- Email: [email protected]
Chain length dependence of antimicrobial peptide–fatty acid conjugate activity
Journal of Colloid and Interface Science 345 (2010) 160–167
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Chain length dependence of antimicrobial peptide–fatty acid conjugate activity Alexander F. Chu-Kung a, Rose Nguyen a, Kristen N. Bozzelli a, Matthew Tirrell a,b,* a b
Department of Chemical Engineering, University of California, Santa Barbara, CA 93106, United States Department of Bioengineering, University of California, Berkley, CA 94720, United States
a r t i c l e
i n f o
Article history: Received 28 September 2009 Accepted 21 November 2009 Available online 2 December 2009 Keywords: Antimicrobial peptide Fatty acid Conjugation
a b s t r a c t The rise of resistant bacteria has prompted the search for new antimicrobial agents. Antimicrobial membrane lytic peptides have potential as future microbial agents due to their novel mode of action. Recently conjugation of a fatty acid to antimicrobial peptides has been explored as a method to modulate the activity and selectivity of the peptide. Our work further explores these phenomena by testing two peptides, YGAAKKAAKAAKKAAKAA (AKK) and LKKLLKLLKLLKL (LKK), conjugated to fatty acids of varying length for their activity, structure, solution assembly properties and the ability to bind model membranes. We found that increasing the length of fatty acids conjugated to peptide AKK, up to a 16 carbons in length, increases the antimicrobial activity. Peptide AKK appears to lose activity when the minimal active concentration is higher than the critical miscelle concentration (CMC) of the molecule. Thus, if the CMC of the peptide conjugate is too low the activity is lost. Peptide LKK has no activity when conjugated to lauric acid and appears to aggregate at very low concentrations. Conjugation of AKK with a fatty acid increases its affinity to model supported lipid membranes. It appears that the increased hydrophobic interaction imparted by the fatty acid increases the affinity of the peptide to the surface thus increasing its activity. At concentrations above the CMC, solution self-assembly inhibits binding of the peptide to cell membranes. Ó 2010 Published by Elsevier Inc.
1. Introduction Antimicrobial peptides are naturally occurring molecules that provide the first line of defense against infection in many animals. In multi-cellular organisms antimicrobial peptides are secreted constantly at low levels, often on the skin or mucus membranes [1]. Over eight hundred antimicrobial peptides have been isolated and a listing has been compiled at http://www.bbcm.univ.trieste.it/~tossi/amsdb.html. Many of these peptides have been shown to have broad spectrum antibiotic and antifungal activity as well as anti-viral and anti-tumor activity [2,3]. Unlike many antibiotics currently on the market antimicrobial peptides function by a non-receptor mediated mechanism. There is evidence that antimicrobial peptides target the lipid membrane of cells and exploit the fundamental differences between eukaryotic and prokaryotic cell membranes [4–6]. These differences provide the basis for selectivity of antimicrobial peptides. It has been suggested that for bacteria to develop resistance to antimicrobial peptides, major changes to their membrane composition would be necessary [1]. Due to the growing problem of bacterial resistance to currently available antibiotics, antimicrobial peptides are being studied as a possible alternative. * Corresponding author. Address: Department of Bioengineering, University of California, Berkley, CA 94720, United States. E-mail address: [email protected] (M. Tirrell). 0021-9797/$ - see front matter Ó 2010 Published by Elsevier Inc. doi:10.1016/j.jcis.2009.11.057
Despite their diversity, antimicrobial peptides tend to share some common characteristics. Antimicrobial peptides tend to be capable of adopting an amphipathic structure, a structure having distinct hydrophobic and hydrophilic regions, allowing the peptide to bind and insert into the hydrophobic core of the cell membrane. [7–10]. The hydrophobic forces contribute to nonselective binding since the peptide cannot differentiate eukaryotic and prokaryotic cell membranes on the basis of hydrophobicity. Highly hydrophobic peptides have been shown to be highly toxic to eukaryotic cells as well as prokaryotic cells [11,12]. Most antimicrobial peptides are composed of a disproportionately high number of cationic amino acids as opposed to anionic amino acids, resulting in a net positive charge on the peptide [1,8]. Positive peptide charge is important for the binding of peptides to anionic lipids in the bacterial cell membrane. Reduction of positive charge on the peptide has been shown to reduce the activity and selectivity of antimicrobial peptides [13,14]. Recently, researchers have found that addition of a fatty acid tail to antimicrobial peptides (Fig. 1) can modify both the activity and selectivity of the peptide [15–21]. In earlier work from our lab we determined that conjugation of a peptide to a fatty acid can increase the antimicrobial activity of amphipathic peptides [17,20]. We also showed that conjugation of a naturally derived peptide containing highly hydrophobic amino acids, SC-4, shows increased activity and reduced selectivity [17]. From our studies we hypothesized that fatty acid conjugation enhances activity by
A.F. Chu-Kung et al. / Journal of Colloid and Interface Science 345 (2010) 160–167
Fig. 1. Chemical structure of a fatty acid conjugated peptide. The first two amino acids of the peptide are shown with side chains R1 and R2.
161
35 is a lactose permease-deficient strain and was obtained from Robert Hancock at the University of British Columbia. E. coli DH5a, a common lab strain used for transformations and was provided by the biology laboratory at University of California Santa Barbara. S. epidermidis (ATCC#12228) were used for antimicrobial activity assays. 2.2. Peptide amphiphile synthesis
increasing the hydrophobic interaction between the cell membrane and peptide. Highly hydrophobic peptides become toxic to eukaryotic cells because the increased hydrophobicity causes nonselective binding to any cell membrane. Weakly hydrophobic peptides appear to bind both eukaryotic and prokaryotic cell membranes but are unable to fold into their active conformations upon binding, possibly due to the strong electrostatic repulsions on the peptide. Studies on the antimicrobial peptide Lactoferrin show that a twelve carbon fatty acid is the optimal length of tail to improve activity [19]. Studies on peptide SC-4 demonstrate that increasing the fatty acid length from 12 carbons to 18 carbons can reduce activity against some strains of bacteria [20]. Our current research tries to further understand the effect of fatty acid conjugation on antimicrobial peptide activity, including the origin of the reduction in activity above a critical tail length. In order to do so, we conjugated our previously studied peptide, YGAAKKAAKAAKKAAKAA (AKK) to fatty acids ranging from 12 to 20 carbon units in length. In order to study the effect of peptide hydrophobicity we used a peptide with the same sequence motif as AKK but incorporating the more hydrophobic leucine residues in place of the alanine residues, LKKLLKLLKKLLKL (LKK). Peptide LKK has previously been studied and is known to have antimicrobial activity before conjugation to a fatty acid. Both peptides are capable of forming amphipathic helices (Fig. 2). We tested these peptides and their fatty acid conjugates for antimicrobial activity, structure, aggregation state, and binding affinity to model membranes in order to determine the effect of fatty acid conjugation on antimicrobial peptide activity. 2. Experimental 2.1. Materials AKK and LKK peptides were synthesized by Synpep Corporation. 1-hydroxybenzotriazole (HOBt), 2-(1H-benzotriazole-1yl)1,1,3,3tetramethyluronium hexafluorophosphate (HBTU) were purchased from Nova Biochem. All phospholipids were purchased from Avanti Polar Lipids. 111 Silicon wafers (native oxide layer) were purchased from MEMC Electronics. All other chemicals and reagents were purchased from Sigma–Aldrich. Bacteria strains E. coli ML-
Peptide was received from the Synpep Corporation bound to resin, with side chains completely protected. Peptides were characterized by Synpep Corporation and accepted with a certificate of authenticity. The N-terminal Fmoc protecting group of resin-bound peptide was removed with 20 vol.% piperidine in dimethylformamide (DMF) for 2 h, followed by three washes with DMF. The peptide was then coupled to either lauric acid (C12), myristic acid (C14), palmitic acid (C16), 2-hexyldecanoic acid (bC16), stearic acid (C18), or arachidic acid (C20) using a four-fold molar excess of HBTU, HOBt, N,N-diisopropylethylamine, and the appropriate fatty acid. The peptide amphiphile was cleaved, purified, and characterized according to the method below. 2.3. Peptide cleavage and purification All peptides were deprotected and cleaved using a mixture of 95/5 vol.% trifluoroacetic acid (TFA)/water solution. The peptides were then precipitated in cold methyl-tert-butyl-ether. Peptides were purified by reverse phase high performance liquid chromatography on a Vydac C4 column using 99.9/0.1 vol.% water/TFA and 99.9/0.1 vol.% acetonitrile/TFA as the mobile phases. All samples were lyophilized and analyzed for molecular mass using a Thermo Bioanalysis matrix-assisted laser desorption/ionization time of flight system (MALDI-TOF). All peptides eluted as a single peak and were 100% pure within the detection limit of MALDITOF analysis. 2.4. Minimum bactericidal concentration (MBC) determination Gram-negative E. coli (strains DH5a or ML-35) or Gram-positive S. epidermidis (ATCC#12228) test bacteria were grown in overnight cultures in tryptic soy broth (TSB) at 37 °C for at least 18 h. The overnight cultures were subcultured and grown to log phase (OD600 of 0.5), then diluted to a concentration of 1 105–1 106 colony forming units (CFU)/mL. Stock solutions of test peptides were prepared at concentrations of 1 mg/mL in Millipore water. Stock peptide solutions were added to polypropylene tubes and diluted with TSB to the desired test concentration and allowed at least 30 min to reach equilibrium. Sample tubes were inoculated with a bacterial stock solution to a final bacteria concentration between 1 104 and 1 105 CFU/mL and then incubated with rotation for 18 h at 37 °C. The following day 50 lL aliquots were plated out and grown overnight. The lowest concentration where no colonies grew was taken as the MBC. 2.5. Vesicle preparation
Fig. 2. Helical wheel diagrams for YGAAKKAAKAAKKAAKAA (left) LKKLLKLLKKLLKL (right) the dark filled circles show the hydrophilic cationic lysine residues.
Powdered lipids, either 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or 70/30 wt.% 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE)/1,2-dilauroyl-sn-glycero-3-[phosphor-rac(1-glycerol)] (DLPG), were dissolved in chloroform then dried under nitrogen to form a thin film. The film was then further dried under vacuum for a minimum of 12 h. The lipids were hydrated in 10 mM sodium phosphate buffer, pH 7.4, to a concentration of 2.4 mM, then extruded five times through a 100 nm Millipore polycarbonate filter. The vesicle size was confirmed using light scattering.
