dissection of antibacterial and toxic activity of melittin ... · neeta asthana‡, sharada prasad...

Dissection of antibacterial and toxic activity of melittin: A leucine zipper motif plays

crucial role in determining its hemolytic activity but not antibacterial activity#

Neeta Asthana‡, Sharada Prasad Yadav‡ and Jimut Kanti Ghosh*

Molecular and Structural Biology Division

Central Drug Research Institute

Lucknow 226 001

India ‡Authors contributed equally to this work

Running Title: Leucine zipper motif in a naturally occurring antimicrobial peptide

*To whom correspondence should be addressed

Telephone: 2212412 (Ext. 4282)

Fax: 091-522-223405/223938

E-mail: [email protected] #CDRI communication No. 6622

JBC Papers in Press. Published on October 8, 2004 as Manuscript M408881200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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Leucine zipper motif in a naturally occurring antimicrobial peptide 2

Summary:

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 a goal to understand 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 comparable antibacterial activity to melittin. Mutations caused

significant decrease in the membrane permeability of melittin in only 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 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 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 probably cell selectivity in

some antimicrobial peptides can be introduced by modulating their assembly in aqueous

environment.


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Introduction: Melittin, the major component of European bee venom, 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 melittin’s hemolytic and antimicrobial activities.

Replacement of the first twenty amino acids by another helix forming sequence did not

alter antimicrobial and hemolytic activity of melittin (5). However, the deletion of L (6),

K (7), V (8), L (9), L (13), L (16), I (17), W (19) and I (20) dramatically reduced

melittin’s hemolytic activity and to a lesser extent its 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 diastereomers 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 like its self-association and phospholipid

membrane-interaction have been studied extensively in order to understand its mode of

action (14-17). Although in water melittin exists as monomer with mostly random coil


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conformation, an increase in peptide concentration, or addition of salt results the

transformation of monomer to tetramer with 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 by carpet mechanism

(25,24).

Despite a lot of 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, ii) is the

same amino acid sequence responsible for both the hemolytic and antibacterial activities?

Towards 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. Since 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 synthesized. Hemolytic activity of single (MM-1) and

double alanine mutant (MM-2) against human red blood cells was significantly less than

that of melittin. Interestingly, MM-1 and MM-2 exhibited comparable antibacterial

activity to melittin. Both 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.


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Experimental Procedures:

Materials:

Rink amide MBHA resin (loading capacity, 0.63 mmol/g) and all the N-α Fmoc and side-

chain protected amino acids were purchased from Novabiochem, Switzerland. Coupling

reagents for peptide synthesis like 1-hydroxybenzotriazole (HOBT), N, N′-di-

isopropylcarbodiimide (DIC), 1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and

N, N′-diisopropylethylamine (DIPEA) were purchased from Sigma, USA.

Dichloromethane, N, N′ dimethylformamide (DMF) and piperidine were of standard

grades and procured from reputed local companies. Acetonitrile (HPLC grade) was

procured from Merck, India while trifluoroacetic acid (TFA) was purchased from Sigma.

Egg phosphatidylcholine (PC), egg phosphatidylglycerol (PG) and dimyristoyl

phosphatidylethanolamine (PE) were procured from Northern Lipids Inc., Canada while

cholesterol (Chol) was purchased from Sigma. 3,3′-Dipropylthiadicarbocyanine iodide

(diS-C3-5) and NBD-fluoride (4-fluoro-7-nitrobenz-2-oxa-1, 3-diazole) were purchased

from Molecular probes (Eugene, OR). Rests of the reagents were of analytical grade and

procured locally; buffers were prepared in milli Q water.

Peptide Synthesis, Fluorescent labeling and Purification: All the peptides were

synthesized manually on solid phase. Stepwise solid phase syntheses were carried out on

rink amide MBHA resin (0.15 mmole) utilizing the standard Fmoc chemistry, employing

DIC/HOBT or TBTU/HOBT/DIPEA coupling procedure (30-32) with checking of de-

protection of α-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


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(35,36,30,32) and after sufficient labeling; the labeled peptides were cleaved from the

resins and precipitated. All the peptides were purified by RP-HPLC on an analytical

Vydac C18 column using a linear gradient of 0-80% acetonitrile in 45 min with a flow

rate of 0.6 ml/min. Both acetonitrile and water contained 0.05% TFA. The purified

peptides were ~95% homogeneous as shown by HPLC. Experimental molecular mass of

the peptides, detected by ES-MS analysis, corresponded to the desired values within 0.5

Da.

