sulfate, pH 6.8), were mixed with isotonic (K⁺-free) Na⁺-buffer followed by the addition of the potential sensitive dye 3,3'-dipropylthiadicarbocyanine iodide. After the addition of valinomycin to the vesicle suspension when fluorescence of the dye exhibited a steady level, the respective peptide was added. The peptide-induced dissipation of diffusion potential, as detected by an increase in fluorescence (at 670 nm with excitation at 620 nm), was measured in terms of percentage of fluorescence recovery (F_t), defined by F_t = [(I_t – I₀)/(I_f – I₀)] x 100%, where I_t is fluorescence after the addition of a peptide at time t, I₀ is fluorescence after the addition of valinomycin, and I_f is the total fluorescence observed before the addition of valinomycin.

Enzymatic Cleavage Experiments—To detect the location of the peptides in their membrane-bound states, enzymatic cleavage experiments were performed with their NBD-labeled versions as reported earlier (30, 32). In brief, lipid vesicles made of either PC/Chol, PC/PG/Chol, or PE/PG were first added to the NBD-labeled peptide. When all the peptide was bound to lipid as detected by the saturation of the fluorescence level, proteinase K (final concentration, 12.0 µg/ml) was added. In this experiment fluorescence of NBD-labeled peptide was recorded at 527 nm with respect to time (s) with excitation wavelength set at 467 nm.

Hemolytic Activity Assay—The hemolytic activity of melittin and its analogs was assayed by a standard procedure (10, 12). In brief, fresh human red blood cells (hRBCs) that were collected in the presence of an anti-coagulant from a healthy volunteer were washed three times in PBS. Peptides, dissolved in water, were added to the suspension of red blood cells (6% final in v/v) in PBS to the final volume of 200 µl and incubated at 37 °C for 35 min. The samples were then centrifuged for 10 min at 2000 rpm, and the release of hemoglobin was monitored by measuring the absorbance (A_sample) of the supernatant at 540 nm. For negative and positive controls hRBCs in PBS (A_blank) and in 0.2% (final concentration v/v) Triton X-100 (A_Triton) were used, respectively. The percentage of hemolysis was calculated according to the equation, Percentage of hemolysis = [(A_sample – A_blank)/(A_Triton – A_blank)] x 100.

Assay of Antibacterial Activity of the Peptides—The antibacterial activity of the peptides was assayed in LB medium under aerobic conditions (10, 12, 42). Different concentrations of each of the peptides, dissolved in water, were added in duplicate to 100 µl (final volume) of medium containing the inocula of the test organism (10⁶ colony forming units) in the midlogarithmic phase of growth. The inhibition of growth of microorganisms was assessed by measuring the absorbance at 492 nm after an incubation of 18–20 h at 37 °C. The antibacterial activity of the peptides was expressed as the minimal inhibitory concentration, the concentration at which 100% inhibition of microbial growth was observed after 18–20 h of incubation. The microorganisms used were Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus, and Gram-negative bacterium, Escherichia coli.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Design of Analogs of Melittin—Melittin has an interesting sequence with 26 amino acids including five cationic residues. A leucine zipper motif, with every seventh amino acid as leucine/isoleucine (leucine at position 6 and 13 and isoleucine at position 20) and also leucine in two "d" positions, was identified (Fig. 1). To look into the possible role of this important structural motif, two analogs of melittin were designed. In one of which leucine at position 13 (MM-1) was replaced with alanine and in the other (MM-2) leucine at positions 6 and 13 were replaced with two alanines (Fig. 1A). Alanine was chosen for its helix propensity and similar hydrophobicity to leucine so that the amphipathic character of melittin was retained in MM-1 and MM-2.

View larger version (28K): [in this window] [in a new window]   FIG. 1. A, designations and sequences of melittin and its analogs used in this study. Heptadic amino acids are marked in bold, whereas mutated amino acids are marked as bold and underlined. B, helical wheel projections of the first 21 amino acids of melittin, and MM-2 with the first amino acid glycine placed in the c position of the heptad.