162
A.F. Chu-Kung et al. / Journal of Colloid and Interface Science 345 (2010) 160–167
2.6. Circular dichroism Circular dichroism (CD) spectra were recorded with a 5 mm quartz cell on an Olis-RSM CD at 37 °C in 10 mM sodium phosphate buffer, pH 7.4. Data was collected from 193 nm to 250 nm at 1 nm intervals. All reported spectra were the result of averaging at least three scans. Circular dichroism measurements of peptides in vesicle solutions were made in 10 mM sodium phosphate buffer, pH 7.4, containing vesicles composed of either DLPC or DLPE/DLPG lipids at a concentration of 0.3 mM. There are a number of different methods to calculate peptide structure from CD spectra [22,23]. We selected a least squares fit method that was performed using a linear combination of standard a-helix and random coil spectra to estimate secondary structure contribution [24]. All fits were done utilizing the 195–250 nm region of the CD spectra since the noise below 195 nm was large. 2.7. Aggregation assays Two methods were utilized to determine the critical micelle concentration of each peptide-fatty acid conjugate. The first method, pyrene solubilization, was used to determine the aggregation state of the peptide amphiphiles [25]. Pyrene is a polycyclic, aromatic hydrocarbon with low solubility in aqueous solutions. Pyrene can be solubilized within the hydrophobic core of micelles, providing a method to detect the presence of micelles. Test peptide was dissolved in 10 mM sodium phosphate buffer, pH 7.4, containing 100 mM NaCl and added to an excess of pyrene at various concentrations. The suspension was then sonicated for a minimum of 8 h and then centrifuged. The supernatant was added to a 50 mM solution of sodium dodecyl sulfate and the absorbance at 336 nm was recorded. Tensiometry was also used to assess the critical micelle concentration (CMC) of our peptide–fatty acid conjugates. The surface tension at the air–water interface is highly dependent on concentration of surfactants in solution. An increase in surfactant concentration at concentrations below the CMC results in decreasing surface tension. Above the CMC, the surface tension is independent of surfactant concentration. We measured surface tension using the Wilhelmy plate method. Peptides for tensiometry were prepared in 10 mM sodium phosphate, 100 mM sodium chloride solution, pH 7.4, at a concentration of 1 mg/ml for peptide C14-AKK, C16-AKK, C18-AKK and C20-AKK. Peptide C12-LKK was prepared in water at a concentration 0.5 mg/ml to avoid precipitation. The peptide solution was then added to 15 ml of buffer solution and allowed to equilibrate before the surface tension was measured. 2.8. Preparation of supported lipid layers Large silicon wafers were cut into small (1 1 cm) squares and cleaned according to the method in the Supplementary data. After cleaning the silicon wafers were functionalized with a hydrophobic monolayer of octadecyltrichlorosilane (OTS) using a method adapted from Ge et al. [26]. 40 lL of a 2 103 M solution of OTS in dry benzene is added dropwise to a water subphase on the Langmuir Blodgett trough at room temperature. The benzene is allowed to evaporate for 1 min and then the layer was compressed to a pressure of 45 mN/m. The layer was allowed to equilibrate for 30 min before the silicon surface was drawn upward through the OTS layer at a speed of 1 mm/min. The layer was allowed to dry under a nitrogen atmosphere. A lipid monolayer was then deposited on the OTS–Si surface using the Langmuir–Blodgett technique. Either 1,2-distearoyl-snglycero-3-phosphocholine (DSPC) or 70/30 wt.% DSPC/1,2-distearoyl-sn-glycero-3-[phosphor-rac-(1-glycerol)] (DSPG) was dissolved in chloroform to a concentration of 1 mg/ml. 40 lL of this
solution were added dropwise to a clean aqueous subphase and the chloroform is allowed 15 min to evaporate. The lipid monolayer was compressed to a surface pressure of 50 mN/m and held for 30 min. The OTS functionalized silicon surfaces are dipped down through the interface at a rate of 1 mm/min. After the dip down the surfaces were stored underwater. 2.9. Peptide binding kinetics A solution of 30–50 ml is prepared in buffer at the desired peptide concentration at least 30 min prior to use. The rate of peptide deposition is measured using a Beaglehole picometer ellipsometer. 20 ml of peptide solution is injected into a sample cell containing a lipid functionalized surface at a rate of 100 ml/min. The remaining peptide solution is recirculated at a rate of 5 ml/min to avoid peptide depletion and diffusion effects. We tested our system at higher flow rates, 10–20 ml/min, and with a larger reservoir, 100 ml, to ensure that neither peptide depletion nor diffusion was influencing our result. 3. Results We used two peptides for this study. YGAAKKAAKAAKKAAKAA (AKK) is a cationic peptide capable of adopting a helical structure. Previous studies on peptide AKK show that lauric acid conjugation to AKK greatly increases its antimicrobial activity [17]. The second peptide, LKKLLKLLKKLLKL (LKK), has the same hydrophobic/hydrophilic sequence motif as AKK, but is shorter and replaces the weakly hydrophobic alanine residues with highly hydrophobic leucine residues. LKK was previously studied and was shown to have antimicrobial activity both in solution and bound to a polymer resin [27]. We conjugated lauric acid to peptide LKK and conjugated fatty acids of varying length to peptide AKK to study their antibacterial properties. Initially we tested our peptides using the MBC test against Gram-negative bacteria E. coli DH5a and ML-35 and Gram positive bacteria S. epidermidis (Table 1). Previous studies on peptide AKK indicate that conjugation to lauric acid increases the antimicrobial activity of the peptide. Conjugation of C14 (myristric acid) to peptide AKK further improves its activity against all the test strains. Conjugation of C16 to AKK improves the peptide activity against the two Gram-negative bacteria strains, however C16AKK loses activity against the Gram positive strain. C18-AKK has slightly lower activity against the two E. coli strains, and also shows no activity against the S. epidermidis. C20-AKK has no measured activity against any strain of bacteria by the MBC test. To determine the effect of branching of peptide antimicrobial activity bC16-AKK was synthesized and tested. It was shown that bC16AKK has slightly lower MBC than C16-AKK against Gram-negative bacteria. Unlike C16-AKK, bC16-AKK shows measurable activity against S. epidermidis. Peptide LKK is known to have some degree of antimicrobial activity before fatty acid conjugation [27] which we confirm using the MBC test. However, lauric acid conjugated LKK loses its activity against all bacterial strains. Two methods, tensiometry and pyrene absorption, were used to measure the CMC of the fatty acid conjugated peptides (Table 1). Because of the low value of the CMC, tensiometry was not effective with C18-AKK and C20-AKK. The pyrene method was not employed with C12-AKK because of the high amount of peptide required. The CMC values as determined by tensiomtery were about 20% higher than those determined by the pyrene method. As expected, the CMC for the Cx-AKK series of peptide amphiphiles shows a logarithmic, decreasing dependence on chain length. bC16-AKK shows a CMC roughly twice that of C16-AKK. There was no detectable CMC using either tensiometry or the pyrene
163
A.F. Chu-Kung et al. / Journal of Colloid and Interface Science 345 (2010) 160–167
Table 1 Properties of tested peptides and peptide conjugates. The first three columns show the peptide activity against various bacteria. The second set of three columns shows the helical content of each peptide in various environments. The final two columns show the CMC of the peptides as measured by either tensiometry or pyrene adsorption. MBC (lM) ± 2.5 lM
AKK C12-AKK C14-AKK C16-AKK bC16-AKK C18-AKK C20-AKK LKK C12-LKK
CMC (lM) ± 10%
Helical content (% Helicity)
E. Coli DH5a
E. Coli ML-35
Staph Ep
Buffer
DLPC
DLPG/DLPE
Tensiometry
Pyrene
>65 11 4 2 6 3 65 7 >65
>65 11 5 4 11 5 >65 10 >65
>65 29 26 >65 24 >65 >65 24 >65
12 17 14 19 8.5 16 44 60 –
5 0 8 12 10 14 24 – –
26 73 74 79 61 81 51 – –
– 790 168 25 – – – – –
– – 130 20 28 8 1 – –
absorption method at low concentrations ([25 lM) for C12-LKK At 25 lM C12-LKK precipitated from solution. It is likely that the critical association concentration for C12-LKK is too low to measure using the methods we employed. Circular dichroism was used to determine the structure of peptides and fatty acid conjugated peptides under various conditions. The structure of peptides were measured at a peptide concentration of 20 lM in phosphate buffer, or phosphate buffer solutions containing either 0.3 mM eukaryotic membrane mimicking DLPC vesicles, or 0.3 mM prokaryotic membrane mimicking DLPE/DLPG vesicle. The resulting spectra were then fit to determine the amount of helicity (Table 1). Peptides C14-AKK, C16-AKK, and C18-AKK behaved in a similar fashion to previously studied C12AKK (Fig. 3) [17]. All these conjugated peptides show very little (<20%) helical structure in solution. In the presence of DLPC vesicles, there was a slight decrease in helical structure. In the presence of vesicles composed of DLPE/DLPG, all the peptide conjugates from C12 to C18 adopt a helical conformation, with >70% helicity. C20-AKK has markedly different behavior. In buffer, C20-AKK adopts a partially helical structure. While in the presence of vesicles containing DLPC, C20-AKK, like the other AKK peptides, adopts a more random coil conformation. However, in the presences of DLPE/DLPG vesicles C20-AKK does not show the significant increase in helicity demonstrated by other AKK peptide conjugates. As expected, bC16-AKK behaves in a similar fashion to C16-AKK. It should be noted that the Cx-AKK peptide series shows an isodichroic point at 208 nm. This is indicative of a two state equilibrium between the helical and disordered states [28]. To study if self-assembly influences the solution peptide secondary structure, we also studied C16-AKK and C18-AKK in phosphate buffer at concentrations above and below the CMC. C16-AKK was studied at concentrations of 10, 20 and 40 lM and the fit of the CD showed 16%, 19% and 19% helical content respectively. C18AKK was studied at 5, 8, and 20 lM and the resulting fit showed 19%, 18% and 16% helical content. The CD spectra at the varying concentrations are nearly identical and thus not shown. Surprisingly neither C16-AKK nor C18-AKK shows significant differences in their structure above and below the CMC. LKK and C12-LKK have interesting behavior when investigated using CD (Fig. 4). Both C12-LKK and LKK precipitated in the presence of DLPE/DLPG vesicles. LKK in buffer adopts a partially helical conformation. In the presence of DLPC vesicles, LKK shows a marked change in structure. The spectrum shows two minima which resembles a helix. However, the resulting spectrum is not an exact match to a classical a-helix. In plain buffer, however C12-LKK shows a spectrum that is neither random coil, a-helix, nor b-sheet and also distinctly different than the LKK spectra in either buffer or with DLPC vesicles. C12-LKK adopts a similar structure as LKK in the presence of DLPC vesicles. Regardless of the exact structure of LKK and C12-LKK in the presence of DLPC vesicles,
the peptides have a distinct change in conformation from the buffer solution, and the peptides adopt a well ordered confirmation. To determine the extent of peptide binding to model lipid membranes we tested peptides AKK, C14-AKK and C16-AKK for their ability to bind a hybrid bilayer (an OTS monolayer covered by a lipid monolayer) surface. We used ellipsometry to monitor the extent of peptide binding to the surface. Fig. 5a shows the concentration dependence of C14-AKK adsorption to DSPC/DSPG and DSPC model membranes. Fig. 5b shows the concentration dependence of AKK adsorption to DSPC/DSPG and DSPC model membranes. Although C14-AKK shows a greater affinity for model membranes than AKK, there are a number of similarities in the behavior of the peptides. With both AKK and C14-AKK we see a fast rise in the signal followed by a transition to a slower rise in the signal. In addition, AKK and C14-AKK show concentration dependence in both rate and extent of binding. Both peptides preferentially bind layers containing DSPG. Fig. 6 shows the adsorption of C16AKK to DSPC/DSPG model membranes. At low concentrations, below the CMC, the peptide amphiphile behaves like AKK and C14AKK. At concentrations above the CMC the peptide rapidly adsorbs to the membrane then ellipsometry signal decreases slowly.