Preparation of Small Unilamellar Vesicles (SUVs): SUVs were prepared by a standard

procedure (35,36,32) 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 re-suspended 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 sulphonic acid. The samples were scanned at room temperature (~300 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) as already reported (30,32). [θ] 222 − [θ]0

222 Fh =[θ]100

222 − [θ]0222


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Where [θ]222 was the experimentally observed mean residue ellipticity at 222 nm. The

values for [θ]100222 and [θ]0

222 that correspond to 100 and 0% helix contents were

considered to have mean residue ellipticity values of –32,000 and –2,000 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 (41,30, 32). 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 diS-C3-5. 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 (Ft), defined by:

Ft = [(It - I0)/(If - I0)] x 100 %

Where It = fluorescence after the addition of a peptide at time t, I0 = fluorescence after the

addition of valinomycin and If = the total fluorescence observed before the addition of

valinomycin.

Enzymatic Cleavage Experiments: In order 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,


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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 (in

sec) with excitation wavelength set at 467 nm.

Hemolytic activity assay: 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 0C for 35

min. The samples were then centrifuged for 10 min at 2000 r.p.m. and the release of

hemoglobin was monitored by measuring the absorbance (Asample) of the supernatant at

540 nm. For negative and positive controls hRBCs in PBS (Ablank) and in 0.2% (final

concentration v/v) Triton X-100 (Atriton) were used respectively. The percentage of

hemolysis was calculated according to the following equation.

Percentage of hemolysis = [(Asample-Ablank)/(Atriton-Ablank)] 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 CFU) in

mid-logarithmic 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 0C.

The antibacterial activity of the peptides was expressed as the minimal inhibitory

concentration (MIC), the concentration at which 100% inhibition of microbial growth

was observed after 18-20 h of incubation. The microorganisms used were Gram-positive


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bacteria, Bacillus subtilis and Staphylococcus aureus and Gram-negative bacterium,

Escherichia coli.

Results:

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, panel A and B). In order 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. 1, panel A).

Alanine was chosen for its helix propensity and similar hydrophobicity to leucine so that

the amphipathic character of melittin is retained in MM-1 and MM-2.

Mutation in the leucine zipper motif drastically reduced the hemolytic activity of

melittin: In order to look into the role of the identified leucine zipper motif in the

functional activities of melittin, hemolytic activity of melittin and its designed analogs

were assayed. The substitution of leucine with alanine resulted in a drastic reduction of

hemolytic activity of melittin against human red blood cells (hRBCs) (Fig. 2). MM-1

exhibited 10-20 % hemolytic activity of melittin while MM-2 showed around 1-2 %

activity. The results indicate that substitution of heptadic amino acids by alanine

markedly inhibited the hemolytic activity of melittin probably suggesting a role of this

motif in maintaining the lytic activity of the peptides towards the RBCs.

The designed analogs exhibited comparable anti-bacterial activity to melittin:

Melittin and its analogs were tested for growth inhibiting activity in liquid cultures of two

Gram-positive and one Gram-negative bacteria. Tetracycline was used as a positive


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control. In contrast to the hemolytic activity, the designed analogs of melittin (MM-1 and

MM-2) exhibited very similar antibacterial activities to that of melittin (Table-1). Thus

the results clearly indicate that substitution of leucine by alanine in the heptadic positions

did not affect the antibacterial activity of melittin unlike its hemolytic activity.

Contrasting difference in membrane permeability of the designed analogs of

melittin in zwitterionic and negatively charged membrane: The ability of melittin to

destabilize the phospholipid bilayer is believed to be associated with its mechanism of

action. Therefore, to understand the basis of decreased hemolytic activity and almost

unaltered antibacterial activity of the designed melittin analogs, membrane permeability

of melittin, MM-1 and MM-2 were examined by determining their efficacy to dissipate

the diffusion potential across the phospholipid vesicles with different lipid composition.

