Contribution of a central proline in model amphipathic a-helical peptides to self-association, interaction with phospholipids, and antimicrobial mode of action Sung-Tae Yang 1 , Ju Yeon Lee 1 , Hyun-Jin Kim 1 , Young-Jae Eu 1 , Song Yub Shin 2 , Kyung-Soo Hahm 2 and Jae Il Kim 1 1 Department of Life Science, Gwangju Institute of Science and Technology, Korea 2 Department of Bio-Materials, Graduate School and Research Center for Proteineous Materials, Chosun University, Gwangju, Korea Antimicrobial peptides are produced as components of the innate immune system by a wide variety of insects, amphibians, and mammals, including humans [1–4]. In recent decades, the structures and functions of many antimicrobial peptides have been extensively studied to elucidate their mode of action. Typically, antimicrobial Keywords aggregation; amphipathic helix; antimicrobial peptides; membrane depolarization; proline Correspondence J. Kim, Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea Fax: +82 62 970 2484 Tel: +82 62 970 2494 E-mail: jikim@gist.ac.kr (Received 9 February 2006, revised 28 June 2006, accepted 5 July 2006) doi:10.1111/j.1742-4658.2006.05407.x Model amphipathic peptides have been widely used as a tool to determine the structural and biological properties that control the interaction of pep- tides with membranes. Here, we have focused on the role of a central Pro in membrane-active peptides. To determine the role of Pro in structure, antibiotic activity, and interaction with phospholipids, we generated a ser- ies of model amphipathic a-helical peptides with different chain lengths and containing or lacking a single central Pro. CD studies showed that Pro-free peptides (PFPs) formed stable a-helical structures even in aqueous buffer through self-association, whereas Pro-containing peptides (PCPs) had random coil structures. In contrast, in trifluoroethanol or SDS mi- celles, both PFPs and PCPs adopted highly ordered a-helical structures, although relatively lower helical contents were observed for the PCPs than the PFPs. This structural consequence indicates that a central Pro residue limits the formation of highly helical aggregates in aqueous buffer and cau- ses a partial distortion of the stable a-helix in membrane-mimetic environ- ments. With regard to antibiotic activity, PCPs had a 2–8-fold higher antibacterial activity and significantly reduced hemolytic activity compared with PFPs. In membrane depolarization assays, PCPs passed rapidly across the peptidoglycan layer and immediately dissipated the membrane potential in Staphylococcus aureus, whereas PFPs had a greatly reduced ability. Fluorescence studies indicated that, although PFPs had strong binding affinity for both zwitterionic and anionic liposomes, PCPs interacted weakly with zwitterionic liposomes and strongly with anionic liposomes. The selective membrane interaction of PCPs with negatively charged phospholipids may explain their antibacterial selectivity. The difference in mode of action between PCPs and PFPs was further supported by kinetic analysis of surface plasmon resonance data. The possible role of the increased local backbone distortion or flexibility introduced by the proline residue in the antimicrobial mode of action is discussed. Abbreviations DiSC 3 (5), 3,3¢-dipropylthiadicarbocyanine iodide; PamOlePtdCho, 1-palmitoyl-2-oleoylphosphatidylcholine; PamOlePtdGro, 1-palmitoyl-2- oleoylphosphatidylglycerol; PCPs, proline-containing peptides; PFPs, proline-free peptides; SPR, surface plasmon resonance. 4040 FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS peptides contain multiple basic amino acids and am- phipathic structures with clusters of hydrophobic and hydrophilic residues [5–9]. Although their precise mechanism of action is not yet fully understood, it is widely accepted that cationic antimicrobial peptides interact with negatively charged bacterial membranes by electrostatic interactions and then cause cell death by permeabilizing cell membranes by forming barrel- stave or toroidal pores [10–14] or by disrupting the membrane via a ‘carpet’ mechanism [15–17]. It is also known that in some cases peptides inhibit the macro- molecular synthesis by penentrating into the bacterial cytoplasm followed by DNA ⁄ RNA binding without causing membrane permeabilization [18–21]. Some antimicrobial peptides can lyse not only microbial but also eukaryotic cells [22]. This activity against eukary- otic cells should be eliminated so that the antimicrobial peptides can be used therapeutically. Thus, consider- able attention has been focused on the design of new antimicrobial peptides with good selectivity for bacter- ial cells. Structure–function studies of antimicrobial peptides have shown that a number of variables modulate antibiotic activity, including chain length, helical pro- pensity, amphipathicity, net positive charge, hydro- phobicity, and hydrophobic moment [23–29]. A variety of model amphipathic a-helical peptides and artificial membranes have been used to analyze the molecular structure–function relationships, understand the gen- eral aspects of peptide–lipid interactions, and deter- mine the variables that control cell selectivity. For example, to investigate the effect of