Báo cáo khoa học: Effects of a tryptophanyl substitution on the structure and antimicrobial activity of C-terminally truncated gaegurin 4 doc

Effects of a tryptophanyl substitution on the structure andantimicrobial activity of C-terminally truncated gaegurin 4 Hyung-Sik Won1, Sang-Ho Park1, Hyung Eun Kim1, Byongkuk Hyun2, Miji

Effects of a tryptophanyl substitution on the structure and

antimicrobial activity of C-terminally truncated gaegurin 4

Hyung-Sik Won1, Sang-Ho Park1, Hyung Eun Kim1, Byongkuk Hyun2, Mijin Kim2, Byeong Jae Lee2and Bong-Jin Lee1

1

College of Pharmacy, Seoul National University, Seoul, South Korea;2Institute of Molecular Biology and Genetics,

Seoul National University, Seoul, South Korea

Gaegurin 4 (GGN4), a 37-residue antimicrobial peptide,

consists of two amphipathic a helices (residues 2–10and

16–32) connected by a flexible loop region (residues 11–

15) As part of an effort to develop new peptide antibiotics

with low molecular mass, the activities of C-terminally

truncated GGN4 analogues were tested D24)37GGN4, a

peptide analogue with 14 residues truncated from the

C-terminus of GGN4, showed a complete loss of

anti-microbial activity However, the single substitution of

aspartic acid 16 by tryptophan (D16W) in the D24)37

GGN4 completely restored the antimicrobial activity,

without any significant hemolytic activity In contrast,

neither the D16F nor K15W substitution of the D24)37

GGN4 allowed such a dramatic recovery of activity In

addition, the D16W substitution of the native GGN4 significantly enhanced the hemolytic activity as well as the antimicrobial activity The structural effect of the D16W substitution in the D24)37GGN4 was investigated by CD, NMR, and fluorescence spectroscopy The results showed that the single tryptophanyl substitution at position 16 of the D24)37GGN4 induced an a helical conformation in the previously flexible loop region in intact GGN4, thereby forming an entirely amphipathic a helix In addition, the substituted tryptophan itself plays an important role in the membrane-interaction of the peptide

Keywords: antimicrobial peptide; GGN4 analogues; try-ptophanyl substitution; CD; NMR

Membrane-active peptides exhibit many interesting

biolo-gical and pharmacolobiolo-gical activities, and they can also serve

as model systems for large membrane proteins [1]

Partic-ularly, many organisms, including fungi, insects,

amphibi-ans, and humamphibi-ans, produce hydrophobic and amphipathic

peptides that exhibit antibiotic, fungicidal, hemolytic,

viru-cidal, and tumoricidal activities Now, it is becoming clear

through many studies that the antimicrobial peptides are an

important component of the innate defenses of all species of

life [2–8] Presently, more than 100 molecules with this

property have been isolated from various vertebrates as well

as invertebrates These antimicrobial peptides can be

grouped into three classes, depending on their structural

properties [9]: a helicoidal peptides, peptides with one to

several disulfide bridges, and peptides rich in certain amino

acids such as Proline or Tryptophan Most of these peptides

share some common characteristics, such as their low

molecular mass (2–5 kDa), the presence of multiple lysine

and arginine residues, and their amphipathic nature

Although the exact mechanism by which they kill bacteria is

not clearly understood, it has been shown that peptide–lipid

interactions leading to membrane permeation play a role in their activity

The best understood group includes the linear amphi-pathic a helical antimicrobial peptides [1,10–13] Although most of these peptides dissolve well in aqueous solutions, they also show a strong affinity for phospholipid mem-branes Generally, they adopt a highly ordered helical structure in hydrophobic or membrane-mimetic environ-ments, whereas they assume a random coil conformation in aqueous solutions It has been demonstrated that the structural and physico-chemical properties, such as the amino-acid composition, helical length, and amphipathic nature, etc of the peptides, rather than the primary sequence similarity or specific receptor–ligand interactions, are responsible for their biological activity [1] Two plausible models for the membrane permeation mechanism by amphipathic a helical peptides have been proposed [10]: the
barrel-stave mechanism¢ and the
carpet-like mechan-ism In the former, the transmembrane amphipathic a heli-ces form bundles, producing a transmembrane pore The latter describes membrane disintegration by disruption of the bilayer curvature, leading to micellization In this model,

in contrast to the barrel-stave mechanism, the peptides do not penetrate into the hydrophobic core of the membrane, but rather bind to the phospholipid headgroups