As shown in Fig. 3, panel A, membrane permeability (expressed as the percentage of

fluorescence recovery), of MM-1 was significantly lower than melittin in zwitterionic

PC/Chol vesicles, which decreased further with MM-2. However, interestingly, both

MM-1 and MM-2 exhibited very similar membrane permeability to that of melittin in

negatively charged PC/PG/Chol lipid vesicles (Fig. 3, panel B). Experimental profiles

for each of the peptides in both kinds of membranes are shown in the insets of panel A

and B of Fig. 3. Moreover, the designed analogs induced membrane permeation as

efficiently as melittin (Figure not shown) in another kind of negatively charged

membrane namely PE/PG (7:3 w/w). The results indicate that although the substitution

of leucine by alanine appreciably impaired the membrane permeability of melittin in

zwitterionic membrane, it had almost negligible effect in negatively charged membrane.

The designed analogs exhibited similar secondary structures to melittin in

negatively charged membrane but not in zwitterionic membrane: Circular dichroism

experiments were performed in order to determine the secondary structures of melittin


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and its analogs in aqueous environment (phosphate buffered saline, PBS, pH 7.4) and in

the presence of zwitterionic and negatively charged phospholipid vesicles. The

corresponding mean residue ellipticity values of the individual peptides at 222 nm were

used to determine their helical contents at a particular environment. Although all three

peptides exhibited low helical structure in PBS (Fig. 4, panel A), the extent of helicity

was slightly more in melittin (~15%) than MM-1 (~8.5%) and MM-2 (~6.5%). However,

in the presence of increasing amount of negatively charged PC/PG/Chol lipid vesicles

significant amount of helicity was induced in all three peptides as shown by a

representative CD spectrum for each of these peptides with a fixed lipid/ peptide (L/P)

molar ratio (~32) (Fig. 4, panel B). Melittin (69%), MM-1 (67.5%) and MM-2 (68.5%)

exhibited very similar extent of helical structures indicating that the substitution of

leucine by alanine did not affect the secondary structure of melittin in the presence of

negatively charged lipid vesicles.

However, very contrasting results were obtained in the presence of zwitterionic

PC/Chol lipid vesicles (Fig. 4, panel C). Although melittin adopted a significant amount

of helical structure (47% at L/P ~24) in the presence of PC/Chol lipid vesicles, the extent

of induction of helicity was much less in MM-1 (~15%) and MM-2 (8.5%) with the same

amount of lipid vesicles.

Mutation in the leucine zipper motif affected the localization of melittin in

zwitterionic membrane but not in negatively charged membrane: In order 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 of 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


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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. 5, panel A) resulted in a

sharp increase in fluorescence due to their binding to the lipid vesicles with a 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 (profile a,

panel A, Fig. 5) 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

around 1900 sec, probably suggesting that a part of the N-terminal of melittin was inside

the lipid bilayer of 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 to that of NBD-melittin as evidenced by the sharp decrease in

fluorescence after the addition of proteinase-k (profiles b and c, panel A Fig. 5). The

corresponding 50% decrease in fluorescence from the membrane-bound level for NBD-

MM-1 and -MM-2 took place at ~ 120 and 40 sec respectively. The results suggest that

probably the N-termini of both the analogs were more exposed on the surface of

zwitterionic membrane than melittin with MM-2 being the most exposed one. Taken

together, the results indicate that mutations in the leucine zipper motif although 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 (panel B, Fig. 5). Since very

similar results were obtained with PE/PG vesicles, profiles not presented.


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The localization of N-terminal of melittin and its analogs onto the membrane were

also detected by recording the emission maxima of their NBD-labeled versions in the

presence of lipid vesicles (panel C and D, Fig. 5). In the presence of zwitterionic

PC/Chol vesicles melittin exhibited emission maximum at 526-527 nm, which was much

shorter than 533 nm, observed for the NBD probe, located on the surface of the

membrane (43). Interestingly, NBD-labeled MM-1 and MM-2 with zwitterionic

membrane displayed fluorescence maxima at 529-530 nm (panel C, Fig. 5), which was

close to the characteristic value for the probe, located onto the membrane surface.

However, in the presence of negatively charged PC/PG/Chol lipid vesicles, NBD-labeled

melittin and both of its analogs exhibited emission maxima in the range of 525-526.5 nm

(panel D, Fig 5), indicating the location of their N-terminal slightly inside the bilayer of

this kind of membrane. Altogether, supporting the enzymatic cleavage experiments,

fluorescence emission maxima of NBD-labeled melittin and its analogs suggested that the

localization of N-terminal of melittin was affected by the mutation of leucine zipper

motif only in zwitterionic but not in negatively charged membrane.