hydrophobic– hydrophilic balance on biological and membrane-lytic activities, Kiyota et al. [30] synthesized five 18-residue model peptides composed of nonpolar (Leu) and basic (Lys) residues of varying hydrophobic–hydrophilic balance. In addition, to determine the proper chain length for potent antimicrobial peptides, Blondelle & Houghten [31] prepared a series of 8–22-residue model amphipathic peptides comprising Leu and Lys. Also, Papo et al. [32] generated several short model peptides and their diastereomeric analogs to study the structural and functional effects of d-amino acids in amphipathic a-helices. These systematic analyses have helped to clarify the characteristics needed for the design of potent, selective antimicrobial peptides as antibiotics. The presence of Pro residues in a-helices generally cre- ates a bend or kink in the peptide backbone because of the lack of an amide proton, which normally provides a hydrogen bond donor, and they are commonly found within the amphipathic a-helices of antimicrobial pep- tides. Pro residues in amphipathic a-helical peptides have been the focus of extensive research because they are functionally important in peptide–lipid interactions. For example, several recent studies have investigated the effect of Pro substitutions on the biological activity and structure of naturally occurring antimicrobial peptides such as PMAP-23 [33], melittin [34], gaegurin [35], tri- trpticin [36], and maculatin [37]. These studies revealed that the replacement of a Pro with an Ala maintained or decreased the antimicrobial activity but significantly increased the hemolytic activity. In addition, Oh et al. [38] reported that a cecropin A–magainin II hybrid pep- tide and its analog P2, which have amphipathic a-helical structures with a central hinge region due to the presence of Gly or Pro, have potent and selective antimi- crobial activity. We also reported that the replacement of a Pro with Leu or Ala in the hybrid analog P18 decreases its antibacterial activity and increases its hemolytic activity [39]. In general, introduction of a Pro near the central region of a-helical antimicrobial peptides reduces the a-helical structure. This partial disruption of the struc- ture appears to contribute to selective cytotoxicity. On the other hand, Pro residues are also found in ion- channel-forming peptides. Alamethicin, for example, has a Pro-kink helical structure which is important for its insertion into lipid bilayers. Once in the lipid bilay- ers, they form transmembrane helices that contribute to transmembrane pores or voltage-induced channels [40,41]. In addition, statistical analysis of transmem- brane helices has established the significance of Pro- containing motifs in transmembrane a-helices [42], and several studies have investigated the structural and dynamic role of Pro residues in transmembrane helices [43]. Although there is growing evidence that the Pro residues largely contribute to the ability of antimicro- bial peptides to kill various types of microbial cells and to form transmembrane helices, the role of the internal kink induced by Pro in amphipathic a-helices has not been systematically studied, and the kinetic significance of this structure remains unknown. Here, we have systematically examined the role of a central Pro residue by using model 17–25-residue am- phipathic a-helical peptides that either contain or lack a Pro residue. We also applied biosensor technology to distinguish the kinetics of membrane binding by Pro-containing peptides (PCPs) and Pro-free peptides (PFPs). We found that the synthetic PCPs have much more potent antibacterial activity and significantly reduced hemolytic activity than the PFPs. In addition, the PCPs were able to selectively bind and strongly permeabilize negatively charged liposomes. We further discuss the role of the helix–bend–helix structure induced by a central Pro residue in the mechanism of selective antimicrobial activity. S T. Yang et al. Central proline in amphipathic a-helix FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS 4041 Results Peptide design To investigate the influence of a central Pro on the biological activity, structure, membrane binding, and membrane-disrupting activity of antimicrobial pep- tides, we generated amphipathic a-helical peptides with different chain lengths (17, 21, and 25 residues) and containing or lacking a Pro residue. The model pep- tides are composed of repeats of hydrophobic (Leu) and basic (Lys) residues to create perfect amphipathic a-helices. A single Trp residue was introduced in posi- tion 2 of these peptides to allow fluorescent deter- mination of their concentration and peptide–lipid interactions. The Pro-free peptides (PFPs) included M17, M21, and M25, and their counterpart central Pro-containing peptides (PCPs) were M17P, M21P, and M25P, respectively (Table 1). Comparison of antimicrobial and hemolytic activities of the peptides The model amphipathic a-helical peptides were studied for their ability to inhibit the growth of Gram-negative and Gram-positive bacteria as well as for their cyto- toxicity against human erythrocytes. The minimal inhibitory concentrations for the peptides against bacteria are summarized in Table 2, and the dose– response