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J. Phys. Chem. B 2007, 111, 6280-6287
Dicynthaurin (ala) Monomer Interaction with Phospholipid Bilayers Studied by Fluorescence Leakage and Isothermal Titration Calorimetry Shaoying Wen,† Monika Majerowicz,† Alan Waring,‡ and Frank Bringezu*,†,§ Institute of Medical Physics & Biophysics, UniVersity of Leipzig, 04107 Leipzig, Germany, and Departments of Medicine, UCLA, Los Angeles, California 90095 ReceiVed: NoVember 23, 2006; In Final Form: March 26, 2007
The interaction of the antimicrobial peptide dicynthaurin (ala) monomer with model membranes of zwitterionic and negatively charged lipids and mixtures thereof was studied by means of isothermal titration calorimetry (ITC), fluorescent leakage, and dynamic light scattering (DLS) measurements. For the ITC analysis, we have applied the surface partitioning equilibrium model which shows that the interaction is predominately driven by hydrophobic effects (Kb between 2 × 104 and 1 × 105 M-1). Under low salt conditions, the enhanced electrostatic interaction leads to larger peptide concentrations immediately above the vesicle surface, which initiates the insertion of the peptide into the bilayer more effectively. Fluorescent leakage measurements have shown a fast leakage of the fluorescent dye within seconds after peptide addition. The analysis of the leakage kinetics was performed in terms of an initial pore formation model (up to t ) 1000 s) that takes the reversible surface aggregation of bound peptide monomers into account. From this analysis, a minimum aggregation number of n ) 7 ( 2 per pore is obtained.
Introduction Antimicrobial peptides (AP) have been identified as being the native line of defense in nature, for example, in animals, plants, and also single-cell organisms. The immune response is governed by the expression of the peptides that show a high toxicity against diverse species including both Gram-negative and Gram-positive bacteria, but they often also exhibit activity against fungi and some also show antiviral activity.1-4 A wealth of active sequences has been described; most of them are relatively short length peptides containing positively charged residues in polar side chains. The increasing scientific interest on the antimicrobial activity is mainly driven by the relevance of the APs to intrinsic host defense and the enormous potential in future drug development.5 There is a strong line of evidence that the activity of many APs is directly connected with the interaction between the peptides and the membrane bilayer.6 This interaction affects the integrity of the bacterial cell membrane by different mechanisms (e.g., pore formation, curvature stress, and final disruption of the membrane), which leads to cell death.7-9 In recent studies, an unusual peptide, dicynthaurin, was isolated from the hemocytes of the tunicate Halocynthia aurantium that contains an unpaired cysteine and forms covalent homodimers. In a membrane mimetic environment, an R-helical conformation was found for both the peptide monomer and the dimer. Dicynthaurin’s broad spectrum activity encompassed Gram-positive (Micrococcus luteus, Staphylococcus aureus, Listeria monocytogenes) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) but not Candida albicans, * Corresponding author. Address: Institute of Medical Physics and Biophysics, University of Leipzig, Ha¨rtelstrasse 16-18, 04107 Leipzig, Germany. E-mail:
[email protected]. † University of Leipzig. ‡ UCLA, Los Angeles. § Current address: MZP, Institute of Biotechnology, Martin-LutherUniversity, Halle-Wittenberg, 06120 Halle, Germany.