A number of peptides with a broad-spectrum of antimi-crobial activities have been isolated from the skin of various amphibians, and six antimicrobial peptides, named gaegu-rins (GGNs), were also isolated from the skin of a Korean frog, Rana rugosa [14] Some of them, particularly those with no or little hemolytic activity, are considered as target molecules for the development of new antibiotic or

Correspondence to B.-J Lee, College of Pharmacy, Seoul National

University, San 56-1, Shillim-Dong, Kwanak-Gu, Seoul 151-742,

South Korea Fax: + 82 2872 3632, Tel.: + 82 28807869,

E-mail: lbj@nmr.snu.ac.kr

Abbreviations: DPC, dodecylphosphocholine; GGN4, gaegurin 4;

MIC, minimal inhibitory concentration; NATA, N-acetyl- L

-tryp-tophanamide; TFE, 2,2,2-trifluoroethanol; [q] M , mean residue molar

ellipticity.

(Received 3 June 2002, accepted 25 July 2002)

anticancer agents by peptide engineering Out of the six

gaegurins, GGN4 has the longest length and is the most

abundant in the frog skin Thus, the peptide is believed to be

crucial in the innate defense system of the frog Our previous

work [15] showed that GGN4 adopts a random structure in

an aqueous solution, but adopts a helical conformation

consisting of two amphipathic a helices (residues 2–10and

16–32) in membrane-mimetic environments Recently, as

part of an effort to develop new potential peptide antibiotics

with lower molecular mass, the antimicrobial activities of

several GGN4 analogues with C-terminal truncations were

analyzed [16] The deletion of up to 14 residues from the

C-terminus of GGN4 almost completely abolished the

antimicrobial activity of the peptide, but the concomitant

single substitution of aspartic acid 16 with tryptophan

showed a nearly complete restoration of activity

In the present work, we further examined the biological

activities of several GGN4 analogue peptides The

struc-tural effect and the functional role of the tryptophanyl

substitution at position 16 was investigated for the

C-terminally truncated GGN4, by CD, fluorescence, and

nuclear magnetic resonance (NMR) spectroscopy We

expect that the present results will not only improve our

understanding of the action mechanism of antimicrobial

peptides, but also present new perspectives for the

develop-ment of new peptide antibiotics

E X P E R I M E N T A L P R O C E D U R E S

Materials, peptide preparation, and activity test

N-Acetyl-L-tryptophanamide (NATA),

2,2,2-trifluoroetha-nol-d3 99.5% (TFE-d3), and sodium dodecyl-d25 sulfate

(SDS-d25) were obtained from Aldrich D2O (99.95%) was

obtained from Sigma, and all other chemicals were either

analytical or biotechnological grade GGN4 analogue

peptides were purchased from ANYGEN (Kwang-ju,

Korea; URL, http://www.anygen.com) The sequence and

purity of the peptides were confirmed by mass spectrometry

and high performance liquid chromatography

Antimicro-bial activities of the peptides were determined by measuring

the minimal inhibitory concentrations (MIC) for diverse

microorganisms, as described previously [14,15] Hemolytic

activities of the peptides were estimated as the percent

hemolysis relative to that by 0.1% Triton X-100, as

described by Park et al [14]