relationship of the hemolytic activity is depic- ted in Fig. 1. As shown in Table 2, the different chain length of the peptides did not significantly affect their activity toward both Gram-negative and Gram-positive bacteria. These results suggest that a long chain length is not required for improved antibacterial activity. Interestingly, compared with PFPs, PCPs had % 2–8- fold greater antibacterial activities. As shown in Fig. 1, however, PFPs (M17, M21, and M25) were relatively strongly hemolytic (63%, 65%, and 75% at 50 lm, respectively), whereas PCPs (M17P, M21P, and M25P) displayed significantly reduced hemolytic activity (4%, 21%, and 11% at 50 lm, respectively). These data suggest that introduction of Pro residues at a central position improved the peptide selectivity for bacterial versus mammalian cells. Structural analysis of the peptides CD spectroscopy was used to monitor the secondary structure of the peptides. The CD spectra of peptides were collected in 50 mm sodium phosphate buffer ⁄ 50% trifluoroethanol ⁄ 30 mm SDS micelles, PamOlePtd- Cho ⁄ PamOlePtdGro (1 : 1) liposomes or PamOlePtd- Cho liposomes (Fig. 2). The CD spectra of all of the synthetic peptides dissolved in water in the absence of salt showed that they were mainly random coils (data not shown). In buffer (50 mm sodium phosphate buffer, pH 7.2), however, PFPs (Fig. 2A, filled Table 1. Amino-acid sequences and molecular masses of the model peptides. Observed mass was from Kratos Kompact MALDI TOF MS. Peptide Sequence Mass Calculated Observed M25 KWKKLLKKLLKLLKKLLKKLKKLLK-NH 2 3114.3 3115.2 M25P KWKKLLKKLLKLPKKLLKKLKKLLK-NH 2 3098.2 3098.8 M21 KWKKLLKKLLKLLKKLLKKLK-NH 2 2631.6 2631.9 M21P KWKKLLKKLLPLLKKLLKKLK-NH 2 2600.5 2601.4 M17 KWKKLLKKLLKLLKKLL-NH 2 2133.9 2134.3 M17P KWKKLLKKPLKLLKKLL-NH 2 2117.8 2118.6 Table 2. Minimal inhibitory concentration (lM) for the peptides. Results indicate the range of three independent experiments, each performed in triplicate. Bacterial strain Peptide M25 M25P M21 M21P M17 M17P E. coli 16–32 4–8 16 4–8 4–8 2–4 S. typhimurium 16 4 8–16 2–4 4–8 2–4 P. aeuroginosa 16–32 4–8 16–32 8 8–16 4–8 B. subtilis 8–16 4–8 8–16 4–8 8 2–4 S. aureus 8–16 2–4 8 2–4 4–8 2–4 S. epidermidis 8–16 4–8 8–16 2–4 4–8 1–4 Fig. 1. Dose–response curves of hemolytic activity of the peptides toward human erythrocytes. Hemolysis assays were carried out for the following peptides: M25 (d), M25P (s), M21 (.), M21P (,), M17 (n), and M17P (h). Results represent the means of duplicate measurements from three independent assays. Central proline in amphipathic a-helix S T. Yang et al. 4042 FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS symbols) exhibited typical a-helical CD spectra, with minimal mean residue molar ellipticity values at 208 and 222 nm, whereas the CD spectra of PCPs (Fig. 2A, empty symbols) had a negative band below 200 nm, indicating a lack of ordered structure. This result supports the idea that proline is an effective a-helix breaker, as previously reported for globular proteins [44]. As expected, the CD spectra of all of the peptides indicated a-helix structures in the presence of trifluoroethanol (Fig. 2B) or SDS micelles (Fig. 2C), but there was little difference in the helical contents between PCPs and PFPs. These results suggest that PCPs have a partially distorted helix structure with a kink around the central Pro in membrane-mimetic environments. Interestingly, the shape of PFP spectra in the presence of PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) liposomes is apparently different from that in the presence of SDS or trifluoroethanol (Fig. 2D). This may point to strong aggregation in this type of mem- brane, which may correlate with the increased cytotox- icity of these peptides. In PamOlePtdCho liposomes (Fig. 2E), PCPs had no distinct secondary structure, but there was a weak shoulder in their spectra, com- pared with aqueous solution, suggesting that some interaction does occur. In contrast, PFPs adopted a-helical structures, indicating that PFPs can strongly interact with zwitterionic liposomes. Next, to determine in detail the effect of salt on the conformational transition from a random coil to an a-helix, the CD spectra were recorded as a func- tion of the NaCl concentration from 0 to 100 mm at a constant peptide concentration (Fig. 3). The results for peptide M21 are shown as an example in Fig. 3A. The CD spectra of M21 in the presence of various NaCl concentrations exhibited an isodichroic point at 203 nm, indicating a two-state equilibrium between a random coil and an a-helix. In pure water, M17 and M25 also became more a-helical as the NaCl concentration was increased (Fig. 3B). Helix formation by the PFPs appears to be accom- panied by self-association. In addition, the ratio of ellipticity values at 222 ⁄ 208 nm is close to 1 in buf- fer, which this is taken to indicate aggregation [45]. The CD spectra of PCPs (M17P, M21P, and M25P), however, did not change as the NaCl concentration was increased. These results suggest that the presence of a kink induced by a Pro residue in amphipathic a-helices is essential for maintaining them as mono- mers in aqueous solution. Peptide-induced dye leakage from liposomes We next measured the