4. Discussion Our previous work [17], along with the work of several other groups [16,18,19,21,29], demonstrates that conjugation of antimicrobial peptides with fatty acids can enhance their membrane lytic activity. We believe the increase in activity is due to the increased hydrophobic attraction to membranes caused by the addition of the fatty acid tail. A longer tail should therefore result in further increased in antimicrobial activity. We tested C14-AKK and determined, as expected, the activity is higher than both C12-AKK and AKK. C16-AKK, when tested against both E. coli strains, shows higher activity than C14-AKK, but surprisingly has no activity against the S. epidermidis strain. C18-AKK shows a slight decrease in activity against the two E. coil strains and no activity against S. epidermidis. C20-AKK shows no activity against any tested bacteria. Comparison of the CMC and MBC of the Cx-AKK peptide series shows loss of activity occurring close to where the MBC will be above the CMC (Fig. 7). We believe the loss in activity occurs due to the self-assembly of the fatty acid conjugated peptides into micelles in solution. C18-AKK shows decreased activity compared to C16-AKK even though the MBC is lower than the CMC. We believe that this is because the CMC of the peptide amphiphile in the growth media may be lower than the CMC measured in the buffer solution. Studies have shown that branched surfactants have a higher CMC than their linear counterparts, possibly due to steric effects [30,31]. ndeed we see the CMC of bC16-AKK is higher than that of C16-AKK. bC16-AKK has activity against both E. coli strains
164
A.F. Chu-Kung et al. / Journal of Colloid and Interface Science 345 (2010) 160–167
Fig. 3. CD spectra of peptides AKK and its fatty acid conjugates C12-AKK, C14-AKK, C16-AKK, C18-AKK and C20-AKK. The symbols on the graph corresponds to CD in sodium phosphate buffer (4) with the addition of DLPC vesicles (h), or DLPE/DLPG vesicles (s).
Fig. 4. CD spectra of peptide LKK and its fatty acid conjugate C12-LKK. The symbols on the graph corresponds to CD in sodium phosphate buffer (4) and with the addition of DLPC vesicles (h). Both samples precipitated in the presence of DLPE/DLPG vesicles.
and S. epidermidis, further suggesting that lack of aggregation or self-assembly in solution prevents inhibition of the activity of antimicrobial peptides. It is known that micellization of peptides conjugated with fatty acid tails can act to stabilize secondary structure of the peptide portion and promote biological activity [32,33]. However, we ob-
served a decrease in activity in some peptide–fatty acid conjugates. We believe that the loss of activity may have been due to the peptides folding together in the micelles, resulting in stable peptidepeptide complexes. These complexes would have a lower affinity for a membrane than an unfolded peptide monomer. The structure of both C16-AKK and C18-AKK was examined by CD at concentra-
A.F. Chu-Kung et al. / Journal of Colloid and Interface Science 345 (2010) 160–167
165
Fig. 5. Adsorption kinetics of C14-AKK (left) and AKK (right) onto model membrane surfaces of either DSPC/DSPG or DSPC. The curves on the left plot correspond to C14-AKK solution concentrations of (from top to bottom) 33 lM onto DSPC/DSPG surfaces, 15 lM onto DSPC/DSPG, 7 lM onto DSPC/DSPG, 60 lM onto DSPC, 40 lM onto DSPC and 15 lM onto DSPC. The curves on the right plot correspond AKK solution concentrations of (from top to bottom) 90 lM onto DSPC/DSPG, 60 lM onto DSPC/DSPG, 30 lM onto DSPC/DSPG and 30 lM onto DSPC.
Fig. 6. Adsorption kinetics of C16-AKK onto DSPC/DPSG at various concentrations. The curves (from top to bottom) correspond to C16-AKK solution concentrations of 60 lM, 50 lM and 15 lM.
Fig. 7. Shows the MBC and CMC of the AKK series of peptides as a function of tail length. The dotted line is the CMC of the peptide. The solid lines show the MBC of the peptide against E. coli DH5a (diamonds), E. coli ML-35 (squares) and S. epidermidis (diamonds).
tions above and below their CMC. No helical content was seen in either peptide. All the peptides were studied at 20 lM, in the presence of vesicles composed of DLPE/DLPG lipids. Peptides C12-AKK, C14-AKK, C16-AKK and C18-AKK show similar structural behavior. Under these conditions, peptides C12-AKK, C14-AKK are well below their CMC, C16-AKK is very close to its CMC, and C18-AKK is above its CMC. This suggests that formation of micelles does not prevent the eventual binding of DLPE/DLPG membranes to some degree. However, micellization may affect both the rate of membrane binding and the final absorbed amount. C20-AKK shows a significant amount of helical structure in solution. Our studies with C16-AKK and C18-AKK suggest that micellization does not stabilize helical structures in solution. For magainin conjugated to a long fatty acid, it has been suggested that helical secondary structure is induced in monomeric helices due to interaction between the amphipathic helix face and the fatty acid chain. The amphipathic helix can then bend to press its amphiphathic faces against each other, with the fatty acid chain in between the faces [15]. C20-AKK shows a reduced amount of structure in the presence of DLPC vesicles indicating that the peptide binds DLPC vesicles but does so in an unstructured fashion. Surprisingly C20-AKK does not show a significant increase in helical structure in the presence of DLPE/DLPG membranes. It is possible that the C20-AKK binds the membrane through electrostatic interactions but never forms a helix with its hydrophobic face imbedded into the membrane. This would explain the lack of antimicrobial activity of C20-AKK. All of the fatty acid conjugates of AKK show a decrease in helical content when in the presence of DLPC vesicles. Only C20-AKK shows a greater than 15% helicity. This suggests the peptides bind the DLPC membrane but do not adopt an active conformation. We would expect none of the fatty acid conjugated AKK peptides to have significant hemolytic activity. Studies show a similar palmitic acid conjugated alanine–lysine peptide does not have significant hemolytic activity [16]. LKK, as expected, shows a significant antimicrobial activity before conjugation to a fatty acid. However, conjugation to lauric acid renders peptide LKK inactive. CD of peptide LKK in solution suggests that it adopts a partially helical conformation. C12-LKK has a significantly different structure in solution. It is likely that conjugation of LKK with lauric acid causes the peptides to aggregate. The highly hydrophobic leucine residues will favor an aggregation and folding in order to bury themselves in a hydrophobic environment.