a fungus.10 The native peptide is composed of two monocysteine-bounded monomers (I L Q K A V L D C L K A A G S S L S K A A I T A I Y N K I T) of 30 amino acids with a formal charge of +3. From the peptide sequence, it is clearly evident that clustering of polar and apolar residues imparts amphipathicity, thus suggesting a high capacity for membrane insertion. The dicynthaurin monomer lacks hemolytic activity and exhibits antimicrobial activity at high salt concentration but optimal activity at lower salt concentrations 99%) and used as obtained without further purification. 5(6)-Carboxyfluorescein was purchased from Sigma-Aldrich (Taufkirchen, Germany, purity g95%). All samples are prepared in phosphate buffered solutions containing 10 mM phosphates with either 140 mM NaCl or no additional salt, at pH 7.4. All aqueous solutions were prepared using Milli-Q deionized water of a specific electrical conductivity of >18 MΩ cm. For the experiments, peptide stocking solutions were prepared by dissolving the solid peptide in phosphate buffer followed by vortexing for 10 min. For the preparation of the lipid mixtures, aliquots of POPC and POPG were mixed via organic solvents and consecutively dried under a stream of nitrogen. Cyclohexane was used for subsequent dissolving the lipid film to lyophilize by freeze-drying overnight. Finally, the dry solid mixture is weighed, suspended in phosphate buffer by intensive vortexing and five freeze-thawing cycles. The lipid concentration was determined on a weight basis of the dry powder mixture obtained after freeze-drying. All samples are stored at 4 °C prior to usage. Extruded samples are obtained by passing the lipid stocking solution 18 times through the Liposo-Fast Basic system (Avestin, Ottawa, Canada) using a membrane with a 100 nm pore size. Methods. Titration Calorimetry. The experiments were performed using an isothermal titration calorimeter VP-ITC (Microcal Inc., Northampton, MA). Before each measurement, the solutions were degassed under vacuum. For the analysis, the dilution data of lipid to buffer titrations were subtracted from the binding heat. Initial data analysis (background subtraction, peak integration) was performed using the MicroCal Origin 5.0 software package. At each injection, 2.5-5 µL of a lipid solution (2.5-5 mM) is titrated into the sample cell, which originally
J. Phys. Chem. B, Vol. 111, No. 22, 2007 6281 contains 1.4337 mL of dicynthaurin (ala) monomer solution (40 µM). All experiments were performed at 45 °C. Final ITC data given in this paper were repeated at least three times. To fit the integrated heat traces, we adopted two procedures; one uses the data handling program that comes with the ITC device and a second one applies the surface partitioning equilibrium model.13,14 The Surface Partitioning Model. To allow a separation of electrostatic effects from chemical binding, a surface partitioning equilibrium model that combines a partitioning model with the Gouy-Chapman theory as derived by Seelig and co-workers must be applied.14-17 In brief, it describes the peptide-lipid interaction by means of a partitioning equilibrium of free and bound peptide between the aqueous phase and the surface of the liposome. The partitioning equilibrium constant is defined as
Kapp )
Pb/γL Pb/γL ) Pfree P - Pb
(1)
where γ is the accessibility of lipid molecules in the vesicle outer surface layer; P, Pb, and Pfree are the total concentration of peptide, the concentration of peptide bound to the membrane surface, and the concentration of unbound peptide, respectively; and L is the total lipid concentration. By this model, the bound peptide concentration, Pb, is further related to PM, the peptide concentration immediately above the membrane surface, enhanced by the electrostatic interaction
Kb )
Pb γLPM
(2)
with Kb being the intrinsic binding constant which describes the chemical binding. Consequently, the apparent binding constant divides into two parts:
(
Kapp ) Kb exp -
)
ZpFψ0 RT
(3)
with the exponential part of Kapp describing the electrostatic interaction in a scaling factor. Here, Zp denotes the effective charge of the peptide and ψ0 is the surface potential of the liposome. As shown by Beschiaschvili and Seelig,14,18 the surface charge density, σ, of a liposome surface with bound peptide can be written as
σ)
e -XPG(1 - fNa) + zP(P/L)b AL 1 + (AP/AL)(P/L)b
(4)
where e is the elementary charge, AL is the area per lipid molecule, Ap is the effective area of the peptide molecule, XPG is the molar fraction of the PG lipid in the mixed POPC/POPG membrane, and fNa is the fraction of POPG associated with Na+. The fraction of POPG lipids provides binding sites for Na+, and the binding can be described using a Langmuir adsorption isotherm.14 A second independent relation between σ and Ψ0 is given by the Gouy-Chapman equation19,20
[ (
∑i Ci exp -
σ2 ) 20000wRT
) ]
ZiFψ0 RT
-1
(5)
where 0 is the permittivity of vacuum and w is the dielectric constant of water and Ci is the concentration of the ith electrolyte in the bulk aqueous phase and Zi. The further analysis follows the approach as published previously that uses a combination
6282 J. Phys. Chem. B, Vol. 111, No. 22, 2007
Wen et al.