membrane-disrupting abilities of the peptides by examining calcein leakage from neg- atively charged PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) or zwitterionic PamOlePtdCho liposomes. Upon addition of the peptides to the liposomes, the entrapped calcein (70 mm) was released into the buf- fer by lysis. This relieves self-quenching of the dye within the liposomes, increasing the fluorescence intensity. Relative lytic efficiencies were determined by comparing the effects of the peptides with those of Triton X-100, which corresponds to the total fluorescence. Dose responses of peptide-induced calc- ein release from the PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) and PamOlePtdCho liposomes are shown in Fig. 4. Compared with PCPs, PFPs released as much or slightly more calcein from the PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) liposomes. All of the amphi- pathic peptides caused an almost total disruption of Fig. 2. CD spectra of the model peptides under various conditions. CD spectra were obtained at 25 °C in (A) 50 mM sodium phosphate buf- fer (pH 7.2), (B) 50% trifluoroethanol, (C) 30 m M SDS micelles, (D) PamOlePtdGro ⁄ PamOlePtdCho (1 : 1) liposomes, or (E) PamOlePtdCho liposomes and in the presence of the following peptides at 25 l M concentration: M25 (d), M25P (s), M21 (.), M21P (,), M17 (n), and M17P (h). S T. Yang et al. Central proline in amphipathic a-helix FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS 4043 the PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) liposomes at 1 : 20 molar ratio of peptide to liposome. In con- trast, the PFPs (M17, M21, and M25) caused relat- ively large calcein leakage (57%, 60%, and 66%, respectively) from PamOlePtdCho liposomes at a peptide to liposome molar ratio of 1 : 10, whereas the PCPs showed a relatively reduced ability to reduce PamOlePtdCho membranes. These results agree well with those from analysis of hemolysis, and they indicate that introduction of Pro into amphi- pathic a-helical peptides confers the ability to selec- tively disrupt anionic versus zwitterionic liposomes. Fig. 3. CD spectra of M21 and [.] 222 for the model peptides at various NaCl concentrations. (A) CD spectra were recorded as a function of the NaCl concentration (from 0 to 50 m M at increments of 5 mM) for 25 lM peptide M21 at 25 °C. (B) Plot of [h] 222 versus NaCl concentra- tion (0–100 m M) for the following peptides at 25 lM concentration: M25 (d), M25P (s), M21 (.), M21P (,), M17 (n), and M17P (h). Fig. 4. Calcein leakage as a function of molar ratio of peptide to lipid. Calcein-containing (A) PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) or (B) PamOlePtdCho liposomes at 25 °C were mixed with the following peptides: M25 (d), M25P (s), M21 (.), M21P (,), M17 (n), or M17P (h). Results represent the means of three independent experiments. Central proline in amphipathic a-helix S T. Yang et al. 4044 FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS Of the PCPs, M17P and M25P showed negligible cyto- toxicity against human red blood cells and a relatively weak ability to disrupt artificial neutral liposomes, whereas M21P had moderate cytolytic activity. In the case of M17P and M25P, Pro replaced the central Leu of the hydrophobic helix face (based on an amphipath- ic helical wheel diagram), whereas in M21P, it replaced the central Lys of the hydrophilic helix face. The mod- erate cytotoxicity of M21P suggests that placement of Pro in the hydrophobic face of amphipathic a-helical peptides is more effective than placement in the hydro- philic region for generating peptides with selectivity for bacterial versus red blood cells. Tryptophan fluorescence To study the interaction of PCPs and PFPs with membranes, we next examined changes in Trp fluor- escence in pure water, aqueous buffer, or anionic PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) or zwitterionic PamOlePtdCho liposomes. As the fluorescence emis- sion characteristics of the Trp are sensitive to its immediate environment, it can be used to monitor the binding of peptides to membranes. All the pep- tides listed in Table 1 have a single Trp residue at position 2. The corresponding maximum emission wavelength (k max ) is plotted as a function of the lipid ⁄ peptide molar ratio in Fig. 5, and the k max val- ues of the peptides at lipid ⁄ peptide molar ratio of 50 : 1 are shown in Table 3. In Tris ⁄ HCl buffer, the k max values for PCPs were % 352 nm, indicating that Trp residues are fully exposed to a hydrophilic envi- ronment. In contrast, the k max value of the Trp resi- due in PFPs was % 343 nm, indicating that Trp was surrounded by a hydrophobic environment through self-association of the peptides in buffer. Addition of PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) liposomes with both PFPs and PCPs results in a large blue shift (22– 24 nm) in the k max and an increase in the fluores- cence quantum yield for all the peptides, indicating that the peptides strongly bind to negatively charged membranes. With zwitterionic PamOlePtdCho lipo- somes, there was an almost constant k max around 349 nm for M17P and M25P at all lipid ⁄ peptide ratios and a small blue shift (9 nm) for M21P, indica- ting a lack of binding to PCPs. In