Substitution of heptadic leucine in the leucine zipper motif impaired the assembly of

melittin in aqueous environment: In aqueous environment at high ionic strength with

increase in concentration, melittin assembles in a tetrameric helical structure as studied

by several biophysical techniques (18-20,15). 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 increase in concentration

in PBS containing 1.5 M NaCl, melittin adopted more and more helical structure (panel

A, Fig. 6), consistent with its helical self-association as also reported by others (18-

20,15). Contrastingly, MM-1 (panel B, Fig. 6) and MM-2 (panel C, Fig. 6) assembled

weakly as evidenced by the shape of the spectra and small increase in CD (in mean


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residue ellipticity values at 222 nm, panel D, Fig. 6) compared to 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 α-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 (Figure not

shown). The results (panels A-D, Fig. 6) clearly suggest that substitution of heptadic

leucine by alanine impaired the helical structure and assembly of melittin in aqueous

environment. Tryptophan fluorescence of each of the peptides was also monitored with

increase in concentration at similar experimental conditions. It was observed that for a

particular concentration, melittin exhibited emission maximum at much shorter

wavelength (~341 nm) compared to that of MM-1 (~352 nm) and MM-2 (~353 nm)

(panel E, Fig. 6). The results indicate that with increase in concentration, tryptophan of

only melittin but not of its analogs moved to a more hydrophobic environment probably

due to its self-association (18), consistent with CD studies.

Mutation in the leucine zipper motif disrupted the oligomerization of melittin in

aqueous environment: In order to get a quantitative picture about the assembly of

melittin at higher concentration and ionic strength after the substitution of heptadic

leucine by alanine, gel filtration of melittin and its analogs were performed using PBS

containing 1.5 M NaCl as the eluent. Fig. 7 shows the plot of elution volumes of known

molecular weight markers and melittin and its analogs after passing through a Sephadex

G-50 column in PBS with 1.5 M NaCl. Melittin eluted (shown in the inset) very close to

cytochrome c (mol. wt., 12,588), indicating a molecular mass, which fairly matched with

its tetrameric molecular weight (11,384), supporting the previous observation (18). There


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was a minor peak, which corresponded to its monomer peak. However, under the same

experimental conditions, both MM-1 and MM-2 eluted at higher volumes (inset Fig. 7),

slightly after aprotinin (mol. wt., 6,500), indicating the presence of their dimeric masses.

Also both MM-1 and MM-2 exhibited prominent peaks of their monomeric masses.

Taking the gel filtration, and the previous CD and fluorescence experiments together, the

results suggest that the tetrameric helical assembly of melittin at higher concentration and

high ionic strength was disturbed as a result of substitution of heptadic leucine by

alanine.

Discussion:

The results presented here indicate the identification of a leucine zipper motif,

which plays a crucial role in the hemolytic activity of melittin. To our knowledge, this is

the first report on the identification and characterization of a leucine zipper motif in this

class of naturally occurring peptides. As a measure of toxic activity, hemolytic activity

of melittin and its analogs against human red blood cells (hRBCs) were assayed. The

data clearly demonstrated that the designed analogs were much less active than the wild

type melittin in lysing the hRBCs (Fig. 2). The synergistic effect of replacement of

heptadic leucine by alanine was evident by the fact that the double alanine mutant (MM-

2) was significantly more inactive than the single alanine mutant (MM-1). However, in

contrast to the hemolytic activity, interestingly, both MM-1 and MM-2 exhibited

comparable antibacterial activity to melittin against the tested Gram positive and negative

bacteria (Table-1).

In order 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,


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secondary structure, localization onto the membrane with different lipid composition and

self-association in aqueous environment were studied with melittin, MM-1 and MM-2.

Considering the lipid component and surface charge of 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 membrane correlate with their hemolytic and antibacterial activities (10,44).

Replacement of heptadic leucine by alanine resulted in an appreciable reduction

in membrane permeability of melittin in PC/Chol vesicles (Fig. 3, panel A), indicating

clearly an essential role of the identified leucine zipper motif in the pore-forming activity

of melittin in zwitterionic membrane. However, contrastingly the melittin analogs

exhibited very similar permeability as 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 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 (1.52 µM, Inset of panel A, Fig. 3), was only slightly slower

than that of melittin although its maximum level of dissipation of diffusion potential was

significantly reduced. Contrastingly, in negatively charged PC/PG/Chol membrane, the

kinetics of membrane permeation induced by the designed melittin analogs (0.76 µM,


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Inset of panel B, Fig. 3) 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 sec.