166
A.F. Chu-Kung et al. / Journal of Colloid and Interface Science 345 (2010) 160–167
It is likely aggregation inhibits antimicrobial action in much the same way that micellization does for Cx-AKK. Both LKK and C12LKK show a significant change in structure from buffer solution when in contact with DLPC vesicles suggesting that they may have high hemolytic activity. Studies show a similar leucine–lysine peptide conjugated to palmitic acid is highly hemolytic [16]. From the activity data we conclude that aggregation inhibits the activity of antimicrobial peptides. There is evidence that the aggregation state of antimicrobial peptides plays an important role in bactericidal activity and selectivity. Studies show a magainin analog dimerized though a covalent cystine linkage show a significant increase in both bactericidal activity and hemolytic activity as a result of dimerization [34]. In another study, analogs of the peptide dermaseptin were modified, either through truncation or substitution of amino acids, to control their aggregation state. It was demonstrated that aggregation reduces the bactericidal activity and increases the hemolytic activity [35]. A third study tethered five helical antimicrobial peptides together through their C-terminus to a short peptide linker. The pentamers show increased helical content, bactericidal activity, and hemolytic activity [36]. These studies demonstrate that aggregation and even folding of antimicrobial peptides in an aggregate does not necessarily lead to loss of bactericidal activity. This suggests that the loss of activity of aggregated fatty acid conjugated peptide is due to the lipid tails inhibiting partitioning into the bacterial membrane. There are numerous examples of surfactants that show increasing biological activity up to a critical chain length; above this chain length the compounds are no longer active. This phenomena has been dubbed the biological surfactant cut-off effect and has been documented in anaesthetics [37], immunosuppressives [38], and antimicrobials [39]. Several theories have been developed to explain the cut-off effect. At the turn of the twentieth century, Meyers and Overton independently proposed the cut-off effect was the result of limited solubility of the surfactant molecule in solution [40,41]. As the chain length of the surfactant increases the aqueous solubility decreases. It is believed the partition coefficient between the lipid and aqueous phase increases less rapidly than the solubility decreases [42]. As a result, the maximum possible concentration of surfactant bound to the lipids, determined by the equilibrium between monomer and lipid-bound peptide, is lower than amount required to elicit the desired biological effect. The contribution of the peptides in micelles towards the maximum amount of bound peptide should be small. The partition function between the micellar peptide and membrane bound peptide should be smaller than the monomer-lipid-bound peptide partition function. This is because the peptide amphiphiles are in a hydrophobic environment in the micelle, thus the driving force to bind the surface and minimize hydrophobic-solvent contacts is reduced. Ellipsometry was employed here to directly measure the binding of peptides and antimicrobial peptides to model lipid membranes. Previously surface plasmon resonance (SPR), another spectroscopic technique to monitor adsorption of molecules to surfaces, has been employed to monitor peptide membrane interactions [43,44]. These systems also incorporate a gold surface covered by a hybrid bilayer (an alkane thiol monolayer covered by a lipid monolayer). AKK and C14-AKK show kinetic behaviors that are qualitatively similar to those observed for magainin and mellitin as observed by SPR [43,44]. As expected C14-AKK bound membranes more strongly than AKK because of the increased hydrophobic interaction between the C14-AKK peptide and the membrane. Additionally we saw that both C14-AKK and AKK bound DSPC/DSPG lipids more strongly than DSPC lipids alone. A close look shows that the peptides, especially C14-AKK, do not follow simple Langmuir adsorption kinetics. There seems
to be a rapid association phase followed by a slower association phase. Data from magainin and mellitin were also found to fit the kinetics of a two state reaction model [43,44]. It was proposed that the two phases correspond to an initial binding of the membrane and the subsequent deeper insertion into the membrane [45]. At concentrations below the CMC C16-AKK shows similar behavior to C14-AKK and AKK, a rapid adsorption phase followed by a slower adsorption phase. At concentrations above the CMC, C16-AKK shows an ‘‘overshoot” then a decrease in the signal. The behavior of C16-AKK above the CMC may give some insight into the cut-off effect observed at high chain lengths. A possible explanation for the overshoot at concentrations above the CMC could be the binding of intact micelles to the surface blocking monomeric peptide from binding. The decrease in signal in signal overtime may correspond to micelles desorbing and being replaced by monomeric peptide. This supports the Meyers and Overton theory of limited solubility [40,41]. Although there are not enough data currently to perform a detailed kinetic analysis, our findings give some insight into the behavior of fatty acid conjugated peptides binding membranes. As we predicted, the conjugation of a fatty acid to a peptides causes to bind membranes more strongly. Ellipsometry and liposome leak studies on synthetic antimicrobial peptides of varying lengths suggest that there is a strong correlation between peptide adsorption and induced liposome leakage [46]. These findings would seem to support the hypothesis that fatty acid conjugation increases the propensity for a peptide to bind a phospholipid membrane which in turn increases the peptide’s antimicrobial activity. There is currently a significant amount of interest in developing more active and selective antimicrobial compounds. Antimicrobial peptides have drawn a significant amount of attention due to their non-receptor based mechanism of action. While naturally occurring antimicrobial peptides have some promise as therapeutic agents a number of techniques have been employed to enhance their potential as therapeutic agents. Some examples of these methods include the optimization of peptide sequence [14,47,48], inclusion of fluorous amino acids [49,50], inclusion of b-amino acids [51], as well as fatty acid conjugation. We have studied fatty acid conjugated antimicrobial peptides to better understand the relationship between fatty acid properties and peptide properties on structure and biological function. It appears that conjugation of fatty acids to peptides imparts the behavior and properties of both antimicrobial peptides and simple antimicrobial surfactants to the peptide-fatty acid complex. The complex shows high lipophilicity due to the fatty acid tail, however the maximum effective length of the fatty acid tail is limited by aggregation or self-assembly of the peptide conjugates. The difference between the peptidefatty acid complex and a simple surfactant lies in the requisite folding of the peptide in order to be active. Although it appears the CxAKK peptides will bind DLPC membranes, they do not fold due to the low hydrophobicity and large electrostatic repulsion of the side chains. Keeping these ideas in mind, it should be possible to maximize the activity and therapeutic window of a fatty acid conjugated peptide. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jcis.2009.11.057. References [1] M. Zasloff, Nature 415 (2002) 389. [2] P.M. Hwang, H.J. Vogel, Biochem. Cell Biol. 76 (1998) 235. [3] D.W. Hoskin, A. Ramamoorthy, Biochim. Biophys. Acta 1778 (2008) 357.
A.F. Chu-Kung et al. / Journal of Colloid and Interface Science 345 (2010) 160–167 [4] D. Wade, A. Boman, B. Wahlin, C.M. Drain, D. Andreu, H.G. Boman, R.B. Merrifield, Proc. Natl. Acad. Sci. USA 87 (1990) 4761. [5] M.R. Yeaman, N.Y. Yount, Pharmacol. Rev. 55 (2003) 27. [6] K. Matsuzaki, O. Murase, K. Miyajima, Biochemistry 34 (1995) 12553. [7] Y. Shai, Z. Oren, Peptides 22 (2001) 1629. [8] K.A. Brogden, Nat. Rev. Microbiol. 3 (2005) 238. [9] U.H.N. Duerr, U.S. Sudheendra, A. Ramamoorthy, Biochim. Biophys. Acta 1758 (2006) 1408. [10] V. Dhople, A. Krukemeyer, A. Ramamoorthy, Biochim. Biophys. Acta 1758 (2006) 1499. [11] M. Dathe, T. Wieprecht, Biochem. Cell Biol. 1462 (1999) 71. [12] T. Wieprecht, M. Dathe, M. Beyermann, E. Krause, W.L. Maloy, D.L. MacDonald, M. Bienert, Biochemistry 36 (1997) 6124. [13] K. Matsuzaki, A. Nakamura, O. Murase, K. Sugishita, N. Fujii, K. Miyajima, Biochemistry 36 (1997) 2104. [14] M. Dathe, H. Nikolenko, J. Meyer, M. Beyermann, M. Bienert, FEBS Lett. 501 (2001) 146. [15] D. Avrahami, Y. Shai, Biochemistry 41 (2002) 2254. [16] D. Avrahami, Y. Shai, J. Biol. Chem. 279 (2004) 12277. [17] A.F. Chu-Kung, K.N. Bozzelli, N.A. Lockwood, J.R. Haseman, K.H. Mayo, M.V. Tirrell, Bioconjugate Chem. 15 (2004) 530. [18] H. Wakabayashi, H. Matsumoto, K. Hashimoto, S. Teraguchi, M. Takase, H. Hayasawa, Antimicrob. Agents Chemother. 43 (1999) 1267. [19] A. Majerle, J. Kidric, R. Jerala, J. Antimicrob. Chemother. 51 (2003) 1159. [20] N.A. Lockwood, J.R. Haseman, M.V. Tirrell, K.H. Mayo, Biochem. J. 378 (2004) 93. [21] S. Thennarasu, D. Lee, A. Tan, U. Prasad Kari, A. Ramamoorthy, Biochim. Biophys. Acta 1711 (2005) 49. [22] G. Bohm, R. Mohr, R. Jaenicke, Protein Eng. 5 (1992) 191. [23] N.R. Kallenbach, P. Lyu, H. Zhou, in: G.D. Fasman (Ed.), Circular Dichroism and the Conformational Analysis of Biomolecules, Plenum Press, New York, 1996, p. 201. [24] J. Reed, T.A. Reed, Anal. Biochem. 254 (1997) 36. [25] J.H. Kim, M.M. Domach, R.D. Tilton, J. Phys. Chem. B 103 (1999) 10582. [26] S. Ge, A. Takahara, T. Kajiyama, J. Vac. Sci. Technol. A 12 (1994) 2530. [27] S.L. Haynie, G.A. Crum, B.A. Doele, Antimicrob. Agents Chemother. 39 (1995) 301. [28] K. Ma, W. Kuan, Biochem. J. (2003) 687.
167
[29] P. Mak, J. Pohl, A. Dubin, M.S. Reed, S.E. Bowers, M.T. Fallon, W.M. Shafer, Int. J. Antimicrob. Agents 21 (2003) 13. [30] J.J.H. Nusselder, T.J. Degroot, M. Trimbos, J. Engberts, J. Org. Chem. 53 (1988) 2423. [31] R. Varadaraj, J. Bock, S. Zushma, N. Brons, Langmuir 8 (1992) 14. [32] R. Tu, Assembly of Peptide Functionalized Molecular Architectures. PhD Thesis, University of California, Santa Barbara, Santa Barbara, 2004. [33] T. Gore, Stabilizing Peptide Triple–Helical Structures with Amphiphile SelfAssembly. PhD Thesis, Universtiy of Minnesota, 2000. [34] C.E. Dempsey, S. Ueno, M.B. Avison, Biochemistry 42 (2003) 402. [35] R. Feder, A. Dagan, A. Mor, J. Biol. Chem. 275 (2000) 4230. [36] N. Sal-Man, Z. Oren, Y. Shai, Biochemistry 41 (2002) 11921. [37] P. Seeman, Pharmacol. Rev. 24 (1972) 583. [38] R.B. Ashman, R.V. Blanden, B.W. Ninham, D.F. Evans, Immunol. Today 7 (1986) 278. [39] C.R. Birnie, D. Malamud, R.L. Schnaare, Antimicrob. Agents Chemother. 44 (2000) 2514. [40] H. Meyer, Arch. Exp. Pathol. Pharmakol. (Naunyn Schmiedebergs) 42 (1999). [41] E. Overton, Studien uber die Narkose, zugleich ein Beitrag zur allgemeinen Pharmakologie. Jenna, Fisher, 1901. [42] M.J. Pringle, K.B. Brown, K.W. Miller, Mol. Pharmacol. 19 (1981) 49. [43] H. Mozsolits, M.I. Aguilar, Biopolymers 66 (2002) 3. [44] N. Papo, Y. Shai, Biochemistry 42 (2003) 458. [45] H. Mozsolits, H.J. Wirth, J. Werkmeister, M.I. Aguilar, Biochim. Biophys. ActaBiomembr. 1512 (2001) 64. [46] L. Ringstad, A. Schmidtchen, M. Malmsten, Langmuir 22 (2006) 5042. [47] I. Zelezetsky, U. Pag, N. Antcheva, H.G. Sahl, A. Tossi, Arch. Biochem. Biophys. 434 (2005) 358. [48] K. Hilpert, M. Elliott, R. Volkmer-Engert, P. Henklein, O. Donini, Q. Zhou, D. Winkler, R. Hancock, Chem. Biol. 13 (2006) 1101. [49] L. Gottler, H. Lee, C.E. Shelburne, A. Ramamoorthy, E.N. Marsh, Biochemistry 47 (2008) 9243. [50] L. Gottler, H. Lee, C.E. Shelburne, A. Ramamoorthy, E.N. Marsh, ChemBioChem 2008 (2008) 3. [51] T.L. Raguse, E.A. Porter, B. Weisblum, S.H. Gellman, J. Am. Chem. Soc. 124 (2002) 12774.