contrast, the three model PFPs displayed a large blue shift (18–19 nm). These results indicate that PCPs interact weakly with zwitterionic phospholipids but strongly with anionic phospholipids. Fig. 5. k max of tryptophan fluorescence as a function of the lipid ⁄ peptide ratio. Fluorescence spectra were recorded at increasing concentra- tions of (A) PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) or (B) PamOlePtdCho liposomes in Tris ⁄ HCl buffer (pH 7.4) at 25 °C and at 3 l M of the following peptides: M25 (d), M25P (s), M21 (.), M21P (,), M17 (n), or M17P (h). The excitation wavelength was 280 nm, the excitation band width was 5 nm, and the emission band width was 3 nm. Results represent the means of three independent experiments. Table 3. k max (nm) of tryptophan fluorescence for the peptides and K SV in the presence of liposomes. Assays were carried out in Tris ⁄ HCl buffer or in the presence of PamOlePtdCho ⁄ PamOlePtd- Gro (1 : 1) or PamOlePtdCho liposomes at a lipid ⁄ peptide molar ratio of 50 : 1. Peptide Pure water Tris ⁄ HCl buffer PtdCho ⁄ PtdGro PamOle- PtdCho K SV (M )1 ) PtdCho ⁄ PtdGro PamOle- PtdCho M25 351 342 328 332 1.96 3.24 M25P 352 351 330 348 2.47 9.14 M21 351 343 329 333 1.83 2.83 M21P 353 351 330 341 2.33 6.29 M17 352 344 330 333 2.20 3.60 M17P 353 352 329 349 2.61 9.88 S T. Yang et al. Central proline in amphipathic a-helix FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS 4045 Quenching of the intrinsic fluorescence by acrylamide To compare the membrane-integrated state of PCPs and PFPs following their interaction with negatively charged PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) or neutral PamOlePtdCho liposomes, we next performed a fluorescence quenching experiment using the neutral fluorescence quencher acrylamide. This quencher can approach Trp more easily when the peptide is free in solution than when it is bound to model membranes. Stern-Volmer plots for fluorescence quenching of Trp by acrylamide in the presence of PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) or PamOlePtdCho liposomes are depicted in Fig. 6, and the apparent K SV values are shown in Table 3. The Trp fluorescence intensity for PFPs decreased in a similar concentration-dependent manner for both types of liposome after the addition of acrylamide, indicating that PFPs are buried in both anionic and neutral liposomes. The tendency of PFPs to self-associate appears to affect their nonselective interaction. However, quenching of Trp fluorescence of PCPs is less efficient with PamOlePtdCho ⁄ PamOlePtd- Gro (1 : 1) than with PamOlePtdCho vesicles, suggest- ing that the Trp residue of PCPs penetrates more efficiently into the hydrophobic core of negatively charged bilayers than zwitterionic bilayers. Membrane depolarization by PFPs and PCPs It is widely believed that many membrane-active anti- microbial peptides pass through the peptidoglycan layer and then kill the target micro-organism by inter- acting with and permeabilizing the cytoplasmic mem- brane. To further study this hypothesis, we examined the ability of PFPs and PCPs to depolarize the mem- brane using the membrane-potential-sensitive dye 3,3¢- dipropylthiadicarbocyanine iodide [DiSC 3 (5)] (Fig. 7). Upon addition to a suspension of S. aureus, the fluor- escence of DiSC 3 (5) (first arrow) is strongly quenched and quickly stabilized. Addition of peptides (second arrow) increased the fluorescence caused by membrane depolarization, and subsequent addition of gramicidin D (third arrow) fully disrupted the membrane poten- tial. Interestingly, PCPs almost completely dissipated the membrane potential at 0.3 lm, but self-associated PFPs showed a largely reduced ability to cause mem- brane depolarization. In addition, all PCPs caused an immediate increase in fluorescence intensity, indicating rapid membrane depolarization, whereas the PFPs caused a gradual increase in the fluorescence. These results suggest that the self-association of PFPs, which have less potent antimicrobial activity, interferes with their passage across the peptidoglycan layer. Analysis of binding using an surface plasmon resonance (SPR) biosensor Finally, we used SPR to monitor the binding of PFPs and PCPs to PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) liposomes immobilized on an L1 sensor chip. Figure 8 shows representative sensorgrams for the binding of M17 and M17P. The sensorgrams for M25 and M21 were similar to that for M17, whereas the sensorgrams for M25P and M21P were similar to that for M17P (data not shown). Examination of the shape of the sensorgrams for M17 and M17P reveals significantly different binding kinetics. In particular, the sensor- grams indicate that the initial association of M17P with the lipid surface starts as a very fast process Fig. 6. Stern-Volmer plots for the quenching of Trp fluorescence by the peptides. Quenching assays were carried out in the presence of 150 l M of either (A) PamOlePtdGro ⁄ PamOlePtdGro or (B) PamOlePtdCho liposomes and the following peptides at 3 l M con- centration: M25 (d), M25P (s), M21 (.), M21P (,), M17 (n), or M17P (h). Results represent the means of three independent experiments. Central proline in amphipathic a-helix S T. Yang et al. 4046 FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS compared with that of M17. In addition, whereas M17P exhibited a distinct association and dissociation, M17 had a very slow dissociation at a low peptide concentration (less 20 lm) and failed to dissociate from the liposomes at high concentrations (more 40 lm). When the sensorgrams are fitted using differ- ent concentrations of M17P, the two-state reaction model fits better than the simple 1 : 1 Langmuir bind- ing model, suggesting that a two-step process mediates the interaction of the peptide with lipid bilayers. How- ever, M17 had similar C 2 values in both fitting models. Only peptide sensorgrams obtained at low peptide concentrations (2.5–20 lm) were used to calculate the association constants for M17 because the peptide was bound irreversibly to the lipid bilayers at high concen- trations. The average values for the rate constants and affinity constants obtained from the two-state model analysis are listed in Table 4. There were striking dif- ferences between PFPs and PCPs in the association rate (k a1 ) for first step and the dissociation rate (k d2 ) for second step. The observations with PFPs and PCPs seem to be in line with those of Zelezetsky et al. [46], using different types of aggregating ⁄ nonaggregating model peptides. Discussion Membrane-active peptides mediate a wide range of biological events, including signal transduction, trans- port through the membrane, membrane fusion and lysis, ion channel formation, and antimicrobial def- ense. These peptides exhibit a structural transition from an extended coil to a well-defined secondary structure upon binding to membrane surfaces. Interac- tion of the peptides with membranes plays an import- ant role in many cellular processes. In particular, Pro residues often appear in the central region of mem- brane-active peptides, and they may control the folding process and affect the membrane translocation or pen- etration [43,47–49]. Recently, model peptides have been intensively stud- ied as tools for determining the structural and biologi- cal properties of antimicrobial peptides. In particular, model amphipathic a-helical peptides have been stud- ied extensively to identify general properties related to peptide–lipid interaction and their relationships with the biological activity of the peptides [28–32]. In the present study, we carried out a systematic structure– activity study on a series of model peptides to deter- mine the role of a central Pro on the biological activ- ity, peptide structure, and interaction with membranes. One interesting finding of this study was that intro- duction of a Pro in the middle position of the sequences of nonselective cytolytic peptides confers high selectivity for bacterial cells. In particular, we found that the depolarization of bacterial membranes caused by PCPs is more potent and rapid than that caused by PFPs. There was a direct correlation between the ability of the peptides to dissipate the membrane potential and their antimicrobial activity. In Fig. 7. Kinetics of membrane depolarization of S. aureus by PFPs and PCPs. DiSC 3 (5) was added to exponential-phase S. aureus cells. once the fluorescence was stable, the peptides (0.3 l M) were added, and membrane depolarization was measured. Gramicidin D (0.22 n M) was used to induce full collapse of the membrane poten- tial. The results are representative of two independent experi- ments. S T. Yang et al. Central proline in amphipathic a-helix FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS 4047 addition, Trp fluorescence measurements indicated that PFPs interacted nonselectively with negatively charged and zwitterionic liposomes, whereas PCPs bound strongly and selectively to anionic liposomes. These results are consistent with the ability of the peptides to induce dye leakage preferentially from negatively charged lipid membranes. The selective membrane interaction of Pro-containing peptides with negatively charged phospholipids may explain the selective anti- bacterial activity because zwitterionic phospholipids are the major constituent of the outer leaflet of red blood cells. Understanding the process of peptide folding in aqueous buffer or in membrane-mimetic environments is critical for elucidating the mechanism of antimicro- Fig. 8. Sensorgrams for the binding of peptides to 1 : 1 PamOlePtdCho ⁄ PamOlePtdGro lipid bilayers. Overlay of the experimental (solid line) and calculated (dotted line) sensorgrams using a two-state model (A and C) or a 1 : 1 Langmuir model (B and D). Lower plot, 5 l M; upper plot, 20 l M. Results are representative of two independent experiments. Table 4. Kinetic interaction of the peptides with PamOlePtd- Cho ⁄ PamOlePtdGro (1 : 1) lipid bilayers. Association (k a1, k a2 ) and dissociation (k d1, k d2 ) kinetic rate constants for the interaction of PFPs and PCPs with PamOlePtdCho ⁄ PamOlePtdGro (1 : 1) were determined by numerical integration using a two-state reaction model. The affinity constant (K) was determined as (k a1 ⁄ k d1 ) (k a2 ⁄ k d2 ). Peptide k a1 (1 ⁄ Ms) k d1 (1 ⁄ s) k a2 (1 ⁄ s) k d2 (1 ⁄ s) K (1 ⁄ M) M25 601 1.41 · 10 )2 3.49 · 10 )2 1.64 · 10 )6 9.07 · 10 8 M25P 4347 5.92 · 10 )2 2.03 · 10 )2 3.17 · 10 )3 4.70 · 10 5 