of the adsorption/binding equation (eq 4) with the GouyChapman equation (eq 5) which yields a self-consistent solution for Ψ0 for each experimental (P/L)B value.13,14,18 Fluorescent Leakage Measurements. The leakage of liposome content to the external medium was kinetically monitored by measuring the release of the 5(6)-carboxyfluorescein (CF) trapped inside the vesicles.21,22 For sample preparation, a certain amount of POPG powder was hydrated using 10 mM 4-(2hydroxyethyl)piperazine-2-ethanesulfonic acid sodium salt (HEPES) buffer with 100 mM NaCl containing 60 mM 5(6)carboxyfluorescein at pH 7.4, and after intensive vortexing, the sample was freeze-thawed for five cycles. The free CF dye was removed by passing the suspension through a Sephadex G-75 column (MP Biomedicals, Inc., Germany). For elution of the vesicles, isotonic buffer was applied. 5(6)-CF at 60 mM inside the POPG vesicles is mostly self-quenched, but when released into the media, such as by pore formation of peptides, the diluted 5(6)-CF fluoresces intensely. The increase in CF fluorescence upon release was followed using a Perkin-Elmer Luminescence Spectrometer LS50B with excitation at 492 nm (5 nm slit width), and the emission intensity was monitored at 518 nm (5 nm slit width). The measurements were performed in quartz cuvettes at a final phospholipid concentration of 100 µM. A baseline measurement of the fluorescence of pure vesicles was made for 5 min prior to adding the appropriate peptide solution. After peptide addition, the samples were mixed by gently stirring and the increase in fluorescence intensity was recorded. Triton X-100 (1% v/v final) addition provided the maximum fluorescence attainable under conditions of totally solubilized vesicles. The percentage of 5(6)-CF released at time t after peptide addition is given by L ) (It - I0)/(Imax - I0) × 100% , where It is the fluorescence intensity at time t after addition of peptide, I0 is the intensity of pure vesicles, and Imax is the intensity after addition of Triton X-100. After this preparation, the vesicle fluorescence, I0, of less than 5% of Imax indicates a high sample quality. The final lipid concentration was determined by quantitative phosphorus analysis.23 Analysis of the Leakage Kinetics Using the Pore Model. The analysis of the leakage was performed using the pore formation mechanism that includes reversible surface aggregation.24 For the analysis, we assumed that the peptide binding to the vesicle surface is rapid and that after pore formation leakage is occurring on a fast time scale.25 In this case, the expression for leakage extent at time t, L(t), is given by S
L(t) )
Nj
∑ ∑ Z(M,i,j,t,KS)Ai,jfj j)1 i)M
(6)
where j is the classes of vesicle size, i is the bound peptide number per vesicle, Nj is the maximum number of bound peptides per j-class vesicle, M is the minimum aggregation number required for pore formation, KS is the equilibrium constant of reversible aggregation, Ai,j is the fraction of the j-class vesicles with i bound peptide, and fj is the enclosed volume fraction of j-class vesicles, respectively. For the sake of simplicity, in our calculations, we have used a single size vesicle with a radius as determined by dynamic light scattering. Therefore, eq 6 becomes N
L(t) )
∑ Z(M,i,t,KS)Ai
(7)
i)M
where the leakage at time L(t) depends on the leakage probability
Figure 1. Typical calorimeter tracings (heat flow vs time) of a titration of negatively charged POPG vesicles (5 mM, 2.5 µL) into dicynthaurin (ala) monomer solution (40 µM). The experiment was performed in phosphate buffer (10 mM phosphates, pH 7.4, 140 mM NaCl) at 45 °C.