View larger version (36K): [in this window] [in a new window]   FIG. 2. Effect of mutation in the leucine zipper motif of melittin on its hemolytic activity. Dose-dependent hemolytic activity of melittin (a), MM-1 (b), and MM-2 (c) against human red blood cells.

View larger version (27K): [in this window] [in a new window]   FIG. 3. Membrane permeability induced by melittin and its analogs as plotted by percent fluorescence recovery induced by the individual peptides. A, plots of fluorescence recovery induced by different concentrations of individual peptides in zwitterionic PC/Chol lipid vesicles (68 µM, final concentration). Closed squares, melittin; closed circles, MM-1; closed triangles, MM-2. Inset depicts the representative experimental profiles for melittin and its analogs at 1.52 µM as marked. B, plots of fluorescence recovery induced by melittin, MM-1 and MM-2 at different concentrations (symbols are the same as in A) in negatively charged PC/PG/Chol lipid vesicles (68 µM, final concentration). Inset depicts the representative experimental profiles for melittin and its analogs as marked at 0.76 µM. Fluorescence recovery was measured at 300 s after the addition of peptides to lipid vesicles.

View larger version (27K): [in this window] [in a new window]   FIG. 4. Determination of secondary structures of melittin (15.2 µM), MM-1 (14.9 µM), and MM-2 (14.5 µM) in PBS (A), in the presence of 482 µM PC/PG/Chol (B), and 362 µM PC/Chol (C). Solid line, melittin; dashed line, MM-1; dotted line, MM-2.

Mutation in the Leucine Zipper Motif Affected the Localization of Melittin in Zwitterionic Membranes but Not in Negatively Charged Membranes—To detect the location of melittin and its analogs onto the membrane, proteolytic cleavage experiments were performed with NBD-labeled peptides in their membrane-bound states. The basis for this experiment is that NBD-labeled peptides, bound onto the membrane surface, are easily cleaved by a proteolytic enzyme like proteinase K, which can be monitored by the decrease in NBD-fluorescence from its characteristic membrane-bound level. On the other hand, a membrane-inserted peptide will not be accessible to proteinase K and therefore its NBD-fluorescence will not decrease. The addition of PC/Chol vesicles to NBD-labeled melittin and its analogs (Fig. 5A) resulted in a sharp increase in fluorescence because of the binding to the lipid vesicles with fast kinetics indicating that mutations in the leucine zipper motif did not impair the membrane-binding ability of melittin. The addition of proteinase K (at time point 3) to melittin (Fig. 5A, a) in its membrane-bound state resulted in a slow decrease in NBD-fluorescence indicating that the N-terminal of NBD-melittin to some extent protects itself from the digestion by the proteolytic enzyme. The 50% decrease in NBD fluorescence, resulting from the cleavage of NBD-melittin in its membrane-bound state took place at 1900 s, suggesting that a part of the N-terminal of melittin was inside the lipid bilayer of the zwitterionic membrane and hence not cleaved easily by proteinase K. Interestingly, NBD-labeled MM-1 and MM-2 were cleaved much faster when bound to PC/Chol vesicles compared with that of NBD-melittin as evidenced by the sharp decrease in fluorescence after the addition of proteinase K (Fig. 5A, b and c). The corresponding 50% decrease in fluorescence from the membrane-bound level for NBD-MM-1 and -MM-2 took place at 120 and 40 s, respectively. The results suggest that probably the N termini of both of the analogs were more exposed on the surface of the zwitterionic membrane than melittin with MM-2 being the most exposed one. Taken together, the results indicate that although mutations in the leucine zipper motif did not inhibit the binding of melittin to zwitterionic membrane, it impaired its localization onto it. However, N termini of melittin and its analogs were appreciably protected in PC/PG/Chol vesicles from proteolytic cleavage as evidenced by the slow decrease in NBD fluorescence following the proteinase K treatment (Fig. 5B). Because very similar results were obtained with PE/PG vesicles, profiles were not presented.

