Aggregation of a Peptide Antibiotic Alamethicin at the Air−Water

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Langmuir 2008, 24, 11770-11777

Aggregation of a Peptide Antibiotic Alamethicin at the Air-Water Interface and Its Influence on the Viscoelasticity of Phospholipid Monolayers Rema Krishnaswamy,* Vikram Rathee, and A. K. Sood† Department of Physics, Indian Institute of Science, Bangalore 560012, India ReceiVed June 24, 2008. ReVised Manuscript ReceiVed August 21, 2008 The aggregation properties of an antibiotic membrane-active peptide alamethicin at the air-water interface have been studied using interfacial rheology and fluorescence microscopy techniques. Fluorescence microscopy of alamethicin monolayers revealed a coexistence of liquid expanded (LE) and solid phases at the surface concentrations studied. Interfacial oscillatory shear measurements on alamethicin monolayers indicate that its viscoelastic properties are determined by the area fraction of the solid domains. The role of zwitterionic phospholipids dioleoylphosphatidyl choline (DOPC) and dioleoylphosphatidyl ethanolamine (DOPE) on the peptide aggregation behavior was also investigated. Fluorescence microscopy of alamethicin/phospholipid monolayers revealed an intermediate phase (I) in addition to the solid and LE phase. In mixed monolayers of phospholipid (L)/alamethicin (P), with increase in L/P, the monolayer transforms from a viscoelastic to a viscous fluid with the increase in area fraction of the intermediate phase. Further, a homogeneous mixing of alamethicin/lipid molecules is observed at L/P > 4. Our studies also confirm that the viscoelasticity of alamethicin/phospholipid monolayers is closely related to the alamethicin/phospholipid interactions at the air-water interface.

1. Introduction The antimicrobial activity of peptides like alamethicin, which do not require specific receptor sites involve membrane disruption.1 This has sparked recently, a lot of interest in developing these antimicrobial peptides, as novel antibiotics2 or as potential therapeutic agents in the treatment of lung infection.3 The physical mechanism which governs the lipid association with peptides and their structural organization is the delicate balance between electrostatic and hydrophobic interactions. Hence a systematic investigation of the peptide/protein interactions with the lipid matrix is crucial in developing a rational drug design for biomedical applications. Interestingly, understanding the aggregation behavior of amphipathic peptides and their viscoelastic behavior at fluid interfaces also allows for the use of synthetic peptides as emulsifying agents or emulsion stabilizers in the food and drug industry. Alamethicin is an antibiotic peptide isolated from the fungus Trichoderma Viride. The peptide consisting of 20 amino acid sequences has a secondary R-helical structure. The hydrophyllic amino acid residues align on one side and the hydrophobic ones along the opposite side of the helix making the molecule amphipathic. Amphipathic nature enables the peptide insertion into the lipid bilayer, close to the lipid-water interfaces, and is correlated with stronger antimicrobial activity. Since the primary action of many of the membrane active peptides involve their interaction with monolayer leaflets of the cell membrane, lipid/ peptide monolayers at air-water interfaces have been successfully used as model systems for the study of their interactions.4 Recently there have been extensive studies on the kinetics and equilibrium properties of alamethicin adsorbed at air-water * Corresponding author. E-mail: [email protected]. † CSIR Bhatnagar Fellow, Department of Physics, Indian Institute of Science, Bangalore 560012, India.

(1) Brogden, K. A. Nature 2005, 3, 238. (2) Robertson, B.; Johansson, J.; Curstedt, T. Molec. Med. Today 2000, 6, 119. (3) Zhang, L.; Parente, J.; Harris, S. M.; Woods, D. E.; Hancock, R. E. W.; Falla, T. J. Antimicrob. Agents Chemother. 2005, 49, 2921. (4) Maget-Dana, R. Biochim. Biophys. Acta 1999, 1462, 109.

interfaces and on lipid monolayers.5,6 However, few studies correlate the morphology of the adsorbed layers at the interface with the surface viscoelastic properties of the monolayer. Interfacial shear rheological measurements can be used to study the peptide-peptide interactions or peptide-lipid interactions at the interface by measuring the force necessary to deform the films under different thermodynamic conditions.7 Viscoelastic behavior of proteins and biopolymers at fluid interfaces have been attributed to their lateral interactions through hydrogen bonding, electrostatic interactions, or covalent bonding. Molecular conformations as well as formation of 2D networklike structures, which may be transient, as in hydrogen bonding between neighboring molecules and physical entanglements, or permanent due to covalent cross-linking, can also enhance the viscosity of the monolayers.7 Interfacial shear viscosity in monolayers of phospholipid or surfactants is also known to arise from the longrange repulsive interactions between the liquid condensed (LC) domains dispersed in a liquid expanded (LE) phase.8 However, many of these monolayers under steady shear exhibit a nonlinear rheological response, as a shear thinning power law fluid with no correspondence between the dynamic and steady shear viscosities.9 Hence it is more appropriate to measure their linear viscoelastic properties than the steady shear viscosity in order to correlate their mechanical properties with their equilibrium structure. In the present work, we study the mechanical properties of alamethicin monolayers with respect to their aggregation behavior at air-water and lipid-water interfaces. These studies were carried out using a biconical bob interfacial shear rheometer and takes into account the correction to the flow field from the bulk (5) Volinsky, R.; Kolusheva, S.; Berman, A.; Jelinek, R. Langmuir 2004, 20, 11084. (6) Ionov, R.; El-Abed, A.; Angelova, A.; Goldmann, M.; Peretti, P Biophys. J. 2000, 78, 3026. (7) Bos, M. A.; van Vliet, T. AdV. Colloid Interface Sci. 2001, 91, 437. (8) Ding, J.; Warriner, H. E.; Zasadzinski, J. A. Phys. ReV. Lett. 2002, 88, 168102. (9) Twardos, M.; Dennin, M. Langmuir 2003, 19, 3542.