of a vesicle, Z, and the number of the vesicles, Ai, containing at least M bound peptides. Extensive studies of the mass action kinetics of small particles binding to large particles and their subsequent fusion allowed numeric solutions for calculating the A(i) distribution to be derived.26,27
Ai )
( )(
)
P0 - P P0 - P i N! 1NG0 i!(N - i)! NG0
N-1
(8)
Here, G0 denotes the molar concentration of vesicles, P0 is the total molar peptide concentration, and P is the free peptide concentration at binding equilibrium conditions. The amount of free peptide was calculated on the basis of the ITC analysis. For the calculation of Z(M,i,t,KS), we have applied the approach published by Rapaport et al. derived from the theory on polymerization kinetics.24,28,29 Dynamic Light Scattering. Dynamic light scattering measurements were carried out at 25 °C using a DLS-SLS 5000 Laser Light Scattering Goniometer (ALV, Langen, Germany) equipped with a 140 mW Nd:YAG laser (diode pumped, frequency doubled to λ0 ) 532 nm; ADLAS, Lu¨beck, Germany) and a ALV-5000/E multiple τ digital correlator. The details of the instrumentation and theory can be found elsewhere.30 For each sample, dynamic light scattering data were acquired typically for a duration of 20-40 min. The intensity autocorrelation function, g2(t), was measured at an observation angle of 90°. The field autocorrelation function, g1(t), was determined from the intensity autocorrelation function using the procedure as described elsewhere.31 The hydrodynamic radii values given in this paper are the mean peak positions using the number weighted radii distribution function, and a normalized peak area. For experiments with independently prepared solutions, the standard deviation of the hydrodynamic radii was found to be (5 nm. Results Figure 1 shows a typical experimental ITC curve obtained from the titration of small aliquots (2.5 µL, 5 mM) of a POPG dispersion into the calorimeter cell (Vcell ) 1.4337 mL) containing dicynthaurin (ala) monomer (40 µM) at a temperature of 45 °C. The exothermic signal arises from the binding of the peptide to the lipid. With ongoing titrations, the binding heat drops because the free peptide concentration in the cell is
Dicynthaurin Monomer Interaction with Phospholipids
Figure 2. Integrated and normalized heat obtained from the calorimeter traces of lipid to peptide titrations is presented. In the experiments, aliquots of POPC/POPG mixtures (2.5 µL, 5 mM) were titrated into the calorimeter cell containing 40 µM dicynthaurin (ala) monomer in 10 mM phosphate buffer with 140 mM NaCl at pH 7.4 (45 °C). The solid lines represent the fit result using the complex formation model. The inset depicts the fitted binding constant, K, as a function of the POPG molar ratio, XPG.