M21 598 1.33 · 10 )2 2.23 · 10 )2 2.55 · 10 )6 3.93 · 10 8 M21P 4822 5.76 · 10 )2 1.56 · 10 )2 3.80 · 10 )3 3.44 · 10 5 M17 634 1.02 · 10 )2 2.95 · 10 )2 1.96 · 10 )6 9.35 · 10 8 M17P 4080 5.92 · 10 )2 1.08 · 10 )2 2.65 · 10 )3 2.81 · 10 5 Central proline in amphipathic a-helix S T. Yang et al. 4048 FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS bial action. CD spectra of the model amphipathic pep- tides revealed that, in buffer, the central Pro residue effectively disrupts the a-helical structure, but, in mem- brane-mimetic environments, the Pro kept a-helical structures, which means that Pro does not always behave as a strong helix breaker in certain surround- ings including membrane-mimetic environments. These findings with amphipathic a-helical peptides agree with those reported by Li et al. [50] for model transmem- brane helical peptides. The CD and Trp fluorescence spectra of PFPs were very sensitive to the salt concentration. In buffer, PFPs are thought to take on an a-helical structure because of self-association. An increase in ionic strength seems to lead to a decrease in the electrostatic repulsive for- ces between the positively charged residues because of the presence of counterions. In contrast, despite the reduced electrostatic repulsion in the presence of a high salt concentration, PCPs had unordered struc- tures. As suggested by Sansom & Weinstein [51], this is presumably due to structural dynamics such as twist- ing and kinking induced by a central Pro residue. The aggregation of PFPs in buffer correlates with the ability of the peptides to cause the lysis of human red blood cells and zwitterionic liposomes. In contrast, the self association of PFPs appears to interfere with their ability to cross the peptidoglycan layer and reach the cytoplasmic membrane. Therefore, PFPs are likely to show less potent membrane depolarization and greatly reduced antimicrobial activity. Despite the cytotoxicity of PFPs, however, it appears that the structural stability and oligomeric form of PFPs in the presence of a high NaCl concentration could be useful for treating cystic fibrosis patients if their anti- microbial versus hemolytic activity is optimized. Kinetic analysis of the sensorgram results suggests that the binding of M17P to the lipid bilayer occurs by a distinct two-step process: the peptides may first bind to the lipid head groups via electrostatic interaction and then insert further into the hydrophobic interior of the membrane via hydrophobic interactions. The largest differences between M17 and M17P were increases in the rate of association (k a1 ; Table 4) in the first step and dissociation (k d2 , Table 4) in the second step. These findings indicate, respectively, that the central Pro of a-helical peptides is important for fast electrostatic interaction with PtdCho ⁄ PtdGro mem- branes and that the Pro is important for effective translocation across the membrane. In addition, the values of k a1 ⁄ k d1 (K 1 ; initial binding) and k a2 ⁄ k d2 (K 2 ; insertion) correspond, respectively, to the affinity con- stants for electrostatic and hydrophobic interaction of the peptides with lipid bilayers. As observed in other amphipathic a-helical peptides such as magainin [52,53], the initial binding of M17P (K 1 ¼ 6.8 · 10 4 m )1 ) was much faster than the following insertion step (K 2 ¼ 4.0). This suggested that the elec- trostatic interaction is a crucial factor for M17P and is responsible for its selective cytotoxicity. In contrast, for M17, the rate of the first step (K 1 ¼ 6.2 · 10 4 m )1 ) was similar to that of the second (K 2 ¼ 1.5 · 10 4 ). The fact that the K 2 value for M17 is much higher than that for M17P indicates that the affinity of the pep- tides for membranes is driven predominantly by hydro- phobic interactions. This may explain the nonselective interaction of M17 with both zwitterionic and negat- ively charged membranes. Clarifying the structural aspects of the peptides that confer selective binding to negatively charged lipid membranes and identification of the driving forces for membrane partitioning are essential for understanding the mechanism of permeabilization and improving antimicrobial selectivity. The interaction of PCPs with negatively charged membranes is thought to confer selective antimicrobial function, but the induction of plasma membrane leakage alone may not be sufficient to explain the action of these peptides. Our results indicate that Pro residues of amphipathic a-helical peptides may promote formation of a bent structure by inducing the formation of a helix turn in mem- brane-mimetic environments. The bending of PCPs is presumably to provide a membrane anchor after their initial interaction with the membrane surface. The overall amphipathic helix of PCPs lies approximately parallel to the bilayer plane, so the bending potential may be the driving force for penetration of the N-ter- minus or C-terminus of the peptides into the core of the bilayer. In other words, partial conformational flexibility may be a prerequisite for import of the pep- tides into membranes or the cytosol. For example, a single Pro residue has been found to be a key struc- tural factor for the penetration of cells by buforin II [20]. Also, the