View larger version (42K): [in this window] [in a new window]   FIG. 5. Determination of localization of melittin and its analogs. Proteolytic digestion of NBD-labeled melittin (a), MM-1 (b), and MM-2 (c) in their membrane-bound states. A and B depict the experimental profiles with PC/Chol and PC/PG/Chol lipid vesicles, respectively. 1, 2, and 3 indicate the addition of NBD-labeled individual peptides (0.20 µM), lipid vesicles (120 µM), and proteinase K (12 µg/ml, final concentration). C and D, recording of fluorescence emission spectra of NBD-labeled melittin and its analogs in the presence of PC/Chol (C) and PC/PG/Chol (D). Peptide and lipid concentrations were the same as the proteolytic cleavage experiments. Solid line, melittin; short dashed line, MM-1; short dash-dotted line, MM-2. Peaks of the spectra in C and D are marked by arrows.

Substitution of Heptadic Leucine in the Leucine Zipper Motif Impaired the Assembly of Melittin in Aqueous Environment—In an aqueous environment at high ionic strength with an increase in concentration, melittin assembles in a tetrameric helical structure as studied by several biophysical techniques (15, 18–20). We have utilized CD, fluorescence, and gel filtration techniques to look into the effect of alanine substitution in the assembly and secondary structure of melittin in aqueous environment. With an increase in concentration in PBS containing 1.5 M NaCl, melittin adopted more and more of a helical structure (Fig. 6A), consistent with its helical self-association as also reported by others (15, 18–20). In contrast, MM-1 (Fig. 6B) and MM-2 (Fig. 6C) assembled weakly, as evidenced by the shape of the spectra and small increase in CD (Fig. 6D, mean residue ellipticity values at 222 nm) compared with that of melittin. At any concentration the helical structures displayed by the designed analogs MM-1 and MM-2 were significantly less than that of melittin. For example, at 50 µM while melittin exhibited a -helix content of 75%, MM-1 and MM-2 displayed helix contents of 15 and 8%, respectively. Very similar results were obtained from concentration-dependent CD experiments with melittin and its analogs in PBS containing 0.6 M NaCl (data not shown). The results (Fig. 6, A–D) clearly suggest that the substitution of heptadic leucine by alanine impaired the helical structure and assembly of melittin in aqueous environment. The tryptophan fluorescence of each of the peptides was also monitored with an increase in concentration at similar experimental conditions. It was observed that for a particular concentration, melittin exhibited emission maximum at a much shorter wavelength (341 nm) compared with that of MM-1 (352 nm) and MM-2 (353 nm) (Fig. 6E). The results indicated that with an increase in concentration, tryptophan of melittin but not of its analogs moved to a more hydrophobic environment probably because of its self-association (18), consistent with CD studies.

View larger version (33K): [in this window] [in a new window]   FIG. 6. Detection of self-association of melittin and its analogs by concentration-dependent CD and fluorescence experiments. A, B, and C are the plots of CD (mdeg, millidegree) for different concentrations of melittin, MM-1, and MM-2, respectively in PBS (pH 7.4) containing 1.5 M NaCl. D, the corresponding plots of mean residue ellipticity values at 222 nm ([]) versus peptide concentration (µM) of melittin (a), MM-1 (b), and MM-2 (c). E, fluorescence spectra of melittin and its analogs as marked in the figure at 18 µM in PBS with 1.5 M NaCl. Excitation wavelength was set at 280 nm.

View larger version (26K): [in this window] [in a new window]   FIG. 7. Effect of alanine substitution in the leucine zipper motif of melittin on its oligomeric state in aqueous environment as detected by gel filtration experiments. The plot of molecular weight of myoglobin (a), cytochrome c (b), aprotinin (c), and insulin B chain (d) with respect to their elution volumes after passing through a Sephadex G-50 (Sigma) column (diameter 1.4 cm, length 38 cm) with PBS containing 1.5 M NaCl on a AKTA FPLC (Amersham Biosciences) system. The elution volumes of melittin, MM-1, and MM-2 are marked with + signs as 1, 2, and 3, respectively. The flow rate was 1.0 ml/min, and absorbance was recorded at 280 nm. The inset shows the gel filtration profiles of melittin, MM-1, and MM-2 as marked. 0.2 ml of 200 µM peptides were introduced into the gel filtration column.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To understand the molecular basis of reduction in hemolytic activity and retention of antibacterial activity of the melittin analogs and to evaluate the structural and functional changes associated with the mutation in this motif, membrane permeability, secondary structure, localization onto the membrane with different lipid composition, and self-association in an aqueous environment were studied with melittin, MM-1, and MM-2. Considering the lipid component and surface charge of the outer membrane of RBC and bacterial membrane, experiments were performed with zwitterionic PC/Chol and negatively charged PC/PG/Chol and PE/PG lipid vesicles. Often the results obtained from the structural and functional studies of membrane-active peptides with these kinds of membranes correlate with their hemolytic and antibacterial activities (10, 44).