10.1021/la8019765 CCC: $40.75  2008 American Chemical Society Published on Web 09/27/2008

Aggregation and Influence of Alamethicin

phases based on the analysis by Oh and Slattery.10,11 We probe the viscoelastic properties of alamethicin monolayers at different surface concentrations where a coexistence of liquid expanded and solid phases is observed. Above a critical area fraction of the solid domains, a transition from a viscous to a viscoelastic behavior is observed, indicated by a sharp increase in the storage and loss moduli. The elastic behavior of the monolayer suggests a long-range correlation of the domains in the monolayer. We have also investigated the monolayer morphology with peptides adsorbed at lipid-water interfaces or in lipid/peptide mixed monolayers. Fluorescence micrographs reveal an intermediate phase distinct from the solid and liquid expanded phases in alamethicin/lipid monolayers. Moreover, a drastic decrease in the storage modulus of the monolayer is observed with increase in the lipid/peptide molar ratio followed by a transition from an elastic to a viscous behavior. This is consistent with a lower intrinsic order of the intermediate phase as compared to the solid domains of alamethicin monolayers. Our studies thus reveal that the viscoelastic behavior of monolayer is closely related to the monolayer morphology. Moreover, the interaction of the lipids with the amphipathic peptides play a crucial role in regulating the viscoelastic properties of monolayers. These studies which characterize the mechanical properties of alamethicin/lipid monolayers and help to correlate their structural and functional aspects can have important biological and technological implications.

2. Experimental Details 2.1. Lipids and Peptides. Alamethicin (Sigma, MW 1959.9) was used as received. It was dissolved in ethanol (BDH), and appropriate amounts of the solution were spread on the aqueous surface (deionized water, Millipore) using a microsyringe (Hamilton) to obtain the desired surface concentration. The aqueous solution (deionized water with resistivity 18 MΩ was used) contained 0.1 M NaCl (pH 7) which was adjusted with 10-3 M phosphate buffer (Na2HPO4:NaH2PO4 ) 1:1, pa grade, Merck). 1,2-Dioleoyl-snglycero-3-phosphatidyl choline (DOPC) or 1,2-dioleoyl-sn-glycero3-phosphatidyl ethanolamine (DOPE) was used to form phospholipid monolayers. To form lipid/peptide monolayers, the requisite amount of DOPC/DOPE dissolved in chloroform to obtain desired surface concentration was spread at the air-water interface. Appropriate concentration of alamethicin was then injected from ethanol solution into the monolayer. To form alamethicin/lipid mixed monolayers, the two components were premixed in a chloroform-methanol (1:1) solution at the required molar ratios and spread at the air-water interfaces. All spread films were left to equilibriate for about 30 min before making further measurements. All measurements were carried out by keeping the temperature fixed at 20 °C. 2.2. Fluorescence Microscopy. Fluorescence microscopy was used to visualize the monolayers at different surface concentrations. Monolayers were imaged using an Olympus fluorescence microscope and a charge-coupled device (CCD) camera (Roper Scientific). The monolayers for fluorescence microscopy observations were formed in a Teflon Petri dish where appropriate amounts of the sample was spread at the air-water interface. Fluorescence microscopy is a useful technique to visualize the coexistence of liquid expanded phases (LE) and solid domains in lipid monolayers. The fluorescent probe Lissamine Rhodamine PE (L1375, Molecular Probes) is amphiphilic and mixes uniformly in the liquid expanded phase of alamethicin or lipid molecules revealing a bright featureless image. Since the dye is expelled from the solid domains, they appear dark. Hence the coexistence of the two phases is indicated by the presence of bright and dark regions in the micrographs. The dye:alamethicin/ lipid ratio was fixed at 1:200. Images were digitized and the image analysis was carried out using NIH Image-J software. The area fraction of the bright and dark regions were estimated at different (10) Oh, S.; Slattery, J J. Colloid Interface Sci. 1978, 67, 516. (11) Erni, P.; Fischer, P; Windhab, E. J ReV. Sci. Instrum. 2003, 74, 4916.