The molecular basis of these observations is not clear. One possible explanation

is that probably melittin needs to adopt a specific assembly in membrane in order 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 was 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 (Inset of panel A,

Fig. 3).

On the other hand, it could be that the assembly of the designed melittin analogs

in PC/PG/Chol lipid vesicles at 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 (Inset of panel B, Fig. 3). However, perhaps,

due to 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 is to be mentioned that at slightly higher


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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 exist between

the designed melittin analogs and melittin in terms of their assembly or even in the

mechanism of permeation of negatively charged membrane that is only at relatively lower

peptide concentration.

Although melittin and its analogs exhibited similar α-helix contents in negatively

charged membrane, the secondary structures exhibited by the mutants were much less

than that of melittin in zwitterionic membrane (Fig. 4). The molecular basis of these

observations is not clear, however it seems that substitution of heptadic leucine by

alanine in the leucine zipper motif affects the secondary structure of melittin in

zwitterionic membrane but not in negatively charged membrane.

Functional activity of a membrane-associated peptide may be influenced by its

localization onto the membrane. Proteolytic cleavage experiments (Fig. 5, panel A and

B) suggest that the substitution of heptadic leucine by alanine had 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 (45,24) 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

dissociate like melittin (46,47) therein and have been postulated to manifest their

antimicrobial activities by carpet/detergent mechanism (48). The results of the

proteolytic cleavage experiments were supported by the emission maxima of NBD-


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labeled peptides in the presence of zwitterionic and negatively charged lipid vesicles

(Fig. 5, panel C and D). While 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 membrane displayed maxima, which 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 (Figure 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 support previous observations

(49) and indicate that the tryptophan residue was located in the shallow interface region

of membrane bilayer as already reported (50). While MM-1 and MM-2 exhibited very

similar emission maxima to melittin in negatively charged membrane, the maxima (338-

339 nm), exhibited by them in the presence of zwitterionic vesicles probably indicate the

location of their tryptophan residues slightly more towards 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 aqueous environment was disrupted following the substitution of heptadic

leucine by alanine. While melittin adopted a tetrameric helical assembly at relatively

higher concentration and at high ionic strength, both MM-1 and MM-2 at similar

conditions exhibited only their dimeric masses (Fig. 7) with much reduced helical

structure. It is important to mention that although under the same experimental

conditions, both the designed melittin analogs exhibited their dimeric masses, the single


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alanine mutant (MM-1) adopted appreciably more helical structure than the double

alanine mutant (MM-2) (Fig. 6, panel B-D). Furthermore, the results suggest that the

identified leucine zipper motif is probably a key structural element, which regulates the

secondary structure and self-association of melittin in 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 for a crucial role of this motif in the assembly of

protein/peptides in aqueous and membrane environments (51,52,30).

A contrasting difference in membrane permeability of melittin and its analogs in

zwitterionic and negatively charged lipid vesicles probably reflect 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 pore via a

barrel stave mechanism in zwitterionic membrane (21-24) and acts as a detergent in

negatively charged membrane (25,24). Probably the results suggest that substitution of

leucine by alanine disturbs only the mechanism of melittin-induced membrane

permeation in zwitterionic but not in negatively charged membrane.

Assembly or conformation of melittin-like membrane lytic peptides in aqueous

environments may influence their activity in membrane also since 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 while N-terminal of melittin could penetrate the bilayer of

zwitterionic membrane, its designed mutants were localized onto the same membrane


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surface (Fig. 5). By disturbing the leucine zipper motif, the helical structure and self-

association and thus the hydrophobic force of melittin was abrogated, which may

contribute to the reduced activity of melittin analogs in 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 were 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 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 more drastic effect (6) on the hemolytic activity due to impairing

its helical assembly as these residues are located at crucial ‘a’ and ‘d’ positions of the

leucine zipper motif. Similarly, it can be explained why the deletion of N-terminal of

melittin had drastic effect on its hemolytic activity compared to its antimicrobial activity

(7,8,12).