Replacement of heptadic leucine by alanine resulted in an appreciable reduction in the membrane permeability of melittin in PC/Chol vesicles (Fig. 3A), clearly indicating an essential role of the identified leucine zipper motif in the pore-forming activity of melittin in the zwitterionic membrane. However, in contrast, the melittin analogs exhibited a very similar permeability to melittin in negatively charged PC/PG/Chol and PE/PG membrane. The results further suggest that the amino acid sequence requirements of a peptide to permeate zwitterionic and negatively charged lipid vesicles are not the same, consistent with a recent study showing that a peptide derived from E. coli toxin hemolysin E efficiently induced permeation in the negatively charged but not in zwitterionic membrane (32).

The kinetics of membrane permeation induced by the designed melittin analogs in zwitterionic and negatively charged membrane showed some interesting features. For example, the kinetics of the zwitterionic PC/Chol membrane permeation induced by the single alanine mutant MM-1 (Fig. 3A, inset, 1.52 µM) was only slightly slower than that of melittin, although its maximum level of dissipation of diffusion potential was significantly reduced. In contrast, in the negatively charged PC/PG/Chol membrane, the kinetics of membrane permeation induced by the designed melittin analogs (Fig. 3B, inset, 0.76 µM) were slower than that of melittin, although the maximum levels of dissipation of diffusion potential induced by MM-1 and MM-2 ultimately reached close to that of melittin in 200–300 s.

The molecular basis of these observations is not clear. One possible explanation is that melittin probably needs to adopt a specific assembly in the membrane to induce permeation in the zwitterionic membrane. Probably MM-1, after substitution of one heptadic leucine by alanine, only partly retained the desired assembly of melittin to induce permeation in zwitterionic membrane. It could be that MM-1 formed pores almost as fast as melittin in zwitterionic membrane, but either it could not form the same number of pores as melittin, or some of the pores formed by MM-1 got closed very fast. So the maximum level of induced membrane permeation was significantly lower than that of melittin, although the kinetics were very similar. However, the effect of substitution of two heptadic leucines was much more prominent in the maximum level of MM-2-induced dissipation of diffusion potential and also in its kinetics (Fig. 3A, inset).

On the other hand, it could be that the assembly of the designed melittin analogs in PC/PG/Chol lipid vesicles, at a relatively lower concentration, was to some extent different from that of melittin. Alternatively, there may be some variation in the mechanism of membrane permeation induced by the designed analogs and melittin in negatively charged lipid vesicles at this peptide concentration. Probably, for any of these possible reasons, the designed analogs induced membrane permeation in PC/PG/Chol lipid vesicles at a slower rate than melittin (Fig. 3B, inset). However, perhaps, because of the presence of the same number of positive charges and almost similar amphipathic character, they induced very similar levels of maximum dissipation of diffusion potential as that of melittin. It should be mentioned that at a slightly higher concentration, for example at 2.28 µM, the kinetics as well as the maximum levels of membrane permeation induced by the melittin analogs were very close to that of melittin (profiles not shown). This result further suggests that whatever difference exists between the designed melittin analogs and melittin, in terms of their assembly or even in the mechanism of permeation of negatively charged membrane, is only at relatively lower peptide concentration.

Although melittin and its analogs exhibited similar -helix contents in the negatively charged membrane, the secondary structures exhibited by the mutants were much less than that of melittin in the zwitterionic membrane (Fig. 4). The molecular basis of these observations is not clear; however, it seems that the substitution of heptadic leucine by alanine in the leucine zipper motif affects the secondary structure of melittin in zwitterionic membranes but not in negatively charged membranes.