Langmuir, Vol. 24, No. 20, 2008 11771 surface concentrations for alamethicin monolayers in the coexistence region. For lipid/alamethicin monolayers, three gray levels bright, dark, and medium are deduced from the images corresponding to the coexistence of liquid expanded, solid, and intermediate phases. 2.3. Interfacial Shear Rheology. All rheological measurements were carried out using an interfacial rheology system (IRS) which consists of a commercial research rheometer (Physica MCR from Anton Paar) with an interfacial rheology cell based on bicone geometry.11 In this shear cell, the interfacial area cannot be varied continuously. However, the surface pressure of the insoluble monolayers can be controlled by varying the interfacial concentration. In general, the flow at the interface cannot be decoupled from the bulk flow. Hence the stress exerted on the probe placed at the interface has contributions from the surface as well as the bulk phases. For the present geometry, Oh and Slattery have provided an exact solution for the velocity distribution in both liquids and in the plane of the interface by solving the Stokes equation.10 Once the velocity distribution is known, the torque exerted on the disk by both liquids and the interface is obtained. The raw data was numerically analyzed (using the software supplied by Anton Paar) based on the analysis by Oh and Slattery after each measurement to determine the interfacial modulus and the interfacial steady shear viscosity and takes into account the correction to the flow field from the bulk phases. For air-water interfaces, the rheometer should not be used below a critical value of the Boussinesq number Bo ) 0.01(Bo ) η/[(η1 + η2)R], where η1 and η2 are the bulk viscosities, η is the interfacial viscosity, and R is a characteristic distance of the flow geometry which in the present case is 40 mm). For a 2D film, the in-plane shear deformation at the interface arises due to a change in the shape of the surface, while keeping the area constant. For a viscoelastic surface, on applying a sinusoidal shear deformation γ ) γo exp(iωt) at an angular frequency ω and strain amplitude γo, an out of phase response is obtained for the deviatoric part of the surface stress tensor defined as σ ) G*γ, where G* ) G′ + iG′′. Here, G′ and G′′ correspond to the interfacial storage and loss modulus which describes the elastic and viscous response of the film, respectively. In amplitude sweep experiments, an oscillatory shear of different strain amplitudes is applied at a constant angular frequency. At low strain amplitudes, G′ and G′′ remain constant and corresponds to the linear viscoelastic regime. By applying an oscillatory shear of low strain amplitude at different angular frequencies, linear viscoelastic measurements probe the structural relaxation of the film. At large strain amplitudes, in amplitude sweep experiments, the viscoelastic response becomes nonlinear when G′ and G′′ are no longer constant, but decay monotonically. Recently, it was seen that at high surface concentrations, Langmuir monolayers of sorbitan tristearate exhibits a distinct nonlinear viscoelastic behavior.12 Under oscillatory shear, at large strain amplitudes, G′ decreases monotonically whereas G′′ exhibits a peak before it decays at larger strain amplitudes with the decay exponents of G′ and G′′ in the ratio 2:1. To follow the film buildup at the interface, an oscillatory shear of strain amplitude 0.05% at an angular frequency of 5 rad/s was applied. All measurements were made on the film only after G′ and G′′ reached the saturation, which is after 1000 s. Different measurements for a given concentration were made on a freshly spread film and the preshear ensures that, at a given concentration and temperature, the initial conditions are identical.

3. Results and Discussion 3.1. Alamethicin Monolayers at Air-Water Interfaces. 3.1.1. Fluorescence Microscopy. The pressure-area isotherms of alamethicin and alamethicin/lipid mixed monolayers formed at air-water interfaces have been well-characterized through film-balance techniques5,6 (Figure 2a, inset). A sharp rise in the surface pressure is observed at a molecular area of 3.2 nm2/ molecule which corresponds to the projected area of an alamethicin helix oriented parallel to the air-water interface. (12) Krishnaswamy, R.; Majumdar, S.; Sood, A. K Langmuir 2007, 23, 12951.

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Figure 1. Fluorescence micrographs of alamethicin monolayers formed at air-water interfaces at different surface concentrations (A) 8, (B) 7, (C) 5.9, (D) 4.4, (E) 3.5, and (F) 0.9 nm2/molecule. The scale bar corresponds to 50 µm.

Figure 2. (a) Storage modulus (G′) obtained at different surface concentrations of alamethicin (expressed in terms of area per molecule A (nm2/molecule)). The P-A isotherm obtained from6 is shown in the inset). (b) Area fraction of solid domains estimated from the fluorescence micrographs at different surface concentration of alamethicin. The dependence of the storage modulus on the area fraction of solid domains are shown in the inset. In these measurements, an oscillatory shear corresponding to the linear viscoelastic regime is applied keeping the strain amplitude and the angular frequency fixed at 0.01% and 5 rad/s, respectively.

This is followed by a plateau region in the P-A isotherm indicating that the peptide monolayer collapses to form multilayers and rules out the reorientation of the R helix perpendicular to the air-water interface. To study the structural organization of alamethicin in the monolayer, the monolayers were visualized using fluorescence microscopy (Figure 1). At low surface concentrations, corresponding to an area per molecule A > 9 nm2/molecule, the monolayer appears uniformly bright. This indicates that, at these surface concentrations, the monolayer forms a liquid expanded (LE) phase. At higher surface concentrations, few dark regions appear on a uniformly bright background (Figure 1A). The dark regions correspond to two-dimensional aggregates of alamethicin which form solidlike domains. Surface X-ray scattering studies have revealed the structural organization of alamethicin molecules in these solid domains. They form a 2D crystalline lattice with an orthorhombic structure,6 with an estimated area per molecule of 3.2 nm2. The size of these aggregates increase with surface concentration, often giving rise to fractal structures (Figure 1B).