CD and fluorescence spectroscopy

For CD spectroscopy, the peptide powder was dissolved to

a final concentration of 50 lM, in various solvents: 20mM

sodium acetate buffer (pH 4.0), TFE/water mixtures, 5 mM

DPC micelles, and 10mM SDS micelles Before the CD

measurement, the pH was adjusted to 4.0by the addition of

0.1MHCl or NaOH CD spectra were obtained at 20C on

a JASCO J-720spectropolarimeter, using a 0.2-cm

path-length cell, with a 1-nm bandwidth and a 4-s response time

CD scans were taken from 250nm to 190nm, with a scan

speed of 50nmÆmin)1and a 0.5-nm step resolution Three

scans were added and averaged, followed by subtraction of

the CD signal of the solvent Finally, the CD intensity was

normalized by the equation as the mean residue molar

ellipticity:

½hkM¼h

k105 lcn where½hkM(deg cm2Ædmol)1) and hk (mdeg) are the mean residue molar elipticity and the observed CD intensity at any wavelength (k), respectively l, c, and n represent the path-length (cm), the concentration (lM), and the number

of residues, respectively

Fluorescence emission was monitored on a Hitachi F-4500 fluorimeter, between 300 and 450 nm at 0.2 nm increments, with an excitation wavelength of 280nm, using

a 10-mm quartz cell at room temperature Scans were taken with a 5-nm excitation and emission bandwidth, a 0.5-s response time, and a scan speed of 40nmÆs)1 All samples contained 8 lM peptide or the same concentration of NATA for control experiments, in water or a 10-mMSDS solution at pH 4.0 All spectra were baseline corrected by subtracting the corresponding solvent spectrum

NMR Spectroscopy and structure calculation Samples for NMR measurements contained 5 mMpeptide

in TFE-d3/H2O (1 : 1, v/v) at pH 4.0, and in 500 mM

SDS-d25 at pH 4.0 NMR spectra were recorded on a Bruker DRX-500 spectrometer, at 298 K in 50% TFE/water and at

313 K in SDS micelles Solvent suppression was achieved using selective low-power irradiation of the water resonance The 2D TOCSY spectra were acquired with an isotropic mixing time of 60ms The 2D NOESY spectra were acquired with mixing times of 150and 200ms, respectively Slowly exchanging amide protons were monitored by the

D2O exchange experiments with a series of 2D NOESY spectra measured immediately after the addition of deuter-ated solvent to a sample lyophilized from nondeuterdeuter-ated solvent, as described previously [15,17] In order to study the interaction between the peptide and SDS micelles, the 2D NOESY spectrum was acquired at 313 K, for 2.5 mMof the peptide dissolved in a solution containing 20mM nondeu-terated SDS micelles at pH 4.0, with a 200-ms mixing time The suppression of the water signal was achieved by the pulsed field gradient method All NMR spectra were processed and analyzed using the NMRPIPE/NMRDRAW software and the NMRVIEW program [18,19] Sequence-specific assignments of the proton resonances were achieved

by spin system identification from the TOCSY and DQF-COSY spectra, followed by sequential assignments through the NOE connectivities [15,17,20] Distance restraints, backbone dihedral angle restraints, and hydrogen bond restraints were obtained and used for the structure calcu-lation by the simulated annealing and energy minimization protocol in the program XPLOR 3.851 [21], as described previously [15] Out of the 50structures calculated by the method demonstrated previously [15], the 49 accepted structures were refined, and finally 20structures with the lowest energies were chosen to represent the solution structure