Functional activity of a membrane-associated peptide may be influenced by its localization onto the membrane. Proteolytic cleavage experiments (Fig. 5, A and B) suggested that the substitution of heptadic leucine by alanine had a remarkable effect on the localization of melittin onto the zwitterionic membrane but not onto the negatively charged membrane. The N-terminal of melittin was located slightly inside the zwitterionic membrane, which is consistent with other studies (24, 45) whereas the mutants were exposed on its surface. Interestingly, the N termini of both melittin and its analogs were located inside the bilayer of negatively charged lipid vesicles. Similar localizations were observed for NBD-labeled dermaseptin-3 and -4, which also dissociated like melittin (46, 47) therein and have been postulated to manifest their antimicrobial activities by the carpet/detergent mechanism (48). The results of the proteolytic cleavage experiments were supported by the emission maxima of NBD-labeled peptides in the presence of zwitterionic and negatively charged lipid vesicles (Fig. 5, C and D). Although NBD-melittin in the presence of both zwitterionic and negatively charged lipid vesicles and NBD-labeled analogs in the presence of negatively charged lipid vesicles exhibited emission maxima, which were much shorter than that observed for the probe located onto the membrane surface, NBD-labeled MM-1 and MM-2 in zwitterionic membranes displayed maxima that were close to the characteristic value (43) for the probe, located on the surface of the membrane. Tryptophan fluorescence of unlabeled melittin and the designed mutants was also recorded in the presence of both kinds of lipid vesicles (data not shown). Melittin exhibited emission maxima at 336–337 and 334–335 nm in the presence of zwitterionic and negatively charged lipid vesicles, respectively, which supports previous observations (49) and indicates that the tryptophan residue was located in the shallow interface region of the membrane bilayer as already reported (50). Although MM-1 and MM-2 exhibited very similar emission maxima to melittin in a negatively charged membrane, the maxima (338–339 nm) exhibited by MM-1 and MM-2 in the presence of zwitterionic vesicles probably indicated that the location of their tryptophan residues was slightly more toward the hydrophilic region of the membrane than that of melittin.

CD, tryptophan fluorescence (Fig. 6), and gel filtration studies (Fig. 7) with melittin and its analogs clearly indicated that secondary structure and assembly of melittin in an aqueous environment were disrupted following the substitution of heptadic leucine by alanine. Although melittin adopted a tetrameric helical assembly at a relatively higher concentration and at a high ionic strength, both MM-1 and MM-2 at similar conditions exhibited only their dimeric masses (Fig. 7) with a much reduced helical structure. It is important to mention that, although under the same experimental conditions, both of the designed melittin analogs exhibited their dimeric masses, the single alanine mutant (MM-1) adopted an appreciably more helical structure than the double alanine mutant (MM-2) (Fig. 6, B–D). Furthermore, the results suggested that the identified leucine zipper motif is probably a key structural element that regulates the secondary structure and self-association of melittin in an aqueous environment. Also the tetrameric crystal structure of melittin is stabilized by the side-chain interaction of the hydrophobic amino acids (13) like leucine and isoleucine, which are present in the a and d positions of the identified leucine zipper motif. These observations are in agreement with studies that suggest a crucial role of this motif in the assembly of protein/peptides in aqueous and membrane environments (30, 51, 52).

A contrasting difference in the membrane permeability of melittin and its analogs in zwitterionic and negatively charged lipid vesicles probably reflects that the mechanisms of membrane permeation of melittin in these two kinds of membranes are different from each other, which is in agreement with the present notion that melittin forms a pore via a barrel-stave mechanism in a zwitterionic membrane (21–24) and acts as a detergent in a negatively charged membrane (24, 25). Probably the results suggest that the substitution of leucine by alanine disturbs only the mechanism of melittin-induced membrane permeation in zwitterionic but not in the negatively charged membrane.

