Effects of a tryptophanyl substitution on the structure and antimicrobial activity of C-terminally truncated gaegurin 4 Hyung-Sik Won 1 , Sang-Ho Park 1 , Hyung Eun Kim 1 , Byongkuk Hyun 2 , Mijin Kim 2 , Byeong Jae Lee 2 and Bong-Jin Lee 1 1 College of Pharmacy, Seoul National University, Seoul, South Korea; 2 Institute 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 2–10 and 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. D 24)37 GGN4, 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 D 24)37 GGN4 completely restored the antimicrobial activity, without any significant hemolytic activity. In contrast, neither the D16F nor K15W substitution of the D 24)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 D 24)37 GGN4 was investigated by CD, NMR, and fluorescence spectroscopy. The results showed that the single tryptophanyl substitution at position 16 of the D 24)37 GGN4 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 pharmacological activities, and they can also serve as model systems for large membrane proteins [1]. Partic- ularly, many organisms, including fungi, insects, amphibi- ans, and humans, 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 ahelical peptides have been proposed [10]: the Ôbarrel-staveÕ mechanism¢ and the Ôcarpet-likeÕ 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 2880 7869, 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) Eur. J. Biochem. 269, 4367–4374 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03139.x 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 2–10 and 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. EXPERIMENTAL PROCEDURES Materials, peptide preparation, and activity test N-Acetyl- L -tryptophanamide (NATA), 2,2,2-trifluoroetha- nol-d 3 99.5% (TFE-d 3 ), and sodium dodecyl-d 25 sulfate (SDS-d 25 ) were obtained from Aldrich. D 2 O (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 l M , in various solvents: 20 m M sodium acetate buffer (pH 4.0), TFE/water mixtures, 5 m M DPC micelles, and 10 m M SDS micelles. Before the CD measurement, the pH was adjusted to 4.0 by the addition of 0.1 M HCl or NaOH. CD spectra were obtained at 20 °Con a JASCO J-720 spectropolarimeter, using a 0.2-cm path- length cell, with a 1-nm bandwidth and a 4-s response time. CD scans were taken from 250 nm to 190 nm, with a scan speed of 50 nmÆmin )1 and 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: ½h k M ¼ h k 10 5 lcn where ½h k M (deg cm 2 Ædmol )1 )andh k (mdeg) are the mean residue molar elipticity and the observed CD intensity at any wavelength (k), respectively. l, c,andn represent the path-length (cm), the concentration (l M ), 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 280 nm, 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 40 nmÆs )1 . All samples contained 8 l M peptide or the same concentration of NATA for control experiments, in water or a 10-m M SDS 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 m M peptide in TFE-d 3 /H 2 O (1 : 1, v/v) at pH 4.0, and in 500 m M SDS- d 25 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 60 ms. The 2D NOESY spectra were acquired with mixing times of 150 and 200 ms, respectively. Slowly exchanging amide protons were monitored by the D 2 O exchange experiments with a series of 2D NOESY spectra measured immediately after the addition of deuter- ated solvent to a sample lyophilized from nondeuterated 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 m M of the peptide dissolved in a solution containing 20 m M 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 50 structures calculated by the method demonstrated previously [15], the 49 accepted structures were refined, and finally 20 structures with the lowest energies were chosen to represent the solution structure. RESULTS AND DISCUSSION Biological activities of the GGN4 analogues Native GGN4 exhibits a broad range of antimicrobial activity against prokaryotic cells, but very little hemolytic 4368 H S. Won et al. (Eur. J. Biochem. 269) Ó FEBS 2002 activity against human red blood cells [14,15]. As shown in Table 1, the C-terminal 14 residue truncated GGN4 (D 24)37 GGN4) showed neither antimicrobial activity against bacterial cells nor hemolytic activity against human red blood cells. Surprisingly, D16W-D 24)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-D 24)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-D 24)37 GGN4 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 m M SDS micelles, and 5m M DPC micelles). For clarity, ½h 222 M jj , the absolute value of the mean residue molar elipticity at 222 nm, which approximately reflects the helical content [13,15,17], is indicatedintheinsetofeachpanel. ½h 208 M =½h 222 M ,theratioof mean residue molar elipticity at 208 nm ( ½h 208 M )tothatat 222 nm ( ½h 222 M ), 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 3.0 to 7.0 in the 50% TFE/water solution (data not shown). The CD spectra in 10 m M SDS and 5 m M DPC 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 from ½h 222 M jj andinthedetailed spectral shape represented by ½h 208 M =½h 222 M .Thesetwoparam- eters, ½h 222 M and ½h 208 M =½h 222 M , correlated well with the biological activities. In membrane mimetic environments (50% TFE, 10 m M SDS, and 5 m M DPC), among the C-terminally truncated GGN4 analogues, D16W-D 24)37 GGN4, which exhibited the largest antimicrobial activity, showed the largest ½h 222 M jj and a relatively small ½h 208 M =½h 222 M , while D 24)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; N 23 , 2358; D16W-N 23 , 2429; D16F-N 23 ,2390;K15W-N 23 , 2416. Microorganism Native D16W N 23 D16W-N 23 D16F-N 23 K15W-N 23 Minimal inhibitory concentration values (lgÆmL )1 ): Micrococcus luteus 2.5 25 > 200 2.5 > 200 > 200 Bacillus subtilis 10 2.5 > 200 25 25 > 200 Klebsiella pneumoniae 25 10 > 200 25 100 > 200 Shigella dysentariae 25 10 > 200 50 50 > 200 Pseudomonas aeruginosa 100 50 > 200 125 200 > 200 Escherichia coli 75 10 > 200 25 25 > 200 Salmonella typhimurium 200 50 > 200 125 > 200 > 200 Serratia marcescens >200 >200 >200 >200 >200 >200 Percent hemolysis values: 10 lgÆmL )1 concentration 0.78% 1.97% 0% 0.02% 0.06% 0% 100 lgÆmL )1 concentration 1.67% 52.9% 0% 0.38% 0.32% 0% Ó FEBS 2002 Structure–activity relationships of GGN4 analogues (Eur. J. Biochem. 