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These structures are reproducible and remain stable up to a few hours. At surface concentrations >6 nm2/molecule, the dark regions corresponding to the solid phase are distributed in the LE phase (Figure 1C). Interestingly the aggregates maintain an overall well-defined separation indicating a repulsive interaction between these domains in the monolayer. Further increase in surface concentration to 4.44 nm2/molecule decreases the extent of LE phase. Fluorescence micrographs reveal bright domains corresponding to LE phase distributed in a dark background (Figure 1D). At a surface concentration of 3.5 nm2/molecule, uniformly dark regions are observed (Figure 1E), confirming the presence of a solid phase alone. At higher surface concentrations, distinctly bright domains in addition to dark regions are observed. These bright regions indicate the collapse of monolayers to form multilayers (Figure 1F). The fluorescence micrographs of alamethicin monolayers are consistent with that reported in earlier studies.5,6 The coexistence region of LE and solid phases correspond to the plateau in the P-A isotherms reported for alamethicin at air-water interfaces. The appearance of the solid domains above a surface concentration of 9 nm2/molecule, reveal the presence of a threshold surface concentration above which the peptide can aggregate. The distribution of the size and shape of these domains are determined by the competing repulsive dipole-dipole interactions and line tension.13 The uniformly dark region above a surface concentration of 3.5 nm2/molecule also suggests that the solid domains are closely packed in the monolayer. 3.2. Interfacial Oscillatory Shear on Alamethicin Monolayer. 3.2.1. Frequency Sweep. To study the viscoelasticity of the monolayer, an oscillatory shear of a strain amplitude of 0.01% is applied at an angular frequency of 5 rad/s. The dependence of the interfacial storage modulus (G′) on the surface concentration (expressed in terms of area per molecule A) of alamethicin is shown in Figure 2a. For A > 9 nm2/molecule, the monolayer exhibits a viscous behavior with G′ ∼ 0. An asymptotic increase in G′ is observed at higher surface concentrations. This is followed by a maxima in the storage modulus at a surface concentration of 2.5 nm2/molecule. In the present system, these values of A correspond to the plateau in the P-A isotherm, beyond the solid phase, with coexisting phases, where the film starts to collapse to a three-dimensional state5,6 to form a multilayer. The increase in storage modulus is also accompanied by a corresponding increase in the loss modulus (data not shown). With further increase in the surface concentration, though G′ decreases, the viscoelastic behavior is retained. In monolayers of alamethicin, the viscoelastic behavior arises not from a physical cross-linking or entanglement of molecules but from the monolayer morphology. The rigidity of the monolayers is closely linked to the presence of solid domains. This is evident from Figure 2b (inset) where an asymptotic increase in G′ is observed above a critical area fraction of solid domains in the monolayer. Hence the monolayers exhibit an elastic behavior at surface concentrations lower than that corresponding to the transition to the solid phase in the P-A isotherm. These studies indicate that the rheological transitions in the monolayers can be quite distinct from the P-A isotherms. The onset of rigidity of the monolayer at a surface concentration of 8 nm2/molecule, well before the domains are in close contact, imply the long-range correlation of the solid domains in the monolayers. At high surface concentrations where a collapse of the monolayer to form a stacked lamellae or multilayers occur, there is a corresponding decrease in the storage modulus. (13) Seul, M.; elman, D. Science 1995, 267, 476.

Aggregation and Influence of Alamethicin

Figure 3. Frequency sweep measurements on alamethicin monolayers at different surface concentrations (a) 8, (b) 3.5, and (c) 0.9 nm2/molecule. The strain amplitude is fixed at 0.01%. Strain amplitude sweep measurements on alamethicin monolayers formed at different surface concentrations (d) 8, (e) 3.5, and (f) 0.9 nm2/molecule are shown. The angular frequency is fixed at 5 rad/s.

Frequency sweep measurements were carried out for probing the structural relaxation in these films by varying the angular frequency in the range 0.01 to 50 rad/s with the strain amplitude fixed at 0.01% (Figure 3). For 8 < A < 4.44 nm2/molecule, the film remains viscoelastic and exhibits an average structural relaxation time (τ) of 10 s which remains nearly independent of surface concentration (Figure 3a). On decreasing the area/ molecule further, up to A ) 2.5 nm2/molecule, the film remains elastic (G′ > G′′) over the range of angular frequencies probed. On examining the frequency spectra at these surface concentrations it is seen that at frequencies 4.4 nm2/molecule, with large area fractions of solid domains in the monolayer, the film exhibits an elastic behavior. Here, the frequency response of the monolayer is strongly reminiscent of soft glassy systems. Soft glassy rheology model14 does predict such a low frequency behavior just below the glass transition with G′ nearly a constant and G′′ ∼ (ωt)x-1 where t is the age of the system and effective temperature “x” ) 1 corresponds to the glass transition. Accordingly, x is 0.77 for the alamethicin monolayer. 3.2.2. Nonlinear Viscoelasticity of Alamethicin Monolayers. To characterize the strength of the monolayers and to study its response to large deformations, the storage and loss modulii are measured as a function of the strain amplitude keeping the angular frequency fixed at 5 rad/s. At low strain amplitudes G′ and G′′ remain nearly constant. This is the linear viscoelastic regime, where the stress is linearly dependent on the strain imposed. As (14) Sollich, P.; Lequex, F.; Hebraud, M.; Cates, M. E. Phys. ReV. Lett. 1997, 78, 2020.