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Chain length dependence of antimicrobial peptide–fatty acid conjugate activity Alexander F. Chu-Kung a, Rose Nguyen a, Kristen N. Bozzelli a, Matthew Tirrell a,b,* a b
Department of Chemical Engineering, University of California, Santa Barbara, CA 93106, United States Department of Bioengineering, University of California, Berkley, CA 94720, United States
a r t i c l e
i n f o
Article history: Received 28 September 2009 Accepted 21 November 2009 Available online 2 December 2009 Keywords: Antimicrobial peptide Fatty acid Conjugation
a b s t r a c t The rise of resistant bacteria has prompted the search for new antimicrobial agents. Antimicrobial membrane lytic peptides have potential as future microbial agents due to their novel mode of action. Recently conjugation of a fatty acid to antimicrobial peptides has been explored as a method to modulate the activity and selectivity of the peptide. Our work further explores these phenomena by testing two peptides, YGAAKKAAKAAKKAAKAA (AKK) and LKKLLKLLKLLKL (LKK), conjugated to fatty acids of varying length for their activity, structure, solution assembly properties and the ability to bind model membranes. We found that increasing the length of fatty acids conjugated to peptide AKK, up to a 16 carbons in length, increases the antimicrobial activity. Peptide AKK appears to lose activity when the minimal active concentration is higher than the critical miscelle concentration (CMC) of the molecule. Thus, if the CMC of the peptide conjugate is too low the activity is lost. Peptide LKK has no activity when conjugated to lauric acid and appears to aggregate at very low concentrations. Conjugation of AKK with a fatty acid increases its affinity to model supported lipid membranes. It appears that the increased hydrophobic interaction imparted by the fatty acid increases the affinity of the peptide to the surface thus increasing its activity. At concentrations above the CMC, solution self-assembly inhibits binding of the peptide to cell membranes. Ó 2010 Published by Elsevier Inc.
1. Introduction Antimicrobial peptides are naturally occurring molecules that provide the first line of defense against infection in many animals. In multi-cellular organisms antimicrobial peptides are secreted constantly at low levels, often on the skin or mucus membranes [1]. Over eight hundred antimicrobial peptides have been isolated and a listing has been compiled at http://www.bbcm.univ.trieste.it/~tossi/amsdb.html. Many of these peptides have been shown to have broad spectrum antibiotic and antifungal activity as well as anti-viral and anti-tumor activity [2,3]. Unlike many antibiotics currently on the market antimicrobial peptides function by a non-receptor mediated mechanism. There is evidence that antimicrobial peptides target the lipid membrane of cells and exploit the fundamental differences between eukaryotic and prokaryotic cell membranes [4–6]. These differences provide the basis for selectivity of antimicrobial peptides. It has been suggested that for bacteria to develop resistance to antimicrobial peptides, major changes to their membrane composition would be necessary [1]. Due to the growing problem of bacterial resistance to currently available antibiotics, antimicrobial peptides are being studied as a possible alternative. * Corresponding author. Address: Department of Bioengineering, University of California, Berkley, CA 94720, United States. E-mail address: [email protected] (M. Tirrell). 0021-9797/$ - see front matter Ó 2010 Published by Elsevier Inc. doi:10.1016/j.jcis.2009.11.057
Despite their diversity, antimicrobial peptides tend to share some common characteristics. Antimicrobial peptides tend to be capable of adopting an amphipathic structure, a structure having distinct hydrophobic and hydrophilic regions, allowing the peptide to bind and insert into the hydrophobic core of the cell membrane. [7–10]. The hydrophobic forces contribute to nonselective binding since the peptide cannot differentiate eukaryotic and prokaryotic cell membranes on the basis of hydrophobicity. Highly hydrophobic peptides have been shown to be highly toxic to eukaryotic cells as well as prokaryotic cells [11,12]. Most antimicrobial peptides are composed of a disproportionately high number of cationic amino acids as opposed to anionic amino acids, resulting in a net positive charge on the peptide [1,8]. Positive peptide charge is important for the binding of peptides to anionic lipids in the bacterial cell membrane. Reduction of positive charge on the peptide has been shown to reduce the activity and selectivity of antimicrobial peptides [13,14]. Recently, researchers have found that addition of a fatty acid tail to antimicrobial peptides (Fig. 1) can modify both the activity and selectivity of the peptide [15–21]. In earlier work from our lab we determined that conjugation of a peptide to a fatty acid can increase the antimicrobial activity of amphipathic peptides [17,20]. We also showed that conjugation of a naturally derived peptide containing highly hydrophobic amino acids, SC-4, shows increased activity and reduced selectivity [17]. From our studies we hypothesized that fatty acid conjugation enhances activity by
A.F. Chu-Kung et al. / Journal of Colloid and Interface Science 345 (2010) 160–167
Fig. 1. Chemical structure of a fatty acid conjugated peptide. The first two amino acids of the peptide are shown with side chains R1 and R2.
161
35 is a lactose permease-deficient strain and was obtained from Robert Hancock at the University of British Columbia. E. coli DH5a, a common lab strain used for transformations and was provided by the biology laboratory at University of California Santa Barbara. S. epidermidis (ATCC#12228) were used for antimicrobial activity assays. 2.2. Peptide amphiphile synthesis
increasing the hydrophobic interaction between the cell membrane and peptide. Highly hydrophobic peptides become toxic to eukaryotic cells because the increased hydrophobicity causes nonselective binding to any cell membrane. Weakly hydrophobic peptides appear to bind both eukaryotic and prokaryotic cell membranes but are unable to fold into their active conformations upon binding, possibly due to the strong electrostatic repulsions on the peptide. Studies on the antimicrobial peptide Lactoferrin show that a twelve carbon fatty acid is the optimal length of tail to improve activity [19]. Studies on peptide SC-4 demonstrate that increasing the fatty acid length from 12 carbons to 18 carbons can reduce activity against some strains of bacteria [20]. Our current research tries to further understand the effect of fatty acid conjugation on antimicrobial peptide activity, including the origin of the reduction in activity above a critical tail length. In order to do so, we conjugated our previously studied peptide, YGAAKKAAKAAKKAAKAA (AKK) to fatty acids ranging from 12 to 20 carbon units in length. In order to study the effect of peptide hydrophobicity we used a peptide with the same sequence motif as AKK but incorporating the more hydrophobic leucine residues in place of the alanine residues, LKKLLKLLKKLLKL (LKK). Peptide LKK has previously been studied and is known to have antimicrobial activity before conjugation to a fatty acid. Both peptides are capable of forming amphipathic helices (Fig. 2). We tested these peptides and their fatty acid conjugates for antimicrobial activity, structure, aggregation state, and binding affinity to model membranes in order to determine the effect of fatty acid conjugation on antimicrobial peptide activity. 2. Experimental 2.1. Materials AKK and LKK peptides were synthesized by Synpep Corporation. 1-hydroxybenzotriazole (HOBt), 2-(1H-benzotriazole-1yl)1,1,3,3tetramethyluronium hexafluorophosphate (HBTU) were purchased from Nova Biochem. All phospholipids were purchased from Avanti Polar Lipids. 111 Silicon wafers (native oxide layer) were purchased from MEMC Electronics. All other chemicals and reagents were purchased from Sigma–Aldrich. Bacteria strains E. coli ML-
Peptide was received from the Synpep Corporation bound to resin, with side chains completely protected. Peptides were characterized by Synpep Corporation and accepted with a certificate of authenticity. The N-terminal Fmoc protecting group of resin-bound peptide was removed with 20 vol.% piperidine in dimethylformamide (DMF) for 2 h, followed by three washes with DMF. The peptide was then coupled to either lauric acid (C12), myristic acid (C14), palmitic acid (C16), 2-hexyldecanoic acid (bC16), stearic acid (C18), or arachidic acid (C20) using a four-fold molar excess of HBTU, HOBt, N,N-diisopropylethylamine, and the appropriate fatty acid. The peptide amphiphile was cleaved, purified, and characterized according to the method below. 2.3. Peptide cleavage and purification All peptides were deprotected and cleaved using a mixture of 95/5 vol.% trifluoroacetic acid (TFA)/water solution. The peptides were then precipitated in cold methyl-tert-butyl-ether. Peptides were purified by reverse phase high performance liquid chromatography on a Vydac C4 column using 99.9/0.1 vol.% water/TFA and 99.9/0.1 vol.% acetonitrile/TFA as the mobile phases. All samples were lyophilized and analyzed for molecular mass using a Thermo Bioanalysis matrix-assisted laser desorption/ionization time of flight system (MALDI-TOF). All peptides eluted as a single peak and were 100% pure within the detection limit of MALDITOF analysis. 2.4. Minimum bactericidal concentration (MBC) determination Gram-negative E. coli (strains DH5a or ML-35) or Gram-positive S. epidermidis (ATCC#12228) test bacteria were grown in overnight cultures in tryptic soy broth (TSB) at 37 °C for at least 18 h. The overnight cultures were subcultured and grown to log phase (OD600 of 0.5), then diluted to a concentration of 1 105–1 106 colony forming units (CFU)/mL. Stock solutions of test peptides were prepared at concentrations of 1 mg/mL in Millipore water. Stock peptide solutions were added to polypropylene tubes and diluted with TSB to the desired test concentration and allowed at least 30 min to reach equilibrium. Sample tubes were inoculated with a bacterial stock solution to a final bacteria concentration between 1 104 and 1 105 CFU/mL and then incubated with rotation for 18 h at 37 °C. The following day 50 lL aliquots were plated out and grown overnight. The lowest concentration where no colonies grew was taken as the MBC. 2.5. Vesicle preparation
Fig. 2. Helical wheel diagrams for YGAAKKAAKAAKKAAKAA (left) LKKLLKLLKKLLKL (right) the dark filled circles show the hydrophilic cationic lysine residues.
Powdered lipids, either 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or 70/30 wt.% 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE)/1,2-dilauroyl-sn-glycero-3-[phosphor-rac(1-glycerol)] (DLPG), were dissolved in chloroform then dried under nitrogen to form a thin film. The film was then further dried under vacuum for a minimum of 12 h. The lipids were hydrated in 10 mM sodium phosphate buffer, pH 7.4, to a concentration of 2.4 mM, then extruded five times through a 100 nm Millipore polycarbonate filter. The vesicle size was confirmed using light scattering.