269) 4369 GGN4, which exhibited no significant activity, showed the least ½h 222 M jj and a relatively large ½h 208 M =½h 222 M .The ½h 222 M jj of D16F-D 24)37 GGN4, which showed moderate activity, was between that of D 24)37 GGN4 and that of D16W-D 24)37 GGN4. The ½h 208 M =½h 222 M of D16F-D 24)37 GGN4 was also relatively small. In contrast, K15W-D 24)37 GGN4, which has no activity, showed the largest ½h 208 M =½h 222 M and a relatively small ½h 222 M jj . 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 jj 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 ½h 222 M jj and a lower ½h 208 M =½h 222 M jj 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 a ½h 208 M =½h 222 M even lower than that of native GGN4, as well as a larger ½h 222 M jj 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 D 24)37 GGN4 and D16W-D 24)37 GGN4 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 D 24)37 GGN4 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 D 24)37 GGN4 showed no significant secondary structure, although some of the initially solved 50 structures randomly showed a short a helical conformation in the C-terminal part. The dis- ordered conformation in the C-terminal region of D 24)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-D 24)37 GGN4 showed a stable helical conformation from residues I2 to V18 in 50% TFE/water, although a few of the initially solved 50 structures 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-D 24)37 GGN4 showed a stable a helical conformation joined to its N- and C-terminal Fig. 1. Conformational preferences of 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), 10 m M SDS micelles (gray lines), and 5 m M DPC micelles (thinlines).B–E:CDspectraofD 24)37 (thin, solid line), D16W-D 24)37 (bold, solid line), D16F-D 24)37 (bold, broken line), and K15W- D 24)37 GGN4 (thin, broken line), in aqueous buffer (B), 50% TFE/water mixture (C), 10 m M SDS micelles (D), and 5 m M DPC micelles(E).Ineachpanel, ½h 222 M jj and ½h 208 M =½h 222 M (in parentheses) of each sample are tabulated in the inset. 4370 H S. Won et al. (Eur. J. Biochem. 269) Ó FEBS 2002 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-D 24)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 D 24)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-D 24)37 GGN4, its structure in SDS micelles was also investigated. The helical structures of D16W-D 24)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-D 24)37 GGN4 in SDS micelles, we observed intraresidue NOEs between the side- chain H e and 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-D 24)37 GGN4 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-D 24)37 GGN4 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-D 24)37 GGN4 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-D 24)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, C a ,andC¢) of the finally refined 20 structures were superimposed, by matching the backbone atoms in the helical region, for D 24)37 GGN4 in the 50% TFE/water mixture (A), D16W-D 24)37 GGN4 in the 50% TFE/water mixture (B), and D16W-D 24)37 GGN4 in SDS micelles (C), respectively. In panel D, the set of D16W-D 24)37 GGN4 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 I2V18, andthemainchain(N,C a ,C¢, and O) and the tryptophan side chain atoms are represented. Fig. 3. Refined average structure of D16W- D 24)37 GGN4. Residues 2–19 in the 50% TFE/ water mixture (A and C) and residues 2–20 in 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- diculartothehelicalaxisinpanelsAandB, and is parallel to the helical axis in panels C and D. Ó FEBS 2002 Structure–activity relationships of GGN4 analogues (Eur. J. Biochem. 269) 4371 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-D 24)37 GGN4 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-D 24)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- D 24)37 GGN4. 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-D 24)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-D 24)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 D 2 O exchange experiments in SDS micelles, the potentially labile proton of the tryptophan indole ring (H e1 ) 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 H e1 is 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 10 nm, 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 360 nm 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-D 24)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- D 24)37 GGN4 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 H e1 atom of the peptide tryptophan. 4372 H S. Won et al. (Eur. J. Biochem. 269) Ó FEBS 2002 positioned among the acyl chains of the SDS molecules. In addition, the large blue shift of more than 10 nm indicates that the tryptophan residue of D16W-D 24)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-D 24)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. 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Yang, S., Zhang, L. & Huang, Y. (2001) Membrane association and conformational change of palmitoylated G o a. FEBS Lett. 498, 76–81. 38. Park, K., Oh, D., Shin, S.Y., Hahm, K S. & Kim, Y. (2002) Structural studies of porcine myeloid antibacterial peptide PMAP- 23 and its analogues in DPC micelles by NMR spectroscopy. Biochem. Biophys. Res. Commun. 290, 204–212. SUPPLEMENTARY MATERIAL The following material is available from http://www.black well-science.com/products/journals/suppmat/EJB/EJB3139/ EJB3139sm.htm. Table S1. Resonance assignments for D 24)37 GGN4 in 50% (v/v) TFE/water mixture at pH 4.0. Table S2. Resonance assignments for D16W-D 24)37 GGN4 in 50% (v/v) TFE/water mixture at pH 4.0. Table S3. Resonance assignments for D16W-D 24)37 GGN4 in 500 m M SDS micelles at pH 4.0. Table S4. NMR restraints and structural statistics of D 24)37 GGN4 and D16W-D 24)37 GGN4. 4374 H S. Won et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . D16W-D 24) 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. 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. Effects of a tryptophanyl substitution on the structure and antimicrobial activity of C-terminally truncated gaegurin 4 Hyung-Sik Won 1 , Sang-Ho Park 1 , Hyung Eun Kim 1 , Byongkuk Hyun 2 ,