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seen from Figure 3, the extent of the linear region is rather low in comparison with monolayers of insoluble surfactants12 and weakly depends on the surface concentration. This is consistent with the predictions of soft glassy rheology model where the size of the linear regime decreases as glass transition is approached. The noteworthy feature of the nonlinear rheological response of these monolayers is the large strain amplitude behavior. The response is distinct from the strain softening observed for example, in cytoplasmic extracts of Xenopus egg,15 or the strain stiffening seen in cytoskeletal networks or red blood cells.16 In a typical strain softening behavior, both G′ and G′′ decay monotonically above a critical strain amplitude. On the other hand under strain stiffening, a peak in G′ is observed above a critical strain amplitude. In the present system, we observe a monotonic decay of the storage modulus above a critical strain amplitude. However, a peak is observed in the loss modulus (G′′), followed by a decrease at higher strain amplitudes. Interestingly, the peak in G′′ is observed for surface concentrations in the range of 8 < A < 3.5 nm2/molecule (Figure 3c and d). This is also accompanied by a shift in the peak position to higher strain amplitudes with increase in surface concentrations. At large strain amplitudes, in addition to the peak in G′′, a power law decay of the storage and loss moduli is observed with G′ ∼ |γ|-2ν and G′′ ∼ |γ|-ν. The power law exponent ν is 0.8 at the highest surface concentration of 3.5 nm2/molecule. The peak in G′′ at high strain amplitudes is proposed to arise from the dependence of the structural relaxation time τ, on the strain rate amplitude,17 a behavior known to be prominent in systems which exhibit slow dynamics including glassy systems. Moreover, the peak in the loss modulus is also consistent with the soft glassy rheology model.14 Simulations of large amplitude sweeps carried out on 2D suspensions of electrorheological fluids, consisting of percolating clusters of particles with repulsive dipole-dipole interactions, have shown that rearrangement of the clusters rather than a large scale breakdown of the structure can give rise to the nonlinear viscoelastic behavior under study.18 Hence it is likely that the nonlinear viscoelastic behavior of Alamethicin monolayers arises from the rearrangement of the solid domains in the monolayer at large strain amplitudes. Successive amplitude sweeps on the same film leads to a decrease in the storage and loss moduli indicating that applying oscillatory shear of large strain amplitude results in the fragmentation of the film with no apparent structural recovery after the imposed shear stress is removed. Hence, the structural rearrangement of these domains at large strain amplitudes results in a viscous shear thinning followed by a fragmentation of the solid domains at higher strain amplitudes. The shift in the peak position to higher strain amplitudes with increase in surface concentration also reveals that as the area fraction of the solid domains in the monolayer increases, the strength of the monolayer increases and a larger strain amplitude is required to induce the rearrangement of solid domains and the subsequent shear thinning of the monolayer. With the increase in surface concentration to 0.9 nm2/molecule (Figure 3f), a monotonic decay of the storage and loss modulus is observed. This indicates that, for multilayers, the plastic flow behavior occurs at lower strain amplitudes, compared to monolayers. (15) Valentine, M. T.; Perlman, Z. E.; Mitchison, T. J.; Weitz, D. A Biophys. J. 2005, 88, 680. (16) Storm, C.; Pastore, J. J.; MacKintosh, F. C.; Lubensky, T. C.; Janmey, P. A. Nature 2005, 445, 191. (17) Miyazaki, K.; Wyss, H. M.; Weitz, D. A.; Reichman, D. A. Europhys. Lett. 2006, 75, 915. (18) Parthasarathy, M.; Klingenberg, D. J. J. Non-Newtonian Fluid Mech. 1999, 81, 83.

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Figure 4. (a) Interfacial shear stress vs shear rate curves for alamethicin monolayers at a surface concentration A ) 4.4 nm2/molecule. The yield stress obtained from the flow curves at different surface concentrations are shown in the inset. (b) Dynamic viscosity as a function of angular frequency (ω) denoted by closed triangles, dynamic viscosity as a function of shear rate amplitude |γ˙ | ) |γ|ω denoted by open triangles, and steady shear viscosity as a function of shear rate (γ˙ ) denoted by closed circles measured on alamethicin monolayers at a surface concentration A ) 4.4 nm2/molecule are shown.

3.3. Alamethicin Monolayers under Steady Shear. We have investigated the flow behavior under steady shear of alamethicin monolayers at different surface concentrations. A typical flow curve obtained at an alamethicin surface concentration of 4.44 nm2/molecule is shown in Figure 4. The shear rates are varied in the range 0.001 to 10 s-1. At low shear rates, a finite yield stress is observed which persists up to 0.05 s-1 beyond which a sharp drop in stress is observed (Figure 4a). Consistent with the nonlinear flow behavior observed over a large range of shear rates in these systems, the steady shear viscosity is distinct from the dynamic viscosity (η*(ω)) (Recall that |η*(ω)| ) [(G′)2 + (G′′)2]1/2/ω) obtained from linear viscoelastic measurements (Figure 3b)). In addition, there is no correlation between steady shear viscosity (η(γ˙ )) and the complex dynamic viscosity η*(|γ|ω) where |γ|ω is the shear rate amplitude. This implies that the behavior of alamethicin monolayers under steady and oscillatory shear is distinct though they involve a large scale deformation. It is also seen that though the yield stress of the monolayer increases with surface concentration of the peptide (Figure 4a, inset), the flow behavior remains insensitive to the monolayer composition. The presence of a finite yield stress indicates the jamming of the solid domains in the monolayer and is consistent with the soft glassy behavior inferred from linear viscoelastic measurements. Moreover, the sharp increase in yield stress at a surface concentration of 3.5 nm2/molecule is consistent with the transition to a solid phase in the P-A isotherm. Under steady shear, the drop in stress at high shear rates indicate a transition from a jammed solidlike state to a fluidlike state when the monolayer begins to flow. These studies reveal that under steady shear, alamethicin monolayers do not exhibit a power law flow behavior that is reported in fatty acid monolayers9 or 2D cross-