TABLE 1: Fitting Results Obtained Using the Complex Formation Model from the Microcal ITC Software Package for the 5 mM POPC/POPG Mixture Titrated into 40 µM Dicynthaurin (ala) Monomer in 10 mM Phosphate Buffer with 140 mM NaCl, pH 7.4 at 45 °C XPG
n
K (104 M-1)
∆HL (kJ/mol)
T∆SL (kJ/mol)
0.33 0.50 0.80 0.91 0.95 0.99 1.0
0.8 ( 0.1 0.8 ( 0.1 0.8 ( 0.1 0.8 ( 0.1 0.6 ( 0.2 0.6 ( 0.2 0.8 ( 0.1
2.2 ( 0.2 2.7 ( 0.3 3.1 ( 0.3 7.2 ( 0.5 13.0 ( 1.0 18.0 ( 2.0 4.9 ( 0.3
-4.6 ( 0.8 -8.0 ( 1.2 -6.6 ( 0.8 -4.4 ( 0.7 -10.2 ( 1.4 -6.9 ( 0.7 -8.8 ( 1.2
22.0 19.0 20.8 25.1 20.8 25.0 19.9
decreased. Integrating the binding heat yields the binding isotherms that can be described with the complex formation model (one set of binding sites) from the Microcal software package to give a binding constant of K ) 4.9 × 104 M-1 and values for enthalpy, entropic contribution, and stoichiometry of ∆HL ) -8.8 kJ/mol, T∆SL ) 19.9 kJ/mol, and n ) 0.8, respectively. Figure 2 summarizes the binding isotherms obtained from ITC measurements of a series of POPC/POPG mixed vesicles titrated into the 40 µM peptide solution at 45 °C together with the fit to the data. Increasing the POPG fraction leads to more negatively charged membrane surfaces and thus modifies the affinity of the peptide for the membrane due to electrostatic interactions. Therefore, starting at low XPG, the first titration steps show only small amplitudes which increase on going to larger XPG values. After several injections, only the dilution heat is recorded and binding of the peptide is completed, as shown for pure POPG (XPG ) 1) at L/P ratios of about 4. The lack in hemolytic activity of dicynthaurin (ala) monomer is reflected in the ITC result, showing no interaction with POPC vesicles, as shown in Figure 2. Table 1 summarizes the results of the fits using the complex formation model to the experimental data. Assuming pure electrostatic binding, increasing the PG component in the lipid mixture would lead to continuously increasing K values. However, the results presented in Table 1 and Figure 2 (inset) clearly depict a maximum in the K values at XPG of about 0.95. During the refinement, the best fit was obtained with the binding stoichiometry (n values) ranging from about 0.6 to 0.8. Taking
J. Phys. Chem. B, Vol. 111, No. 22, 2007 6283
Figure 3. Example of the fit (solid line) of the binding isotherm using the surface partitioning equilibrium model obtained from the titration of POPG vesicles into dicynthaurin (ala) monomer in 10 mM phosphate buffer with 140 mM NaCl. The results of the analysis are summarized in Table 2.
TABLE 2: Fitting Results Obtained by the Surface Partitioning Equilibrium Model for 5 mM POPC/POPG Titrated into 40 µM Dicynthaurin (ala) Monomer in 10 mM Phosphate Buffer with 140 mM NaCl, pH 7.4 at 45 °C (γL ) 0.6) Kb XPG (104 M-1) e-(zPFψ0/RT) 0.33 3.0 ( 0.4 0.50 2.3 ( 0.4 0.80 2.4 ( 0.4 0.91 4.2 ( 0.5 0.95 9.6 ( 0.8 0.99 11.6 ( 1.0 1.0 2.7 ( 0.5 a
1.9 2.3 3.0 3.0 2.3 2.3 3.2
∆Hb (kJ/mol) -3.0 ( 0.5 -6.2 ( 0.7 -4.3 ( 0.5 -3.3 ( 0.4 -4.8 ( 0.5 -3.4 ( 0.4 -9.4 ( 0.7
T∆Sb ∆Gba Kapp (kJ/mol) (104 M-1) (kJ/mol) -37.9 -37.1 -37.3 -38.7 -40.9 -41.4 -37.6
5.7 5.3 7.2 12.6 22.1 26.7 8.6
34.8 30.9 33.0 35.4 36.1 38.1 28.2
∆Gb ) -RT ln(Kb × 55.5); ∆Sb ) (∆Hb - ∆Gb)/T.