R E S U L T S A N D D I S C U S S I O N

Biological activities of the GGN4 analogues Native GGN4 exhibits a broad range of antimicrobial activity against prokaryotic cells, but very little hemolytic

activity against human red blood cells [14,15] As shown in

Table 1, the C-terminal 14 residue truncated GGN4 (D24)37

GGN4) showed neither antimicrobial activity against

bacterial cells nor hemolytic activity against human red

blood cells Surprisingly, D16W-D24)37 GGN4, a GGN4

analogue with both the C-terminal 14 residue truncation

and the substitution of the aspartic acid at position 16 by

tryptophan, showed antimicrobial activity comparable to

that of native GGN4 and less hemolytic activity than that of

native GGN4 These results are consistent with the previous

report by Kim et al [16], in which the antimicrobial

activities were checked against only two species of bacteria

(Micrococcus luteus and Escherichia coli) In this previous

report, the antimicrobial activities of several C-terminally

truncated GGN4 analogues with a substituted tryptophan

were analyzed The single tryptophanyl substitution of the

C-terminally truncated GGN4, at position 3, 17, 18, or 19,

did not increase the activity Likewise, in the present work,

the tryptophanyl substitution at position 15 (K15W-D24)37

GGN4) did not restore the antimicrobial activity Taken

together, these results suggest that position 16 is the most

effective position for a single tryptophanyl substitution to

increase the antimicrobial activity of the C-terminally

truncated GGN4 In addition, in this work, the single

phenylalanine substitution at position 16 of the C-terminally

truncated GGN4 moderately restored the activity of the

peptide, but less than that by tryptophan This suggests that

the single tryptophan introduced at position 16 of the

D16W-D24)37GGN4 would have an amino-acid specific

role in the biological action of the peptide Finally, the effect

of the tryptophanyl substitution at position 16 on the

biological activity was confirmed for the native GGN4

Consistent with the results of the C-terminally truncated

GGN4, the D16W substitution in the native GGN4 also

significantly increased the antimicrobial activity of the

peptide However, a remarkable increase of the hemolytic

activity was observed concomitantly

Conformational preferences of the GGN4 analogues

Figure 1 summarizes the CD results of the GGN4 analogue

peptides in aqueous buffer and membrane-mimetic

envi-ronments (50% TFE/water, 10 mM SDS micelles, and

5 mMDPC micelles) For clarity, ½h 222

M

j j, the absolute value

of the mean residue molar elipticity at 222 nm, which approximately reflects the helical content [13,15,17], is indicated in the inset of each panel.½h 208

M =½h 222

M, the ratio of mean residue molar elipticity at 208 nm (½h208M) to that at

222 nm (½h222M), is also included in parentheses, in order to reflect the spectral shape In aqueous buffer, the CD spectra

of the GGN4 analogues, including the native GGN4, showed a strong negative band near 200 nm and a weak and broad band around 222 nm, indicating a predominantly random-coil conformation with a slight helical propensity [17,22,23] Especially, the D16W GGN4 showed a rather significant helical content, even in the aqueous buffer However, in a 50% TFE/water mixture, the CD spectra changed dramatically, with a strong positive band near

192 nm and strong negative bands centered at 208 and

222 nm, which are indicative of a highly a helical confor-mation [22–25] The signals at 193, 208, and 222 nm were intensified with increasing percentages of TFE, which indicates that the helicity of the peptides increased within more hydrophobic environments The spectral change induced by the increased concentration of TFE was nearly complete at about 40–60% TFE/water, and no significant spectral change occurred upon the change of pH from 3.0to 7.0in the 50% TFE/water solution (data not shown) The

CD spectra in 10mMSDS and 5 mMDPC micelles, which are above their critical micellar concentrations [12,26,27], showed shapes similar to those in 50% TFE/water, also indicating a typical a helix pattern This conformational change from a random-coil in aqueous buffer to an a helix

in membrane-mimetic environments is common to many membrane-binding peptides [1,10–13,17]

Although all of the GGN4 analogues tested in this work showed the same conformational preferences in various solvents, they differed remarkably from one another in their helical contents deduced fromj½h222Mj and in the detailed spectral shape represented by ½h208M=½h222M These two param-eters,½h222M and½h208M=½h222M, correlated well with the biological activities In membrane mimetic environments (50% TFE, 10mM SDS, and 5 mM DPC), among the C-terminally truncated GGN4 analogues, D16W-D24)37GGN4, which exhibited the largest antimicrobial activity, showed the largest j½h 222j and a relatively small ½h 208 =½h 222, while D24)37

Table 1 Antimicrobial activity (a) and hemolytic activity (b) of GGN4 analogue peptides Percent hemolysis is relative to that by 0.1% Triton X-100 Molecular masses (in Da) are: Native, 3748; D16W, 3819; N23, 2358; D16W-N23, 2429; D16F-N23, 2390 ; K15W-N23, 2416.