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linked Actin networks.19 The surface viscosity of the monolayers in the fluid phase is found to be 10-5 Pa · s · m (2 orders of magnitude higher than the instrumental limit which is 10-7 Pa · s · m at the air-water interface). This is higher than that reported for LE phases of phospholipid/fatty acid monolayers which is ∼10-10 Pa · s · m.20 The change in the morphology of the liquid condensed (LC) domains in the monolayers of fatty acids under shear have been examined in channel flow experiments.21 It was proposed from these studies that, in addition to a change in the shape of domains under shear, the shear thinning behavior of the monolayer arises from the slippage of the neighboring domains in multidomain groups, which increases with the shear rate. Moreover, on cessation of shear, the domain clusters fail to recover their initial configuration, after the slippage. We suspect that a similar mechanism gives rise to the shear thinning behavior of alamethicin monolayers. 3.4. Surface Rheology of Alamethicin/Lipid Monolayers. The penetration of antimicrobial peptides into lipid monolayers and their interactions in mixed lipid/peptide monolayers have been investigated extensively from pressure-area isotherms.4 However, there are very few reports on the surface rheology of these films. We examine the correlation of the aggregation behavior of alamethicin on lipid monolayers with the viscoelastic properties of alamethicin/lipid monolayers. Elastic properties of monolayer consisting of aggregates of antimicrobial peptides on the lipid monolayer determine its deformation behavior. Hence the study of mechanical properties of alamethicin/lipid monolayers are also relevant in probing the permeability of lipid monolayer for ions in its function as ion channels in lipid bilayers.6 We have investigated systematically the viscoelastic properties of alamethicin/lipid monolayers. The monolayers are formed by the adsorption of alamethicin at lipid-water interfaces or by premixing the two components in an appropriate spreading solvent and spreading them at air-water interfaces. 3.4.1. Alamethicin Adsorbed on Lipid Monolayers. In the present study, to examine the kinetics of adsorption of peptide on lipid monolayers, we monitor the time dependent changes of the storage modulus under an oscillatory shear of low strain amplitude at a fixed angular frequency (Figure 5, inset). For DOPC monolayers formed at the air-water interface, no time dependent changes in the surface viscoelastic behavior was observed (inset, Figure 5, curve a). The monolayers remain viscous up to a surface concentration of 0.6 nm2/molecule. The kinetics of aggregation of alamethicin at air-water interfaces and on lipid monolayers has also been studied. An oscillatory shear is applied, keeping the strain amplitude and angular frequency fixed at 0.01% and 5 rad/s, respectively. On injecting the peptide into the water subphase, an increase in G′ is seen after 150 or 70 s depending on the surface concentration (inset, Figure 5, curves b and c). This indicates that the alamethicin spread at the airwater interface, aggregate to form stable monolayers with solid domains after a time delay of ∼150 s with the storage modulus saturating after about 1000 s. To form adsorbed alamethicin/ lipid monolayers, the peptide was injected into the subphase beneath the DOPC monolayers where the surface concentration of the lipid was varied from 6 to 0.6 nm2/molecule. The surface activity of alamethicin on lipid-water interfaces is evident (inset, Figure 5, curves d and e) where G′ increases rapidly to reach a plateau. These monolayers remain stable up to a couple of hours. However at high surface concentrations of lipids corresponding (19) Walder, R.; Levine, A. J.; Dennin, M. Phys. ReV. E 2008, 77, 11909. (20) Sickert, M.; Rondelez, F.; Stone, H. A. Europhys. Lett. 2007, 79, 660005. (21) Ivanova, A. T.; Ign e s-Mullol, J.; Schwartz, D. K. Langmuir 2001, 17, 3406.