the accessibility of the lipid in the vesicle of γL ) 0.6 into account, these n values yield about 0.4-0.5 binding sites per peptide. In addition, it is immediately evident that the driving force for the interaction is entropy, as absolute numbers of T∆S exceed the ∆H values in the whole XPG range. These findings suggest that multiple factors that contribute to the interaction (e.g., hydrophobic effect, peptide conformational change, sodium binding equilibrium at the lipid head groups, etc.) must be taken into account. To attribute for the charge independent affinity of the peptide to the lipid membrane, we have therefore adopted the surface partitioning equilibrium model to extract the thermodynamic information from the ITC data and to allow the separation of the electrostatic effect from the intrinsic binding factors. Figure 3 depicts a typical example of the fitting result, and all fitting parameters are given in Table 2. Best fit results yielded an effective charge of the peptide of only +0.5, which is significantly smaller than the formal charge of +3. The analysis further yields that the exponential term dominates over the Kb values in the whole XPG range. The large binding entropy indicates that the interaction is entropy driven, which is in agreement with these findings. Consequently, the classical and nonclassical hydrophobic effects, for example, water and counterion release from the peptide upon binding and membrane incorporation, must be considered as major driving forces for dicynthaurin (ala) monomer binding.17,32,33 As the peptide was shown to have a higher activity at low salt concentration ( 1000 s cannot be explained by the pore formation model, suggesting a second mechanism that leads to leakage upon binding on the long time scales. The inset depicts the distribution, Ai, of bound peptides per vesicle at this P/L ratio.
peptide, there are vesicles containing an insufficient number of peptides required to form a single pore. Consequently, only vesicles containing more than six peptides have a certain probability to show leakage due to pore formation, which is reflected in the final leakage extent of about 65%. The complete leakage of all vesicles as found in the experiment suggests that just binding of the peptide to the membrane also destabilizes the lipid bilayers. Here, we propose that the initial binding of the peptide replaces the small counterions bound at the lipid head groups. This replacement by the large peptide requires space between neighboring lipid molecules; thus, a significant expansion of the polar region must be expected. On the other hand, such an asymmetric expansion that is not compensated by the hydrophobic chains induces curvature stress on the membrane. This effect could be responsible for instabilities that cause slow leakage effects found experimentally. Discussion The ITC experiments have shown a strong interaction of the peptide with negatively charged membranes with an intrinsic binding constant between 2 × 104 and 1 × 105 M-1 depending on the surface potential of the model system. Similar values have also been reported for pore forming peptides, for example, Melittin and for PGLa or Penetratin binding to lipid model membranes composed of charged and zwitterionic lipid vesicles and mixtures thereof.13,18,34,35 Membrane selectivity was almost exclusively caused by electrostatic contributions but not by hydrophobic interactions. For dicynthaurin (ala) monomer, a similar behavior of almost constant intrinsic binding constants is also present at XPG < 0.85 (see Table 2); however, especially under high salt conditions and high PG content, a maximum in the Kb values at about XPG ) 0.95 is observed. These findings suggest that adding a small amount of PC to a completely negatively charged PG membrane induces defects in the lipid head group organization. As the lipid tail regions are identical, complete miscibility is suggested, but the charge density profile at the interface must be modulated and, consequently, the surface charge density is affected. Alteration of the surface charge density could modify the peptide’s ability to fold into the R-helical secondary structure
6286 J. Phys. Chem. B, Vol. 111, No. 22, 2007 and thus modify the chemical binding potential. Another effect that would enhance the chemical binding potential is given by improved insertion into the hydrophobic region of the bilayer. Such an insertion requires transfer through the lipid head group region. Therefore, the data obtained suggest that, for pure POPG, the charge density is too high for a very effective insertion, as the polar amino acids cannot go through the highly charged surface. Addition of POPC yields a drop in the surface charge density and thus increases the ability of the peptide to insert into the hydrophobic core. At the same time, this reduction in the surface charge density decreases the peptide accumulation at the bilayer surface due to electrostatic interactions. Consequently, the two concurrent effects, namely, the drop in the surface bound peptide concentration and the increased ability of the peptide to insert and thus go through the polar part, yield an optimum for insertion. Strong binding also displaces the adsorbed counterions bound at the lipid head groups. This would alter the entropic effects and thus explain the decrease found for T∆S on going from 140 mM NaCl toward no additional salt. A large chemical binding potential, Kb, was found under low and high salt