Minimal inhibitory concentration values (lgÆmL)1):

Serratia marcescens > 20 0 > 20 0 > 20 0 > 20 0 > 20 0 > 20 0

Percent hemolysis values:

GGN4, which exhibited no significant activity, showed the

least j½h222Mj and a relatively large ½h208M=½h222M The j½h222Mj of

D16F-D24)37GGN4, which showed moderate activity, was

between that of D24)37GGN4 and that of D16W-D24)37

GGN4 The ½h208M=½h222M of D16F-D24)37 GGN4 was also

relatively small In contrast, K15W-D24)37GGN4, which

has no activity, showed the largest½h 208

M =½h 222

M and a relatively small ½h 222

M

j j

Generally, SDS micelles, which have negatively charged

surfaces, mimic the bacterial cell membrane with its

negatively charged surface, while DPC micelles, which

have zwitterionic surfaces, mimic the eukaryotic cell

membrane with its zwitterionic surface [10,17,28] In the

case of the C-terminally truncated GGN4 analogues, the

maximum ½h 222

M

j j and the minimum ½h 208

M =½h 222

M were com-monly observed in SDS micelles However, both the

native GGN4 and D16W GGN4 showed a higher j½h222Mj

and a lowerj½h208M=½h222Mj in DPC micelles than those in SDS

micelles In particular, in DPC micelles, D16W GGN4,

which was the only peptide with significant hemolytic

activity as well as the largest antimicrobial activity, yielded

a½h 208

M =½h 222

M even lower than that of native GGN4, as well

as a larger ½h 222

M

j j than that of native GGN4

In summary, the CD results showed that the differences

in the activities between the GGN4 analogue peptides are

deeply related to their conformational properties and helical

contents in various environments In addition, it became

clear that the D16W substitution of both the native and the

C-terminally truncated GGN4 increased the helical

propensity of the peptides, which would have a key role in

increasing their biological activities

Solution structures of GGN4 analogues

In order to reveal the detailed structural effects of the D16W substitution, the solution structures of D24)37GGN4 and D16W-D24)37GGN4 were investigated by NMR spectros-copy The structure of the native GGN4 in 50% TFE/water consists of two a helices extending from residues I2 to A10 and from residues D16–32, respectively [15] The final selected structures (Fig 2A) and the refined average struc-ture (figure not shown) of D24)37GGN4 in 50% TFE/water reveal the well-ordered N-terminal a helix composed of residues from I2 to K11, which is in good agreement with the corresponding part of the native GGN4 However, the C-terminal part (residues 12–23) of D24)37GGN4 showed

no significant secondary structure, although some of the initially solved 50structures randomly showed a short

a helical conformation in the C-terminal part The dis-ordered conformation in the C-terminal region of D24)37 GGN4 is probably due to the break of the peptide bond at position 23 In contrast, the finally selected structures (Fig 2B) and the refined average structure (Fig 3A) of D16W-D24)37GGN4 showed a stable helical conformation from residues I2 to V18 in 50% TFE/water, although a few

of the initially solved 50structures randomly showed a rather loosened conformation in the C-terminal part In the previous work [15], the loop region (residues 11–15) of native GGN4 exhibited a flexible, but helix-like conforma-tion in the membrane-mimetic environment, although it could not be defined as a stable a-helix However, the corresponding region in D16W-D24)37 GGN4 showed a stable a helical conformation joined to its N- and C-terminal