Aggregation and Influence of Alamethicin

Figure 5. Storage modulus of monolayers formed by the adsorption of alamethicin present in the water subphase onto DOPC (closed circles) and DOPE (open triangles) monolayers at different surface concentrations. An oscillatory shear corresponding to the linear viscoelastic regime is applied keeping the strain amplitude and the angular frequency fixed at 0.01% and 5 rad/s, respectively. In the inset, the evolution of the storage modulus with time of (a) DOPC monolayers for surface concentrations ranging from 6 to 0.6 nm2/molecule, spread alamethicin monolayers at a surface concentration of (b) 8 and (c) 4.4 nm2/molecule, and monolayers formed by the adsorption of alamethicin present in the water subphase onto lipid monolayers at a surface concentration (d) 6, (e) 3, and (f) 0.6 nm2/molecule is shown. An oscillatory shear corresponding to the linear viscoelastic regime is applied keeping the strain amplitude and the angular frequency fixed at 0.01% and 5 rad/s, respectively.

to 0.6 nm2/molecule, no increase in G′ is observed (inset, Figure 5, curve f). As seen from Figure 5, the surface activity of alamethicin monolayers increase on lipid-water interfaces. Here the alamethicin/lipid monolayers are obtained by the insertion of alamethicin into the lipid monolayers. At high lipid surface concentration, alamethicin no longer penetrates the lipid monolayer. This is also consistent with the earlier studies where the alamethicin insertion into phospholipid monolayers was found to depend on the initial surface pressure.5 From linear viscoelastic measurements on these monolayers, one can also study the dependence of the monolayer rigidity on the surface concentration of lipid. As seen from Figure 5, with increase in surface concentration of DOPC or DOPE, a steady decrease in G′ occurs, indicating the transition of the monolayers from a viscoelastic to a viscous behavior. In amplitude sweep measurements, where the large strain amplitude behavior of the monolayer is examined at a constant angular frequency, the peak in G′′ is absent as the lipid surface concentration is increased above 4 nm2/molecule. The surface viscoelastic properties of the alamethicin/lipid monolayer is closely related to the morphology of the monolayer. As seen from Figure 6A-F, a marked difference in the morphology of the monolayers is observed as the surface concentration of DOPC is increased. The morphology of the monolayers where alamethicin is inserted on the lipid monolayers (B) is distinct from that seen for alamethicin monolayers at the air-water interface (Figure 6A) or for lipid monolayers (Figure 6F) at air-water interfaces. In the former (Figure 6A), we observe dark regions corresponding to the solid domains dispersed in a uniformly bright back ground (LE phase) whereas in the latter (Figure 6F), a uniformly bright background is observed corresponding to the LE phase. However when alamethicin aggregates on lipid monolayers (Figure 6B), we observe gray regions, in addition to the bright and dark domains. A closer inspection of the gray regions reveal a highly cross-linked filamentous aggregates (Figure 6B, inset). In addition, long aggregates typically 2-3 µm in width and with lengths varying from 40-100 µm are also observed (Figure 6B, inset). The area fraction of the gray regions decrease with increase in lipid surface concentration

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from 6 nm2/molecule (Figure 6C and D). At a lipid surface concentration of 2 nm2/molecule (Figure 6E), surprisingly, the gray region disappears and a few dark domains consisting of elongated aggregates dispersed in an LE phase reappears. Consistent with the earlier studies,5 fluorescence microscopy and the viscoelastic measurements on alamethicin/lipid monolayers indicate that an increase in the lipid surface concentration leads to a decrease in the number of alamethicin molecules inserted in lipid monolayers. This further inhibits alamethicin aggregation on lipid monolayers at high lipid surface concentrations. Moreover, the absence of a peak in loss modulus at high lipid surface concentrations indicate that the formation of a filamentous network (corresponding to gray regions in the fluorescence micrographs) increases the fluidity of the monolayers. Similar structures have been seen in monolayers of hydrophobic surfactant peptides SP-B and SP-C with DPPC and DPPG,22 SP-A with pulmonary surfactant lung extracts,23 and PL-A2 with phospholipids.24 These studies reveal that lipid-peptide interactions can induce rearrangement of solid domains in the monolayer leading to the formation of a meshlike network. This loosely organized network of gray domains are absent at high lipid surface concentration. The absence of dark domains at high surface concentration of lipid could be due to the exclusion of alamethicin from the monolayer, analogous to the pressure-induced exclusion in such systems.4,5 The appearance of a network, which has less contrast as compared to the dark, probe excluded solid domains and the bright liquid expanded phase can be identified as the new intermediate phase consisting of both lipids and alamethicin proposed in many lipid/peptide or lipid/phospholipase monolayers.22-24 It is likely that the fluorescence probe is partially soluble in this phase unlike the dark solid domains indicating the disordered nature or fluidity of this phase. This is further enforced by the viscoelastic measurements where a drastic reduction in the storage modulus to form a viscous monolayer is observed with increase in the area fraction of gray domains. The decrease in monolayer rigidity and the monotonic decay of storage and loss moduli in amplitude sweep measurements at large strain amplitudes also bring out the absence of long-range correlation of these domains in the monolayer. Recall that, in alamethicin monolayers formed at air-water interfaces, the peak in loss modulus is observed at all area fractions of solid domains in the monolayer. To further affirm the fluidity of the intermediate phase we have also examined the viscoelastic behavior and morphology of mixed alamethicin/lipid monolayers. 3.4.2. Alamethicin/Lipid Mixed Monolayers. Earlier studies on the alamethicin/lipid mixed monolayers have concluded the complete immiscibility of the two components from the analysis of their pressure-area isotherms at different lipid-peptide compositions.4-6 This is surmised from the absence of deviation from an ideal mixing behavior6 as well as the invariance of collapse pressure with surface composition of the two components. We have examined the mechanical properties of alamethicin/ lipid mixed monolayers keeping the surface concentration of alamethicin fixed at 6 nm2/molecule and varying the lipid/peptide (L/P) molar ratios in the range of 0.5-10. For ideal mixing, the area/molecule of the mixed monolayer would vary from 2.6 to 1.1 nm2/molecule. The linear viscoelastic properties of the monolayers formed at different peptide-lipid compositions have been examined under oscillatory shear, keeping the strain (22) Kruger, P.; Schalke, M.; Wang, Z.; Notter, R. H.; Dluhy, R. A.; Losche, M. Biophys. J. 1999, 77, 903. (23) Worthman, L. A.; Nag, K.; Rich, N.; Ruano, M. L. F.; Casals, C.; PerezGil, J.; Keough, K. M. W. Biophys. J. 2000, 79, 2657. (24) Grainger, D. W.; Reichert, A.; Ringsdorf, H.; Salesse, C. Biochim. Biophys. Acta 1990, 1023, 365.