conditions. Insertion of the peptide into the hydrophobic membrane core would require the release of water from the surface of the nonpolar amino acids, an entropic phenomenon known as the hydrophobic effect.32 Indeed, comparing the enthalpy of binding with the T∆S value shows larger entropy contributions to the total free energy of binding, suggesting that the hydrophobic effect must be taken into account. In addition, a change in the conformation of the peptide upon binding could be relevant for the large Kb value. However, for the peptide under investigation, such a change in conformation was studied recently by comparing the CD data of bulk peptide solution with IRRAS data obtained in lipid monolayers.11 The result suggests largely R-helical contributions in both, peptide bulk solution and peptide at the interface after adsorption toward a DPPG monolayer, thus suggesting that a dramatic change in the secondary structure can be excluded. Consequently, we must assume that the water release from nonpolar sites of the peptide is a major driving force for the insertion of the peptide. The ITC analysis yields the effective charge of the peptide that is being reduced from the formal charge by about 80% (zP/z ∼ 0.17). Such a reduction has also been found for other peptides in the literature.13,36,37 However, taking the nonideal additivity of the hydrophobic and the electrostatic free energies in membrane partitioning of indolicidin as derived by Ladokhin et al.37 into account, a binding free energy of about -40 kJ/mol (see Table 2) would lead to a zP/z value between 0.18 and 0.40, thus strongly supporting our findings. In recent studies, the stoichiometry obtained in the complex formation model has been related to the effective charge obtained from the surface partitioning equilibrium model.36 Using this approach for our system could give additional evidence for this effective charge reduction. Taking both the stoichiometry of L/P ∼ 0.6-0.8 obtained in the complex formation model and the accessibility of the lipid of about 0.6 into account gives 0.48, a value that closely matches the effective charge obtained in the fit (zP ) 0.5). Dicynthaurin displays a high broad spectrum activity at low and high salt concentrations with increased effects under low salt conditions.10 The binding parameters obtained from the ITC study show enhanced electrostatic contributions under low salt conditions, indicating that the critical surface concentration of peptides required for membrane insertion could be reached
Wen et al.
Figure 9. Hydrophobicity plot of dicynthaurin (ala) monomer obtained using the whole residue free energy of transfer from water to the POPC interface.
already at lower peptide bulk concentration. This could already explain the enhanced in vivo activity at lower salt concentrations. On going to low salt conditions, the increase in the electrostatic contribution is larger than the change in the hydrophobic interactions; therefore, the partitioning constant is enhanced (see the Kapp values in Tables 2 and 3). Obviously, at low salt concentration, optimal binding conditions are not limited to a very narrow concentration window (0.9 < XPG < 1); instead, a broad optimal concentration range starting from XPG ) 0.8 (see Tables 2 and 3) exists. It should be noted that such a high content of negatively charged lipids is indeed found in bacterial cytoplasmic membranes.38 Comparing the intrinsic binding constants under low and high salt conditions yields similar results that are in the same order of magnitude, thus providing significant evidence for membrane insertion in both cases. Such membrane insertion could explain the results from the activity study showing a high capacity for killing bacteria by targeting at the cell membrane. Given the sequence of dicynthaurin (ala) monomer, one could calculate the free energy of transfer of the sequence from the bulk to the membrane surface using the whole residue interfacial hydrophobicity scale.39 The final location of the peptide within the membrane will depend on the balance between hydrophobic interactions, electrostatic interactions, bilayer effects, other interactions, and the conformational states of the peptide. Figure 9 shows the hydrophobicity plot of dicynthaurin (ala) monomer obtained using the whole residue free energy of transfer from water to the POPC interface of Wimley and White.39 This model gives a total free energy of transfer of +15.5 kJ/mol; thus, membrane binding could only be possible assuming an R-helical conformation. Indeed, a detailed CD study10 has shown that dicynthaurin exhibits largely R-helical conformations (residues 3 up to 25) in the bulk, and IRRAS studies have confirmed an R-helical conformation of the peptide bound to a PG lipid monolayer.11 Secondary structure formation is driven by the reduction in the free energy of partitioning of peptide bonds upon hydrogen bond formation. For melittin, it was shown that the reduction is about 1.7 kJ/mol per peptide bond for R-helix formation.40 Taking these findings into account, dicynthaurin (ala) monomer shows an accumulative effect of this modest reduction of -38.5 kJ/mol and therefore a free energy of binding of about -23 kJ/mol is obtained. ITC has yielded a free energy of the interaction of about -38 kJ/mol under high salt conditions. Given the fact that the interaction involves binding, folding, and insertion of the peptide, a free energy of insertion of -15 kJ/mol can be estimated.