Fig 1 Conformational preferencesof GGN4 analogues in various solvents A: CD spectra of native (empty symbols and broken lines) and D16W GGN4 (filled symbols and solid lines)

in aqueous buffer (triangle symbols), 50% TFE/water mixture (bold lines), 10m M SDS micelles (gray lines), and 5 m M DPC micelles (thin lines) B–E: CD spectra of D24)37(thin, solid line), D16W-D24)37(bold, solid line), D16F-D24)37(bold, broken line), and

K15W-D24)37GGN4 (thin, broken line), in aqueous buffer (B), 50% TFE/water mixture (C), 10m M SDS micelles (D), and 5 m M DPC micelles (E) In each panel, ½h 222

M

j j and ½h 208

M =½h 222 M

(in parentheses) of each sample are tabulated

in the inset.

portions Position 16 is at the border between the loop

region (residues 11–15) and the C-terminal helix (residues

16–32) in the intact GGN4 Thus, it can be inferred that the

W16 residue of D16W-D24)37 GGN4 would stabilize the

potential helical propensity of the previous loop region as

well as the C-terminal helix destabilized by truncation In

line with the CD results, the solution structures clearly

support the idea that the D16W substitution of D24)37

GGN4 contributed to the restoration of the antimicrobial

activity, at least by changing its structure

In order to elucidate the structure-function relationship

of D16W-D24)37GGN4, its structure in SDS micelles was

also investigated The helical structures of D16W-D24)37

GGN4 in SDS micelles and in TFE/water were very similar

to each other (Fig 2), and showed several structural

features that are characteristic of many membrane-binding

peptides To begin with, as shown in Fig 3, the peptide

adopts a typical amphipathic helix structure, with the

hydrophobic residues on one side and the hydrophilic

residues on the other side of the helical axis In particular, all

of the lysine residues are oriented to the same side Thus, it

can be deduced that the positively charged hydrophilic side

would easily recognize and bind to the negatively charged

membrane surface of microorganisms Indeed, in the

NOESY experiment of D16W-D24)37 GGN4 in SDS

micelles, we observed intraresidue NOEs between the

side-chain Heand Hz atoms of lysine residues (data not shown),

which could not observed in the TFE/water mixture,

probably due to the high mobility of the side-chain or the

rapid exchange of the Hz amino protons This observation indicates that the lysine side-chains are immobilized in SDS micelles, probably by the electrostatic interaction between their positively charged amino groups and the negatively charged surfaces of the SDS micelles In addition, consistent with the CD results, D16W-D24)37GGN4 displayed a more lengthened a-helix (from I2 to G20) in SDS micelles than that in 50% TFE/water (Fig 2) The relatively more stable C-terminal helical structure of D16W-D24)37GGN4 in SDS micelles than in TFE/water is also attributable to the possible interaction between the K19 residue and the SDS micelles

In many cases, the amphipathic nature of a helical peptide is known to be important for its membrane binding [1,10] Along with the positively charged side-chains from the hydrophilic face, the nonpolar residues in the hydro-phobic face of D16W-D24)37GGN4 seem to contact the SDS micelles by hydrophobic interactions with the acyl chains of the micelles This possible interaction, which has been proposed for other amphipathic peptides [1,10,17,24],

is also supported in this work by the intermolecular NOEs between several hydrophobic residues of the peptide and the acyl chains of the SDS molecules In the NOESY experi-ment of D16W-D24)37 GGN4 in the nondeuterated SDS micelles (Fig 4), a strong resonance at about 1.19 p.p.m., which originates from the methylene protons of SDS [29], was observed Figure 4 clearly depicts the intermolecular NOE cross-peaks between the SDS methylene protons and the peptide backbone amide protons of the F9, V13, and

Fig 2 Solution structures of GGN4 analogues Backbone atoms (N, Ca, and C¢) of the finally refined 20structures were superimposed, by matching the backbone atoms in the helical region, for D24)37GGN4 in the 50% TFE/water mixture (A), D16W-D24)37GGN4 in the 50% TFE/water mixture (B), and D16W-D24)37GGN4 in SDS micelles (C), respectively In panel D, the set of D16W-D24)37GGN4 structures in the 50% TFE/ water mixture (gray lines) was superimposed over that in 500 m M SDS micelles (black lines), by matching the backbone atoms in residues I2V18, and the mainchain (N, C a , C¢, and O) and the tryptophan side chain atoms are represented.