162
A.F. Chu-Kung et al. / Journal of Colloid and Interface Science 345 (2010) 160–167
2.6. Circular dichroism Circular dichroism (CD) spectra were recorded with a 5 mm quartz cell on an Olis-RSM CD at 37 °C in 10 mM sodium phosphate buffer, pH 7.4. Data was collected from 193 nm to 250 nm at 1 nm intervals. All reported spectra were the result of averaging at least three scans. Circular dichroism measurements of peptides in vesicle solutions were made in 10 mM sodium phosphate buffer, pH 7.4, containing vesicles composed of either DLPC or DLPE/DLPG lipids at a concentration of 0.3 mM. There are a number of different methods to calculate peptide structure from CD spectra [22,23]. We selected a least squares fit method that was performed using a linear combination of standard a-helix and random coil spectra to estimate secondary structure contribution [24]. All fits were done utilizing the 195–250 nm region of the CD spectra since the noise below 195 nm was large. 2.7. Aggregation assays Two methods were utilized to determine the critical micelle concentration of each peptide-fatty acid conjugate. The first method, pyrene solubilization, was used to determine the aggregation state of the peptide amphiphiles [25]. Pyrene is a polycyclic, aromatic hydrocarbon with low solubility in aqueous solutions. Pyrene can be solubilized within the hydrophobic core of micelles, providing a method to detect the presence of micelles. Test peptide was dissolved in 10 mM sodium phosphate buffer, pH 7.4, containing 100 mM NaCl and added to an excess of pyrene at various concentrations. The suspension was then sonicated for a minimum of 8 h and then centrifuged. The supernatant was added to a 50 mM solution of sodium dodecyl sulfate and the absorbance at 336 nm was recorded. Tensiometry was also used to assess the critical micelle concentration (CMC) of our peptide–fatty acid conjugates. The surface tension at the air–water interface is highly dependent on concentration of surfactants in solution. An increase in surfactant concentration at concentrations below the CMC results in decreasing surface tension. Above the CMC, the surface tension is independent of surfactant concentration. We measured surface tension using the Wilhelmy plate method. Peptides for tensiometry were prepared in 10 mM sodium phosphate, 100 mM sodium chloride solution, pH 7.4, at a concentration of 1 mg/ml for peptide C14-AKK, C16-AKK, C18-AKK and C20-AKK. Peptide C12-LKK was prepared in water at a concentration 0.5 mg/ml to avoid precipitation. The peptide solution was then added to 15 ml of buffer solution and allowed to equilibrate before the surface tension was measured. 2.8. Preparation of supported lipid layers Large silicon wafers were cut into small (1 1 cm) squares and cleaned according to the method in the Supplementary data. After cleaning the silicon wafers were functionalized with a hydrophobic monolayer of octadecyltrichlorosilane (OTS) using a method adapted from Ge et al. [26]. 40 lL of a 2 103 M solution of OTS in dry benzene is added dropwise to a water subphase on the Langmuir Blodgett trough at room temperature. The benzene is allowed to evaporate for 1 min and then the layer was compressed to a pressure of 45 mN/m. The layer was allowed to equilibrate for 30 min before the silicon surface was drawn upward through the OTS layer at a speed of 1 mm/min. The layer was allowed to dry under a nitrogen atmosphere. A lipid monolayer was then deposited on the OTS–Si surface using the Langmuir–Blodgett technique. Either 1,2-distearoyl-snglycero-3-phosphocholine (DSPC) or 70/30 wt.% DSPC/1,2-distearoyl-sn-glycero-3-[phosphor-rac-(1-glycerol)] (DSPG) was dissolved in chloroform to a concentration of 1 mg/ml. 40 lL of this
solution were added dropwise to a clean aqueous subphase and the chloroform is allowed 15 min to evaporate. The lipid monolayer was compressed to a surface pressure of 50 mN/m and held for 30 min. The OTS functionalized silicon surfaces are dipped down through the interface at a rate of 1 mm/min. After the dip down the surfaces were stored underwater. 2.9. Peptide binding kinetics A solution of 30–50 ml is prepared in buffer at the desired peptide concentration at least 30 min prior to use. The rate of peptide deposition is measured using a Beaglehole picometer ellipsometer. 20 ml of peptide solution is injected into a sample cell containing a lipid functionalized surface at a rate of 100 ml/min. The remaining peptide solution is recirculated at a rate of 5 ml/min to avoid peptide depletion and diffusion effects. We tested our system at higher flow rates, 10–20 ml/min, and with a larger reservoir, 100 ml, to ensure that neither peptide depletion nor diffusion was influencing our result. 3. Results We used two peptides for this study. YGAAKKAAKAAKKAAKAA (AKK) is a cationic peptide capable of adopting a helical structure. Previous studies on peptide AKK show that lauric acid conjugation to AKK greatly increases its antimicrobial activity [17]. The second peptide, LKKLLKLLKKLLKL (LKK), has the same hydrophobic/hydrophilic sequence motif as AKK, but is shorter and replaces the weakly hydrophobic alanine residues with highly hydrophobic leucine residues. LKK was previously studied and was shown to have antimicrobial activity both in solution and bound to a polymer resin [27]. We conjugated lauric acid to peptide LKK and conjugated fatty acids of varying length to peptide AKK to study their antibacterial properties. Initially we tested our peptides using the MBC test against Gram-negative bacteria E. coli DH5a and ML-35 and Gram positive bacteria S. epidermidis (Table 1). Previous studies on peptide AKK indicate that conjugation to lauric acid increases the antimicrobial activity of the peptide. Conjugation of C14 (myristric acid) to peptide AKK further improves its activity against all the test strains. Conjugation of C16 to AKK improves the peptide activity against the two Gram-negative bacteria strains, however C16AKK loses activity against the Gram positive strain. C18-AKK has slightly lower activity against the two E. coli strains, and also shows no activity against the S. epidermidis. C20-AKK has no measured activity against any strain of bacteria by the MBC test. To determine the effect of branching of peptide antimicrobial activity bC16-AKK was synthesized and tested. It was shown that bC16AKK has slightly lower MBC than C16-AKK against Gram-negative bacteria. Unlike C16-AKK, bC16-AKK shows measurable activity against S. epidermidis. Peptide LKK is known to have some degree of antimicrobial activity before fatty acid conjugation [27] which we confirm using the MBC test. However, lauric acid conjugated LKK loses its activity against all bacterial strains. Two methods, tensiometry and pyrene absorption, were used to measure the CMC of the fatty acid conjugated peptides (Table 1). Because of the low value of the CMC, tensiometry was not effective with C18-AKK and C20-AKK. The pyrene method was not employed with C12-AKK because of the high amount of peptide required. The CMC values as determined by tensiomtery were about 20% higher than those determined by the pyrene method. As expected, the CMC for the Cx-AKK series of peptide amphiphiles shows a logarithmic, decreasing dependence on chain length. bC16-AKK shows a CMC roughly twice that of C16-AKK. There was no detectable CMC using either tensiometry or the pyrene
163
A.F. Chu-Kung et al. / Journal of Colloid and Interface Science 345 (2010) 160–167
Table 1 Properties of tested peptides and peptide conjugates. The first three columns show the peptide activity against various bacteria. The second set of three columns shows the helical content of each peptide in various environments. The final two columns show the CMC of the peptides as measured by either tensiometry or pyrene adsorption. MBC (lM) ± 2.5 lM
AKK C12-AKK C14-AKK C16-AKK bC16-AKK C18-AKK C20-AKK LKK C12-LKK
CMC (lM) ± 10%
Helical content (% Helicity)
E. Coli DH5a
E. Coli ML-35
Staph Ep
Buffer
DLPC
DLPG/DLPE
Tensiometry
Pyrene
>65 11 4 2 6 3 65 7 >65
>65 11 5 4 11 5 >65 10 >65
>65 29 26 >65 24 >65 >65 24 >65
12 17 14 19 8.5 16 44 60 –
5 0 8 12 10 14 24 – –
26 73 74 79 61 81 51 – –
– 790 168 25 – – – – –
– – 130 20 28 8 1 – –
absorption method at low concentrations ([25 lM) for C12-LKK At 25 lM C12-LKK precipitated from solution. It is likely that the critical association concentration for C12-LKK is too low to measure using the methods we employed. Circular dichroism was used to determine the structure of peptides and fatty acid conjugated peptides under various conditions. The structure of peptides were measured at a peptide concentration of 20 lM in phosphate buffer, or phosphate buffer solutions containing either 0.3 mM eukaryotic membrane mimicking DLPC vesicles, or 0.3 mM prokaryotic membrane mimicking DLPE/DLPG vesicle. The resulting spectra were then fit to determine the amount of helicity (Table 1). Peptides C14-AKK, C16-AKK, and C18-AKK behaved in a similar fashion to previously studied C12AKK (Fig. 3) [17]. All these conjugated peptides show very little (<20%) helical structure in solution. In the presence of DLPC vesicles, there was a slight decrease in helical structure. In the presence of vesicles composed of DLPE/DLPG, all the peptide conjugates from C12 to C18 adopt a helical conformation, with >70% helicity. C20-AKK has markedly different behavior. In buffer, C20-AKK adopts a partially helical structure. While in the presence of vesicles containing DLPC, C20-AKK, like the other AKK peptides, adopts a more random coil conformation. However, in the presences of DLPE/DLPG vesicles C20-AKK does not show the significant increase in helicity demonstrated by other AKK peptide conjugates. As expected, bC16-AKK behaves in a similar fashion to C16-AKK. It should be noted that the Cx-AKK peptide series shows an isodichroic point at 208 nm. This is indicative of a two state equilibrium between the helical and disordered states [28]. To study if self-assembly influences the solution peptide secondary structure, we also studied C16-AKK and C18-AKK in phosphate buffer at concentrations above and below the CMC. C16-AKK was studied at concentrations of 10, 20 and 40 lM and the fit of the CD showed 16%, 19% and 19% helical content respectively. C18AKK was studied at 5, 8, and 20 lM and the resulting fit showed 19%, 18% and 16% helical content. The CD spectra at the varying concentrations are nearly identical and thus not shown. Surprisingly neither C16-AKK nor C18-AKK shows significant differences in their structure above and below the CMC. LKK and C12-LKK have interesting behavior when investigated using CD (Fig. 4). Both C12-LKK and LKK precipitated in the presence of DLPE/DLPG vesicles. LKK in buffer adopts a partially helical conformation. In the presence of DLPC vesicles, LKK shows a marked change in structure. The spectrum shows two minima which resembles a helix. However, the resulting spectrum is not an exact match to a classical a-helix. In plain buffer, however C12-LKK shows a spectrum that is neither random coil, a-helix, nor b-sheet and also distinctly different than the LKK spectra in either buffer or with DLPC vesicles. C12-LKK adopts a similar structure as LKK in the presence of DLPC vesicles. Regardless of the exact structure of LKK and C12-LKK in the presence of DLPC vesicles,
the peptides have a distinct change in conformation from the buffer solution, and the peptides adopt a well ordered confirmation. To determine the extent of peptide binding to model lipid membranes we tested peptides AKK, C14-AKK and C16-AKK for their ability to bind a hybrid bilayer (an OTS monolayer covered by a lipid monolayer) surface. We used ellipsometry to monitor the extent of peptide binding to the surface. Fig. 5a shows the concentration dependence of C14-AKK adsorption to DSPC/DSPG and DSPC model membranes. Fig. 5b shows the concentration dependence of AKK adsorption to DSPC/DSPG and DSPC model membranes. Although C14-AKK shows a greater affinity for model membranes than AKK, there are a number of similarities in the behavior of the peptides. With both AKK and C14-AKK we see a fast rise in the signal followed by a transition to a slower rise in the signal. In addition, AKK and C14-AKK show concentration dependence in both rate and extent of binding. Both peptides preferentially bind layers containing DSPG. Fig. 6 shows the adsorption of C16AKK to DSPC/DSPG model membranes. At low concentrations, below the CMC, the peptide amphiphile behaves like AKK and C14AKK. At concentrations above the CMC the peptide rapidly adsorbs to the membrane then ellipsometry signal decreases slowly.