Dicynthaurin Monomer Interaction with Phospholipids Conclusion This is the first study on dicynthaurin (ala) monomer interacting with zwitterionic and negatively charged vesicles. The analysis of the ITC data using the surface partitioning equilibrium model has revealed a high capacity for membrane insertion. The improved in vivo activity of the peptide under low salt conditions is attributed to the enhanced electrostatic interaction leading to accumulation of the peptide at the interface. Fluorescent leakage in combinations with DLS measurements have shown leakage of the fluorescent dye upon peptide addition. The analysis of the data suggests that initial pore formation could be taken into account to explain the leakage kinetics up to 1000 s. From the DLS data, a detergentlike mode of action as found for other systems must be excluded.41 The analysis of the final extent of leakage by the reversible surface aggregation model yields a minimum aggregation number per pore of 7 ( 2, a value that is in the range as obtained also for other pore forming peptides.24,42,43 Such pore formation at the bacterial cytoplasmic membrane would cause ion efflux and finally cell death on short time scales. Therefore, our findings suggest that targeting at the cell membrane and subsequent pore formation are responsible for the high in vivo activity of the peptide. Acknowledgment. This work was supported by the Deutsche Forschungsgemeinschaft (Emmy Noether Grant BR 1826/2-3). We gratefully acknowledge Dr. H. Binder, L. Thomas, and T. Sigmund for helpful discussions and experimental support. References and Notes (1) Boman, H. G.; Marsh, J.; Goode, J. A. Antimicrobial Peptides; John Wiley and Sons: Chichester, U.K., 1994; pp 1-272. (2) Wu, Z. B.; Cocchi, F.; Gentles, D.; Ericksen, B.; Lubkowski, J.; DeVico, A.; Lehrer, R. I.; Lu, W. Y. FEBS Lett. 2005, 579, 162-166. (3) Owen, S. M.; Rudolpil, D. L.; Wang, W.; Cole, A. M.; Waring, A. J.; Lal, R. B.; Lehrer, R. I. AIDS Res. Hum. RetroViruses 2004, 20, 11571165. (4) Hancock, R. E.; Scott, M. G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8856-8861. (5) Hancock, R. E. W. Expert Opin. InVest. Drugs 2000, 9, 17231729. (6) Lohner, K. The role of membrane lipid composition in cell targeting of antimicrobial peptides. In DeVelopment of NoVel Antimicrobial Agents: Emerging Strategies; Lohner, K., Ed.; Horizon Scientific Press: Norfolk, U.K., 2001; pp 149-165. (7) Matsuzaki, K.; Sugishita, K.; Ishibe, N.; Ueha, M.; Nakata, S.; Miyajima, K.; Epand, R. M. Biochemistry 1998, 37, 11856-11863. (8) Oren, Z.; Shai, Y. Biopolymers 1999, 47, 451-463. (9) Shai, Y. Biochim. Biophys. Acta 1999, 1462, 55-70. (10) Lee, H. I.; Lee, Y. S.; Kim, H. C.; Kim, C. R.; Hong, T.; Menzel, L.; Boo, L. M.; Pohl, J.; Sherman, M. A.; Waring, A. J.; Lehrer, R. I. Biochim. Biophys. Acta 2001, 1527, 141-148.
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