Pro residue is thought to promote trans- location across lipid bilayers [21]. Many signal peptides also contain a helix-breaking residue and adopt a dynamic helix–break–helix conformation, and this structural motif is thought to be important for the effi- cient initiation of translocation [54–56]. In addition, all enveloped viruses enter cells by peptide-mediated mem- brane fusion. The viral fusion peptides involved in this process interact with and destabilize the target mem- brane. A common feature of many internal viral fusion peptides is the presence of a Pro near the center of their sequence, and it is known that the central Pro residue in fusion peptides is important for the forma- tion of their native structure as well as for the S T. Yang et al. Central proline in amphipathic a-helix FEBS Journal 273 (2006) 4040–4054 ª 2006 The Authors Journal compilation ª 2006 FEBS 4049 [...]... Diversity of antimicrobial peptides and their mechanisms of action Biochim Biophys Acta 1462, 11–28 8 Tossi A, Sandri L & Giangaspero A (2000) Amphipathic, alpha-helical antimicrobial peptides Biopolymers 55, 4–30 9 Hancock RE & Scott MG (2000) The role of antimicrobial peptides in animal defenses Proc Natl Acad Sci USA 97, 8856–8861 10 Huang HW (2000) Action of antimicrobial peptides: two-state model. .. Determination of antimicrobial activity The antimicrobial activity of peptides against a range of micro-organisms was determined by broth microdilution assay Briefly, a single colony of bacteria was inoculated into culture medium (Luria–Bertani broth) and cultured overnight at 37 °C An aliquot of this culture was transferred to 10 mL fresh culture medium and incubated for an additional 3–5 h at 37 °C to. .. tendency of PFPs to self-associate in buffer correlated with their cytotoxicity to human red blood cells and their ability to lyse artificial zwitterionic liposomes In addition, biosensor technology indicated that the interaction of PCPs with membranes was predominantly in uenced by initial electrostatic interactions, whereas the interaction of PFPs with membranes was most affected by hydrophobic interactions... higher bending potential, their ability to cause membrane disruption, and the existence of an intracellular target for the peptides In summary, we have demonstrated that a central Pro in amphipathic a- helical peptides effectively disrupts their a- helical structures and aggregation in buffer but that they maintain a- helical structures in membrane-mimetic environments despite somewhat reduced a- helical contents... II Proc Natl Acad Sci USA 97, 8245–8250 21 Kobayashi S, Takeshima K, Park CB, Kim SC & Matsuzaki K (2000) Interactions of the novel antimicrobial peptide buforin 2 with lipid bilayers: proline as a translocation promoting factor Biochemistry 39, 8648–8654 22 Shai Y (1999) Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell... fitting using numerical integration analysis [64] The sensorgram data obtained at five different concentrations were simultaneously fitted using BIA evaluation software (version 3.2) Because a poor fit was obtained with the simple 1 : 1 binding model, the association and dissociation rate constants were determined using the two-state reaction model For peptide–lipid interaction, this may correspond to: ka1... Calceincontaining vesicles were separated from free calcein by gel filtration chromatography in Tris ⁄ HCl buffer using a Sephadex G-50 column (Pharmacia, Uppsala, Sweden) The concentration of lipid vesicles used in the various assays is Central proline in amphipathic a- helix the lipid concentration initially used for large and small unilamellar vesicle preparation Measurement of peptide-induced dye leakage... 867–873 Lau SY, Taneja AK & Hodges RS (1984) Synthesis of a model protein of defined secondary and quaternary structure Effect of chain length on the stabilization and formation of two-stranded alpha-helical coiled-coils J Biol Chem 259, 13253–13261 Zelezetsky I, Pacor S, Pag U, Papo N, Shai Y, Sahl HG & Tossi A (2005) Controlled alteration of the shape and conformational stability of alpha-helical cell-lytic.. .Central proline in amphipathic a- helix S.-T Yang et al membrane interactions that lead to fusion [57,58] Furthermore, Niidome et al [59] reported that a peptide in which in the central double Pro was replaced with double Ala was less able to promote membrane fusion and was more lytic Therefore, it is plausible that the improved bactericidal activity of PCPs is due to the promotion of translocation... systematically varied hydrophobic-hydrophilic balance and their interaction with lipid- and bio-membranes Biochemistry 35, 13196–13204 Blondelle SE & Houghten RA (1992) Design of model amphipathic peptides having potent antimicrobial activities Biochemistry 31, 12688–12694 Papo N, Oren Z, Pag U, Sahl HG & Shai Y (2002) The consequence of sequence alteration of an amphipathic alpha-helical antimicrobial . Contribution of a central proline in model amphipathic a- helical peptides to self-association, interaction with phospholipids, and antimicrobial mode of. phospholipids, we generated a ser- ies of model amphipathic a- helical peptides with different chain lengths and containing or lacking a single central Pro. CD studies