4. Discussion Our previous work [17], along with the work of several other groups [16,18,19,21,29], demonstrates that conjugation of antimicrobial peptides with fatty acids can enhance their membrane lytic activity. We believe the increase in activity is due to the increased hydrophobic attraction to membranes caused by the addition of the fatty acid tail. A longer tail should therefore result in further increased in antimicrobial activity. We tested C14-AKK and determined, as expected, the activity is higher than both C12-AKK and AKK. C16-AKK, when tested against both E. coli strains, shows higher activity than C14-AKK, but surprisingly has no activity against the S. epidermidis strain. C18-AKK shows a slight decrease in activity against the two E. coil strains and no activity against S. epidermidis. C20-AKK shows no activity against any tested bacteria. Comparison of the CMC and MBC of the Cx-AKK peptide series shows loss of activity occurring close to where the MBC will be above the CMC (Fig. 7). We believe the loss in activity occurs due to the self-assembly of the fatty acid conjugated peptides into micelles in solution. C18-AKK shows decreased activity compared to C16-AKK even though the MBC is lower than the CMC. We believe that this is because the CMC of the peptide amphiphile in the growth media may be lower than the CMC measured in the buffer solution. Studies have shown that branched surfactants have a higher CMC than their linear counterparts, possibly due to steric effects [30,31]. ndeed we see the CMC of bC16-AKK is higher than that of C16-AKK. bC16-AKK has activity against both E. coli strains
164
A.F. Chu-Kung et al. / Journal of Colloid and Interface Science 345 (2010) 160–167
Fig. 3. CD spectra of peptides AKK and its fatty acid conjugates C12-AKK, C14-AKK, C16-AKK, C18-AKK and C20-AKK. The symbols on the graph corresponds to CD in sodium phosphate buffer (4) with the addition of DLPC vesicles (h), or DLPE/DLPG vesicles (s).
Fig. 4. CD spectra of peptide LKK and its fatty acid conjugate C12-LKK. The symbols on the graph corresponds to CD in sodium phosphate buffer (4) and with the addition of DLPC vesicles (h). Both samples precipitated in the presence of DLPE/DLPG vesicles.
and S. epidermidis, further suggesting that lack of aggregation or self-assembly in solution prevents inhibition of the activity of antimicrobial peptides. It is known that micellization of peptides conjugated with fatty acid tails can act to stabilize secondary structure of the peptide portion and promote biological activity [32,33]. However, we ob-
served a decrease in activity in some peptide–fatty acid conjugates. We believe that the loss of activity may have been due to the peptides folding together in the micelles, resulting in stable peptidepeptide complexes. These complexes would have a lower affinity for a membrane than an unfolded peptide monomer. The structure of both C16-AKK and C18-AKK was examined by CD at concentra-
A.F. Chu-Kung et al. / Journal of Colloid and Interface Science 345 (2010) 160–167
165
Fig. 5. Adsorption kinetics of C14-AKK (left) and AKK (right) onto model membrane surfaces of either DSPC/DSPG or DSPC. The curves on the left plot correspond to C14-AKK solution concentrations of (from top to bottom) 33 lM onto DSPC/DSPG surfaces, 15 lM onto DSPC/DSPG, 7 lM onto DSPC/DSPG, 60 lM onto DSPC, 40 lM onto DSPC and 15 lM onto DSPC. The curves on the right plot correspond AKK solution concentrations of (from top to bottom) 90 lM onto DSPC/DSPG, 60 lM onto DSPC/DSPG, 30 lM onto DSPC/DSPG and 30 lM onto DSPC.
Fig. 6. Adsorption kinetics of C16-AKK onto DSPC/DPSG at various concentrations. The curves (from top to bottom) correspond to C16-AKK solution concentrations of 60 lM, 50 lM and 15 lM.
Fig. 7. Shows the MBC and CMC of the AKK series of peptides as a function of tail length. The dotted line is the CMC of the peptide. The solid lines show the MBC of the peptide against E. coli DH5a (diamonds), E. coli ML-35 (squares) and S. epidermidis (diamonds).
tions above and below their CMC. No helical content was seen in either peptide. All the peptides were studied at 20 lM, in the presence of vesicles composed of DLPE/DLPG lipids. Peptides C12-AKK, C14-AKK, C16-AKK and C18-AKK show similar structural behavior. Under these conditions, peptides C12-AKK, C14-AKK are well below their CMC, C16-AKK is very close to its CMC, and C18-AKK is above its CMC. This suggests that formation of micelles does not prevent the eventual binding of DLPE/DLPG membranes to some degree. However, micellization may affect both the rate of membrane binding and the final absorbed amount. C20-AKK shows a significant amount of helical structure in solution. Our studies with C16-AKK and C18-AKK suggest that micellization does not stabilize helical structures in solution. For magainin conjugated to a long fatty acid, it has been suggested that helical secondary structure is induced in monomeric helices due to interaction between the amphipathic helix face and the fatty acid chain. The amphipathic helix can then bend to press its amphiphathic faces against each other, with the fatty acid chain in between the faces [15]. C20-AKK shows a reduced amount of structure in the presence of DLPC vesicles indicating that the peptide binds DLPC vesicles but does so in an unstructured fashion. Surprisingly C20-AKK does not show a significant increase in helical structure in the presence of DLPE/DLPG membranes. It is possible that the C20-AKK binds the membrane through electrostatic interactions but never forms a helix with its hydrophobic face imbedded into the membrane. This would explain the lack of antimicrobial activity of C20-AKK. All of the fatty acid conjugates of AKK show a decrease in helical content when in the presence of DLPC vesicles. Only C20-AKK shows a greater than 15% helicity. This suggests the peptides bind the DLPC membrane but do not adopt an active conformation. We would expect none of the fatty acid conjugated AKK peptides to have significant hemolytic activity. Studies show a similar palmitic acid conjugated alanine–lysine peptide does not have significant hemolytic activity [16]. LKK, as expected, shows a significant antimicrobial activity before conjugation to a fatty acid. However, conjugation to lauric acid renders peptide LKK inactive. CD of peptide LKK in solution suggests that it adopts a partially helical conformation. C12-LKK has a significantly different structure in solution. It is likely that conjugation of LKK with lauric acid causes the peptides to aggregate. The highly hydrophobic leucine residues will favor an aggregation and folding in order to bury themselves in a hydrophobic environment.