In conclusion, the results demonstrate a mechanistic dissection of melittin’s

hemolytic and antibacterial activities. 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 membrane 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 suggest that substitution of heptadic leucine by alanine disturbed


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the helical assembly of melittin in aqueous environment, 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 aqueous

environment.

Acknowledgements: The authors express their sincere thanks to the Director, CDRI for

his support. The authors are thankful to the Head, RSIC, for assistance in recording the

ES-MS spectra and thank the Head, Biochemistry Division for allowing them to use the

lyophilizer. The authors acknowledge the assistance of Dr. G. R. Bhatt and Mr. R. K.

Purshottam in using the HPLC at the initial stage of the work. The authors are very much

thankful to Drs. B. Kundu and S. Sinha for their kind assistance in many ways. Richa

Verma and Aqeel Ahmad are acknowledged for their cooperation during the progress of

the work. N.A. and S.P.Y. acknowledge the receipt of a JRF and SRF respectively from

CSIR, India.


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Figure Legends: Fig. 1: Panel 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. Panel 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.

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.

Fig. 3: Membrane permeability induced by melittin and its analogs as plotted by % of

fluorescence recovery induced by the individual peptides. Panel A, plots of fluorescence

recovery induced by different concentrations of individual peptides in zwitterionic

PC/Chol lipid vesicles (68 µM, final concentration). Symbols- closed squares, melittin;

closed circles, MM-1 and closed triangles, MM-2. Inset depicts the representative

experimental profiles for melittin and its analogs at 1.52 µM as marked. Panel B, Plots of

fluorescence recovery induced by melittin, MM-1 and MM-2 at different concentrations

(Symbols are the same as panel A) in negatively charged PC/PG/Chol lipid vesicles (68

µΜ, final concentration). Inset depicts the representative experimental profiles for

melittin and its analogs as marked at 0.76 µΜ. Fluorescence recovery was measured at ~

300 sec after addition of peptides to lipid vesicles.

Fig. 4: Determination of secondary structures of melittin (15.2 µM), MM-1 (14.9 µM)

and MM-2 (14.5 µM) in PBS (panel A), in the presence of 482 µM PC/PG/Chol (panel

B) and 362 µM PC/Chol (panel C). Symbols-solid line, melittin; dashed line, MM-1 and

dotted line, MM-2.


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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.

Panels 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 µΜ), lipid vesicles (120 µΜ) and proteinase-k (12 µg/ml, final concentration).

Recording of fluorescence emission spectra of NBD-labeled melittin and its analogs in

the presence of PC/Chol (panel C) and PC/PG/Chol (panel D). Peptide and lipid

concentrations were the same as the proteolytic cleavage experiments. Symbols-solid

line, melittin; short dashed line, MM-1 and short dash dotted line, MM-2. Peaks of of the

spectra in panels C and D are marked by arrows.

Fig. 6: Detection of self-association of melittin and its analogs by concentration-

dependent CD and fluorescence experiments. Panels 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. Panel D, the corresponding plots of

mean residue ellipticity values at 222 nm ([θ]) vs. peptide concentration (µΜ) of melittin

(a), MM-1 (b) and MM-2 (c). Panel E, fluorescence spectra of melittin and its analogs as

marked in the figure at ~18 µΜ in PBS with 1.5 M NaCl. Excitation wavelength was set

at 280 nm.

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 (diam. 1.4 cm, length 38 cm) with PBS containing 1.5 M NaCl on a

AKTA FPLC (Amersham Pharmacia Biotech) system. The elution volumes of melittin,


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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.


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Table 1. Antibacterial activity of melittin and its analogs against gram-positive and gram-negative bacteria

4.0 ± 0.20.2 ± 0.11.2 ± 0.2Tetracycline

3.6 ± 0.61.8 ± 0.24.2 ± 0.7MM-2

4.3 ± 0.402.4 ± 0.34.5 ± 0.7MM-1

3.6 ± 0.52.0 ± 0.23.9 ± 0.6Melittin

S. aureusB. subtilisE. coli DH5αPeptide/anti-biotic name

Minimal inhibitory concentration (MIC) in µM


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Figure: 1

Sequences of Melittin and its MutantsMelittin: X-NH-GIGAVLKVLTTGLPALISWIKRKRQQ-CONH2

MM-1: X-NH-GIGAVLKVLTTGAPALISWIKRKRQQ-CONH2

MM-2: X-NH-GIGAVAKVLTTGAPALISWIKRKRQQ-CONH2

X= H or NBD

'd' 'e'