Fig 3 Refined average structure of

D16W-D24)37GGN4 Residues 2–19 in the 50% TFE/

water mixture (A and C) and residues 2–20in

500 m M SDS micelles (B and D) are shown as

space-filling models Hydrophilic,

hydropho-bic, and tryptophan residues are colored

black, gray, and dark gray, respectively The

direction of view is approximately

perpen-dicular to the helical axis in panels A and B,

and is parallel to the helical axis in panels C

and D.

W16 residues, which are oriented toward the same direction,

forming a hydrophobic face of the peptide (Fig 3) In

addition, NOE cross-peaks between the SDS methylene

protons and the aromatic ring protons of F9 and W16 could

be identified Thus, it can be concluded that the

hydropho-bic face of D16W-D24)37GGN4 is in close contact with the

hydrophobic core of the SDS micelles, at least through the

residues F9, V13, and W16

All of the physico-chemical and structural properties of

the D16W-D24)37 GGN4, which are similar to those of

other known amphipathic a helical antimicrobial peptides,

are satisfactory for both the barrel-stave and the carpet-like

action mechanisms [10], although it could not be determined

which one of the two mechanisms is correct for the

D16W-D24)37GGN4

Amino-acid specific role of tryptophan

As the indole side chain has both hydrophobic and

hydrophilic characteristics, tryptophan often plays an

important role by anchoring proteins to the lipid bilayer

surface [30,31] In addition, several antimicrobial peptides

are rich in tryptophan [32,33], which implies a

residue-specific role of tryptophan in their function

The NMR results and the solution structures of

D16W-D24)37 GGN4 suggest that the single tryptophan

residue of the peptide would have a residue-specific role,

as well as the structural effect mentioned above, in the

membrane-interaction of the peptide In both 50% TFE/

water and SDS micelles, the tryptophan residue was located between the hydrophobic face and the hydrophilic face of the amphipathic helix (Fig 3) This location would

be advantageous to facilitate the amphipathic interaction between the peptide and the membrane surface, as the tryptophan side chain is amphiphilic in nature The tryptophan side chain conformation was more clearly defined in both of the environments than those of the other residues (Fig 2D) However, the orientation of the tryptophan side chain from the helical axis was quite different between the two conditions (i.e it slanted more toward the hydrophobic face in SDS micelles than in 50% TFE/water), despite the well-converged backbone confor-mation between the two (Figs 2D and 3) As the SDS micelle more closely mimics the amphiphilic environment

of a biological phospholipid bilayer than TFE does [17,34], the different orientation of the tryptophan side chain seems to imply the anchoring role of the residue in the membrane-binding process of D16W-D24)37 GGN4 This is supported by the intermolecular NOEs between the W16 side chain protons and the SDS methylene protons (Fig 4), which indicate that the tryptophan residue interacts with the hydrophobic core of SDS micelles In addition, in the D2O exchange experiments in SDS micelles, the potentially labile proton of the tryptophan indole ring (He1) remained unchanged even after two hours (data not shown), whereas it was completely exchanged with the solvent deuterium in TFE/water within 25 min This indicates that the atom He1is either involved in a specific interaction, such as hydrogen bonding in SDS micelles, or is buried in a hydrophobic environment, such as the core of the SDS micelle Finally, the fluorescence experiments also confirmed the local environment of the peptide tryptophan residue in SDS micelles It is known that certain indole derivatives interact with detergent micelles [35] For example, trypta-mine, a positively charged indole derivative, interacts with the negatively charged SDS micelles A similar complex is formed between the negatively charged N-acetyltrypto-phan and the positively charged cetyltrimethylammonium bromide In doing so, the fluorescence yield drops in the former, while it increases in the latter case However, these interactions commonly result in significant blue shifts in their fluorescence emissions by more than about 10nm, as the indole ring is positioned close to the hydrophobic tails