Assembly or conformation of melittin-like membrane lytic peptides in aqueous environments may influence their activity in the membrane also, because most likely these peptides bind to the target membrane from the aqueous environment. The results presented here probably indicate that a strong hydrophobic force of melittin is needed to disrupt the zwitterionic lipid bilayer. Proteolytic cleavage experiments and emission spectra of NBD-labeled analogs, which were recorded with peptide concentration as low as 10^–7 M, revealed that although the N-terminal of melittin could penetrate the bilayer of zwitterionic membrane, its designed mutants were localized onto the same membrane surface (Fig. 5). By disturbing the leucine zipper motif, the helical structure and self-association and thus the hydrophobic force of melittin were abrogated, which may contribute to the reduced activity of melittin analogs in the zwitterionic membrane and also against RBCs.

That the identified leucine zipper motif may play a crucial role in regulating the toxic activity of melittin can explain several results in the literature, the molecular basis of which was not clearly understood. For example, a critical look at the sequence of melittin analog, designed by DeGrado et al. (5), reveals that although it lacks appreciable sequence homology to melittin, the identified leucine zipper motif with amino acids at a and d positions remained intact in it. Probably, for this, the analog (5) self-associated (53) with the helical structure and retained the hemolytic activity. The absence of proline may add to its better self-association property and helical structure and thus to the higher hemolytic activity than melittin. Probably, the deletion of amino acids at 6, 9, 13, 16, and 20 of melittin showed a more drastic effect (6) on the hemolytic activity because of impairing its helical assembly as these residues are located at crucial a and d positions of the leucine zipper motif. Similarly, the reason why the deletion of the N-terminal of melittin had drastic effect on its hemolytic activity compared with its antimicrobial activity can be explained (7, 8, 12).

In conclusion, the results demonstrated a mechanistic dissection of the hemolytic and antibacterial activities of melittin. The results probably indicate the presence of different sequence elements, which control the toxic and antibacterial activity of melittin. The ability of melittin to efficiently permeate both the negatively charged and zwitterionic membranes makes it a poor cell-selective molecule and renders it active against both prokaryotic (e.g. antibacterial activity) and eukaryotic cells (e.g. hemolytic activity). The results suggested that the substitution of heptadic leucine by alanine disturbed the helical assembly of melittin in an aqueous environment, the secondary structure, membrane permeability, and localization in zwitterionic membrane, which probably caused the reduction in hemolytic activity of melittin. However, alanine substitution did not alter much the amphipathic character of melittin, showed much less effect on its membrane permeability, secondary structure, and localization in negatively charged membrane and presumably therefore did not affect its antibacterial activity.

Antimicrobial peptides are potential lead molecules for designing novel drugs. However, one of the major issues is their selective lytic activity. The results presented here probably suggest that cell selectivity in some antimicrobial peptides can be introduced by modulating their secondary structures and/or assembly in an aqueous environment.

	FOOTNOTES

 
^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J. K. G. dedicates this article to his mentor Prof. Yechiel Shai of Dept. of Biological Chemistry, Weiznmann Institute of Science, Israel, with deep respect. This is Central Drug Research Institute Communication 6622.

Both authors contributed equally to this work.

Recipient of a Junior Research Fellowship from the Council of Scientific and Industrial Research, India.

^¶ Recipient of a Senior Research Fellowship from the Council of Scientific and Industrial Research, India.

^|| To whom correspondence should be addressed. Tel.: 091-522-2212412 (ext. 4282); Fax: 091-522-223405/223938; E-mail: jighosh{at}yahoo.com.

¹ The abbreviations used are: Fmoc, N-(9-fluorenyl)methoxycarbonyl; PC, phosphatidylcholine; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; Chol, cholesterol; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; NBD, 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole; RBC, red blood cell; hRBC, human RBC.

	ACKNOWLEDGMENTS

 
We thank the Director of the Central Drug Research Institute for his support. We thank the Head of the Sophisticated Analytical Instrument Facility, for assistance in recording the ES-MS spectra and thank the Head of the Biochemistry Division for allowing the use of the lyophilizer. We thank Dr. G. R. Bhatt and R. K. Purshottam for assistance in using the HPLC at the initial stage of the work. We thank to Drs. B. Kundu and S. Sinha for their kind assistance in many ways. Richa Verma and Aqeel Ahmad are thanked for their cooperation during the progress of the work.

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