166
A.F. Chu-Kung et al. / Journal of Colloid and Interface Science 345 (2010) 160–167
It is likely aggregation inhibits antimicrobial action in much the same way that micellization does for Cx-AKK. Both LKK and C12LKK show a significant change in structure from buffer solution when in contact with DLPC vesicles suggesting that they may have high hemolytic activity. Studies show a similar leucine–lysine peptide conjugated to palmitic acid is highly hemolytic [16]. From the activity data we conclude that aggregation inhibits the activity of antimicrobial peptides. There is evidence that the aggregation state of antimicrobial peptides plays an important role in bactericidal activity and selectivity. Studies show a magainin analog dimerized though a covalent cystine linkage show a significant increase in both bactericidal activity and hemolytic activity as a result of dimerization [34]. In another study, analogs of the peptide dermaseptin were modified, either through truncation or substitution of amino acids, to control their aggregation state. It was demonstrated that aggregation reduces the bactericidal activity and increases the hemolytic activity [35]. A third study tethered five helical antimicrobial peptides together through their C-terminus to a short peptide linker. The pentamers show increased helical content, bactericidal activity, and hemolytic activity [36]. These studies demonstrate that aggregation and even folding of antimicrobial peptides in an aggregate does not necessarily lead to loss of bactericidal activity. This suggests that the loss of activity of aggregated fatty acid conjugated peptide is due to the lipid tails inhibiting partitioning into the bacterial membrane. There are numerous examples of surfactants that show increasing biological activity up to a critical chain length; above this chain length the compounds are no longer active. This phenomena has been dubbed the biological surfactant cut-off effect and has been documented in anaesthetics [37], immunosuppressives [38], and antimicrobials [39]. Several theories have been developed to explain the cut-off effect. At the turn of the twentieth century, Meyers and Overton independently proposed the cut-off effect was the result of limited solubility of the surfactant molecule in solution [40,41]. As the chain length of the surfactant increases the aqueous solubility decreases. It is believed the partition coefficient between the lipid and aqueous phase increases less rapidly than the solubility decreases [42]. As a result, the maximum possible concentration of surfactant bound to the lipids, determined by the equilibrium between monomer and lipid-bound peptide, is lower than amount required to elicit the desired biological effect. The contribution of the peptides in micelles towards the maximum amount of bound peptide should be small. The partition function between the micellar peptide and membrane bound peptide should be smaller than the monomer-lipid-bound peptide partition function. This is because the peptide amphiphiles are in a hydrophobic environment in the micelle, thus the driving force to bind the surface and minimize hydrophobic-solvent contacts is reduced. Ellipsometry was employed here to directly measure the binding of peptides and antimicrobial peptides to model lipid membranes. Previously surface plasmon resonance (SPR), another spectroscopic technique to monitor adsorption of molecules to surfaces, has been employed to monitor peptide membrane interactions [43,44]. These systems also incorporate a gold surface covered by a hybrid bilayer (an alkane thiol monolayer covered by a lipid monolayer). AKK and C14-AKK show kinetic behaviors that are qualitatively similar to those observed for magainin and mellitin as observed by SPR [43,44]. As expected C14-AKK bound membranes more strongly than AKK because of the increased hydrophobic interaction between the C14-AKK peptide and the membrane. Additionally we saw that both C14-AKK and AKK bound DSPC/DSPG lipids more strongly than DSPC lipids alone. A close look shows that the peptides, especially C14-AKK, do not follow simple Langmuir adsorption kinetics. There seems
to be a rapid association phase followed by a slower association phase. Data from magainin and mellitin were also found to fit the kinetics of a two state reaction model [43,44]. It was proposed that the two phases correspond to an initial binding of the membrane and the subsequent deeper insertion into the membrane [45]. At concentrations below the CMC C16-AKK shows similar behavior to C14-AKK and AKK, a rapid adsorption phase followed by a slower adsorption phase. At concentrations above the CMC, C16-AKK shows an ‘‘overshoot” then a decrease in the signal. The behavior of C16-AKK above the CMC may give some insight into the cut-off effect observed at high chain lengths. A possible explanation for the overshoot at concentrations above the CMC could be the binding of intact micelles to the surface blocking monomeric peptide from binding. The decrease in signal in signal overtime may correspond to micelles desorbing and being replaced by monomeric peptide. This supports the Meyers and Overton theory of limited solubility [40,41]. Although there are not enough data currently to perform a detailed kinetic analysis, our findings give some insight into the behavior of fatty acid conjugated peptides binding membranes. As we predicted, the conjugation of a fatty acid to a peptides causes to bind membranes more strongly. Ellipsometry and liposome leak studies on synthetic antimicrobial peptides of varying lengths suggest that there is a strong correlation between peptide adsorption and induced liposome leakage [46]. These findings would seem to support the hypothesis that fatty acid conjugation increases the propensity for a peptide to bind a phospholipid membrane which in turn increases the peptide’s antimicrobial activity. There is currently a significant amount of interest in developing more active and selective antimicrobial compounds. Antimicrobial peptides have drawn a significant amount of attention due to their non-receptor based mechanism of action. While naturally occurring antimicrobial peptides have some promise as therapeutic agents a number of techniques have been employed to enhance their potential as therapeutic agents. Some examples of these methods include the optimization of peptide sequence [14,47,48], inclusion of fluorous amino acids [49,50], inclusion of b-amino acids [51], as well as fatty acid conjugation. We have studied fatty acid conjugated antimicrobial peptides to better understand the relationship between fatty acid properties and peptide properties on structure and biological function. It appears that conjugation of fatty acids to peptides imparts the behavior and properties of both antimicrobial peptides and simple antimicrobial surfactants to the peptide-fatty acid complex. The complex shows high lipophilicity due to the fatty acid tail, however the maximum effective length of the fatty acid tail is limited by aggregation or self-assembly of the peptide conjugates. The difference between the peptidefatty acid complex and a simple surfactant lies in the requisite folding of the peptide in order to be active. Although it appears the CxAKK peptides will bind DLPC membranes, they do not fold due to the low hydrophobicity and large electrostatic repulsion of the side chains. Keeping these ideas in mind, it should be possible to maximize the activity and therapeutic window of a fatty acid conjugated peptide. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jcis.2009.11.057. References [1] M. Zasloff, Nature 415 (2002) 389. [2] P.M. Hwang, H.J. Vogel, Biochem. Cell Biol. 76 (1998) 235. [3] D.W. Hoskin, A. Ramamoorthy, Biochim. Biophys. Acta 1778 (2008) 357.
A.F. Chu-Kung et al. / Journal of Colloid and Interface Science 345 (2010) 160–167 [4] D. Wade, A. Boman, B. Wahlin, C.M. Drain, D. Andreu, H.G. Boman, R.B. Merrifield, Proc. Natl. Acad. Sci. USA 87 (1990) 4761. [5] M.R. Yeaman, N.Y. Yount, Pharmacol. Rev. 55 (2003) 27. [6] K. Matsuzaki, O. Murase, K. Miyajima, Biochemistry 34 (1995) 12553. [7] Y. Shai, Z. Oren, Peptides 22 (2001) 1629. [8] K.A. Brogden, Nat. Rev. Microbiol. 3 (2005) 238. [9] U.H.N. Duerr, U.S. Sudheendra, A. Ramamoorthy, Biochim. Biophys. Acta 1758 (2006) 1408. [10] V. Dhople, A. Krukemeyer, A. Ramamoorthy, Biochim. Biophys. Acta 1758 (2006) 1499. [11] M. Dathe, T. Wieprecht, Biochem. Cell Biol. 1462 (1999) 71. [12] T. Wieprecht, M. Dathe, M. Beyermann, E. Krause, W.L. Maloy, D.L. MacDonald, M. Bienert, Biochemistry 36 (1997) 6124. [13] K. Matsuzaki, A. Nakamura, O. Murase, K. Sugishita, N. Fujii, K. Miyajima, Biochemistry 36 (1997) 2104. [14] M. Dathe, H. Nikolenko, J. Meyer, M. Beyermann, M. Bienert, FEBS Lett. 501 (2001) 146. [15] D. Avrahami, Y. Shai, Biochemistry 41 (2002) 2254. [16] D. Avrahami, Y. Shai, J. Biol. Chem. 279 (2004) 12277. [17] A.F. Chu-Kung, K.N. Bozzelli, N.A. Lockwood, J.R. Haseman, K.H. Mayo, M.V. Tirrell, Bioconjugate Chem. 15 (2004) 530. [18] H. Wakabayashi, H. Matsumoto, K. Hashimoto, S. Teraguchi, M. Takase, H. Hayasawa, Antimicrob. Agents Chemother. 43 (1999) 1267. [19] A. Majerle, J. Kidric, R. Jerala, J. Antimicrob. Chemother. 51 (2003) 1159. [20] N.A. Lockwood, J.R. Haseman, M.V. Tirrell, K.H. Mayo, Biochem. J. 378 (2004) 93. [21] S. Thennarasu, D. Lee, A. Tan, U. Prasad Kari, A. Ramamoorthy, Biochim. Biophys. Acta 1711 (2005) 49. [22] G. Bohm, R. Mohr, R. Jaenicke, Protein Eng. 5 (1992) 191. [23] N.R. Kallenbach, P. Lyu, H. Zhou, in: G.D. Fasman (Ed.), Circular Dichroism and the Conformational Analysis of Biomolecules, Plenum Press, New York, 1996, p. 201. [24] J. Reed, T.A. Reed, Anal. Biochem. 254 (1997) 36. [25] J.H. Kim, M.M. Domach, R.D. Tilton, J. Phys. Chem. B 103 (1999) 10582. [26] S. Ge, A. Takahara, T. Kajiyama, J. Vac. Sci. Technol. A 12 (1994) 2530. [27] S.L. Haynie, G.A. Crum, B.A. Doele, Antimicrob. Agents Chemother. 39 (1995) 301. [28] K. Ma, W. Kuan, Biochem. J. (2003) 687.
167
[29] P. Mak, J. Pohl, A. Dubin, M.S. Reed, S.E. Bowers, M.T. Fallon, W.M. Shafer, Int. J. Antimicrob. Agents 21 (2003) 13. [30] J.J.H. Nusselder, T.J. Degroot, M. Trimbos, J. Engberts, J. Org. Chem. 53 (1988) 2423. [31] R. Varadaraj, J. Bock, S. Zushma, N. Brons, Langmuir 8 (1992) 14. [32] R. Tu, Assembly of Peptide Functionalized Molecular Architectures. PhD Thesis, University of California, Santa Barbara, Santa Barbara, 2004. [33] T. Gore, Stabilizing Peptide Triple–Helical Structures with Amphiphile SelfAssembly. PhD Thesis, Universtiy of Minnesota, 2000. [34] C.E. Dempsey, S. Ueno, M.B. Avison, Biochemistry 42 (2003) 402. [35] R. Feder, A. Dagan, A. Mor, J. Biol. Chem. 275 (2000) 4230. [36] N. Sal-Man, Z. Oren, Y. Shai, Biochemistry 41 (2002) 11921. [37] P. Seeman, Pharmacol. Rev. 24 (1972) 583. [38] R.B. Ashman, R.V. Blanden, B.W. Ninham, D.F. Evans, Immunol. Today 7 (1986) 278. [39] C.R. Birnie, D. Malamud, R.L. Schnaare, Antimicrob. Agents Chemother. 44 (2000) 2514. [40] H. Meyer, Arch. Exp. Pathol. Pharmakol. (Naunyn Schmiedebergs) 42 (1999). [41] E. Overton, Studien uber die Narkose, zugleich ein Beitrag zur allgemeinen Pharmakologie. Jenna, Fisher, 1901. [42] M.J. Pringle, K.B. Brown, K.W. Miller, Mol. Pharmacol. 19 (1981) 49. [43] H. Mozsolits, M.I. Aguilar, Biopolymers 66 (2002) 3. [44] N. Papo, Y. Shai, Biochemistry 42 (2003) 458. [45] H. Mozsolits, H.J. Wirth, J. Werkmeister, M.I. Aguilar, Biochim. Biophys. ActaBiomembr. 1512 (2001) 64. [46] L. Ringstad, A. Schmidtchen, M. Malmsten, Langmuir 22 (2006) 5042. [47] I. Zelezetsky, U. Pag, N. Antcheva, H.G. Sahl, A. Tossi, Arch. Biochem. Biophys. 434 (2005) 358. [48] K. Hilpert, M. Elliott, R. Volkmer-Engert, P. Henklein, O. Donini, Q. Zhou, D. Winkler, R. Hancock, Chem. Biol. 13 (2006) 1101. [49] L. Gottler, H. Lee, C.E. Shelburne, A. Ramamoorthy, E.N. Marsh, Biochemistry 47 (2008) 9243. [50] L. Gottler, H. Lee, C.E. Shelburne, A. Ramamoorthy, E.N. Marsh, ChemBioChem 2008 (2008) 3. [51] T.L. Raguse, E.A. Porter, B. Weisblum, S.H. Gellman, J. Am. Chem. Soc. 124 (2002) 12774.