of the detergents In the present work, we measured the fluorescence emission of another indole derivative, NATA, which is often used as a control material for the intrinsic tryptophan fluorescence of proteins [35,36], as it mimics a tryptophan residue involved in peptide bonds more closely than any other available indole derivative The fluores-cence emission peak of NATA only showed a blue shift of about 2 nm, from about 360nm in water to about

358 nm in SDS micelles, although the peak intensity decreased by about 23% (Fig 5) In contrast, the fluorescence emission from the unique tryptophan of D16W-D24)37 GGN4 showed a large blue shift of about

13 nm, from about 357 nm in water to about 344 nm in SDS micelles, with a concomitant decrease of the peak intensity by about 13% (Fig 2) This blue shift is representative of the tryptophan residue partitioning into

a more hydrophobic environment [32,33,35,37], which would be expected if the tryptophan residue were

Fig 4 Selected strips taken from the 2D NOESY spectrum of

D16W-D24)37GGN4 in the nondeuterated SDS micelles The right strip shows

the intermolecular NOEs between SDS methylene protons and several

peptide protons, while the left strip shows the intramolecular NOEs

from the He1atom of the peptide tryptophan.

positioned among the acyl chains of the SDS molecules.

In addition, the large blue shift of more than 10nm

indicates that the tryptophan residue of D16W-D24)37

GGN4 anchors into the hydrophobic core of the SDS

micelle, as shown in the case of tryptamine, more

efficiently or more tightly than NATA Thus, the structure

and/or the physico-chemical property of the peptide seems

to contribute to the effective anchoring of the tryptophan

residue

Concluding remarks

The C-terminal 14 residue truncation of GGN4 abolished

the biological activity of the peptide However, the

tryptophanyl substitution at position 16 of the truncated

GGN4 most effectively restored the antimicrobial activity,

without significant hemolytic activity The substituted

tryptophan not only contributed to stabilizing the

amphi-pathic helical structure of the peptide, but also had the

key role of anchoring in the membrane-binding process of

the peptide The present structural investigations of the

GGN4 analogues not only contribute to a better

under-standing of the structure–activity relationships of this

group of antimicrobial peptides with a linear amphipathic

a-helix, but also suggest that the D16W-D24)37 GGN4

could be considered as a potential target molecule for new

peptide antibiotics

Another example showing the helix-stabilizing role of a

tryptophan residue was reported quite recently [38] The

W21A substitution of a cathelicidin-derived antimicrobial

peptide, PMAP-23, destroyed the C-terminal helix of the

peptide, although the W7A substitution did not disrupt

the N-terminal helix Altogether, the utility of a

trypto-phan insertion is also proposed for peptide engineering

to enhance the helical propensity and/or

membrane-interacting ability

A C K N O W L E D G E M E N T S

This work was supported by a grant (HMP-00-B-20900–0096) from the

Ministry of Health & Welfare, Korea, and in part by the 2001 BK21

project for Medicine, Dentistry, and Pharmacy.

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S U P P L E M E N T A R Y M A T E R I A L

The following material is available from http://www.black well-science.com/products/journals/suppmat/EJB/EJB3139/ EJB3139sm.htm

Table S1 Resonance assignments for D24)37 GGN4 in 50% (v/v) TFE/water mixture at pH 4.0

Table S2 Resonance assignments for D16W-D24)37GGN4

in 50% (v/v) TFE/water mixture at pH 4.0

Table S3 Resonance assignments for D16W-D24)37GGN4

in 500 mMSDS micelles at pH 4.0

Table S4 NMR restraints and structural statistics of D24)37 GGN4 and D16W-D24)37GGN4