'f''c'

'b''g'

'a'

ILLL L I

W G V

GVA

A T S

K P K

G T I

'd' 'e'

'f''c'

'b''g'

'a'

L L I

W G V

GVA

A T S

K P K

G T I

IAA

Melittin MM-2

A

B by guest on July 13, 2018http://w

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0 1 2 3 4 5 6 70

20

40

60

80

100

ccc

b

bbb

a

aa

a

c cb

a

Peptide (in µM)

% o

f Hem

olyt

ic A

ctiv

ity

Figure: 2


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0.0 0.5 1.0 1.5 2.0 2.50

20

40

60

80

100

0 100 200 300 4000

40

80

120

160

Time (sec)

MM-2MM-1

Melittin

Flur

esce

nce (

a.u.)

Peptide (in µM ) % o

f Flu

ores

cenc

e R

ecov

ery

A

B

0.0 0.5 1.0 1.5 2.0 2.50

20

40

60

80

100

0 100 200 300 400 5000

102030405060

Time (sec)

Flur

esce

nce (

a.u.)

MM-2MM-1

Melittin

%

of F

luor

esce

nce

Rec

over

y

Peptide ( in µM )

Figure: 3


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200 210 220 230 240 250-25

-20

-15

-10

-5

0

5

10

Wavelength (nm)[θ] x

10-3

( Deg

ree

x cm

2 x dm

ole-1

)

200 210 220 230 240 250-20

-15

-10

-5

0

5

10

[θ

] x 1

0-3( D

egre

e x

cm2 x

dmol

e-1)

Wavelength (nm)

200 210 220 230 240 250-30-25-20-15-10-50510

Wavelength (nm)[θ] x

10-3

( Deg

ree

x cm

2 x dm

ole-1

)

A B

C

Figure: 4


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500 520 540 560 580 6000

10

20

30

40

50

Wavelength (nm)

Fluo

resc

ence

(a.u

.)

C

500 520 540 560 580 6000

10

20

30

40

50

Wavelength (nm)

Fluo

resc

ence

(a.u

.)

D

0 500 1000 1500 2000 25000

20

40

60 3

21

c

b

a

Fluo

resc

ence

(a.u

.)

Time (sec)

A

0 500 1000 1500 2000 25000

20

40

60

80

cb

a

12

3

Time (sec)

Fluo

resc

ence

(a.u

.) B

Figure: 5


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300 330 360 390 420 4500

160

320

480

640

800 MM-2

MM-1

Melittin

Fl

uore

scen

ce (a

.u.)

Wavelength (nm)

0 20 40 60 80 100 120 140048

1216202428

[θ] x

10-3

( Deg

ree

x cm

2 x dm

ole-1

)Peptide concentration (in µM )

cb

a

200 210 220 230 240 250

-120

-90

-60

-30

0

30

Incr

easin

g pe

ptid

e co

nc.

C

D (m

deg)

Wavelength (nm)

200 210 220 230 240 250

-200

-160

-120

-80

-40

0

40

C

D (m

deg)

Incr

easin

g pe

ptid

e co

nc.

A

200 210 220 230 240 250-150

-120

-90

-60

-30

0

30

Incr

easin

g pe

ptid

e co

nc.

CD

(mde

g)

B

C

E

D

Figure: 6


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24 28 32 36 40 44 48 523.0x103

6.0x103

9.0x103

1.2x104

1.5x104

1.8x104

32

1

++

+

d

c

b

a

20 30 40 50 60

Melittin

MM-1

MM-2

Abs

orba

nce

Elution volume (ml)

Mol

ecul

ar w

eigh

t

Elution volume (ml)

Figure: 7


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Neeta Asthana, Sharada Prasad Yadav and Jimut Kanti Ghoshcrucial role in determining its hemolytic activity but not antibacterial activity

Dissection of antibacterial and toxic activity of melittin: A leucine zipper motif plays

published online October 8, 2004J. Biol. Chem.

10.1074/jbc.M408881200Access the most updated version of this article at doi:

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http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M408881200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2004/10/08/jbc.M408881200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2004/10/08/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2004/10/08/jbc.M408881200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

dissection of antibacterial and toxic activity of melittin ... · neeta asthana‡, sharada prasad...

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