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Figure 6. Fluorescence micrographs of spread alamethicin monolayers at a surface concentration of (A) 6 nm2/molecule, adsorbed on lipid monolayers at surface concentrations of (B) 6, (C) 4, (D) 3, (E) 2, and (F) 0.6 nm2/molecule. The white and black scale bars correspond to 10 and 50 µm, respectively.

Figure 7. Fluorescence micrographs of alamethicin/DOPC mixed monolayers obtained at DOPC/alamethicin (L/P) molar ratios (A) L/P ) 0.5, (B) L/P ) 1, and (C) L/P ) 5. The scale bar in the inset corresponds to 100 µm. (D) Dependence of storage modulus of mixed alamethicin/ lipid monolayers formed at different lipid/alamethicin molar ratios (L/P). The closed circles and the open triangles correspond to the DOPC/ alamethicin and DOPE/alamethicin mixtures, respectively. An oscillatory shear corresponding to the linear viscoelastic regime is applied keeping the strain amplitude and the angular frequency fixed at 0.01% and 5 rad/s, respectively.

amplitude fixed at 0.01% and angular frequency at 5 rad/s. The variation of the storage modulus with the lipid/peptide molar ratio is shown in Figure 7D. At L/P ) 0.5, the storage modulus decreases and a viscoelastic behavior is observed for the monolayer. At L/P ) 1 however a sharp drop in G′ by nearly 2 orders of magnitude is observed with the monolayer exhibiting a viscous behavior over the range of angular frequencies probed. Moreover on carrying out amplitude sweep measurements on lipid/peptide monolayers, no peak in loss modulus is observed. A similar behavior is observed for peptide/lipid mixed monolayers obtained with both DOPE and DOPC.

Fluorescence micrographs obtained at different lipid/peptide molar ratios (Figure 7) indicate the marked change in the morphology of alamethicin monolayers in the presence of lipids. At L/P ) 0.5 (Figure 7A), in addition to dark as well as bright regions, we observe a gray region corresponding to a meshlike network of the domains. At equimolar ratios (Figure 7B), only a homogeneous gray region is observed. The decrease in the storage modulus and the absence of the peak in loss modulus confirms the fluidity of the intermediate phase. At L/P ) 5, a homogeneous bright region (Figure 7C) is observed. 3.5. Role of Surface Rheology in Elucidating Lipid-Peptide Interactions in Monolayers. The present study highlights surface rheology of lipid/peptide monolayers as a valuable tool to identify the parameters required for optimal peptide activity in antimicrobial peptide therapeutics. Though techniques like thermodynamic analysis of surface pressure-area isotherms or fluorescence microscopy which allow the visualization of monolayer morphology are used, these studies become more meaningful only if they can be correlated with the mechanical properties of monolayers. Our studies clearly indicate that the aggregation behavior of alamethicin at air-water or lipid-water interfaces determines the viscoelastic properties of the monolayers. The viscoelastic behavior of the alamethicin monolayer formed at air-water interfaces arises from the long-range repulsive interaction of solid domains in the monolayer. At present, we do not understand the insensitivity of the average structural relaxation time of the monolayers for surface concentrations in the range 8 < A < 4.44 nm2/molecule. However, the flat viscoelastic spectra implies a distribution of relaxation times in the system. It is likely that the structural relaxation observed in the monolayer at low surface concentrations arises from the rearrangement of the solid domains under a shear stress. Due to the absence of a theoretical model for viscoelastic monolayers, the frequency spectra could not be fitted to any model that can yield a more quantitative description of the relaxation mechanism. At surface concentrations >4.44 nm2/molecule, the viscoelastic response is consistent with that proposed in the “soft” glassy rheology model below the glass transition.14 The glassy behavior arises because the solid domains

Aggregation and Influence of Alamethicin

of the monolayer are highly compressed and too closely packed to easily move past each other and rearrange. Hence our studies on the viscoelastic behavior of the monolayer indicate that as the area fraction of domains increase with alamethicin surface concentration, the monolayer behaves like a jammed thermal system. This is further confirmed by the presence of a finite yield stress at low shear rates under steady shear. The glassy behavior of monolayers have been invoked earlier, in order to explain the absence of collapse of lung surfactant monolayers when compressed at a rate higher than a threshold rate.25 However these studies did not provide any rheological or structural evidence of a glassy phase. The aggregation of the peptide at air-water interfaces is driven by its hydrophobicity which is likely to decrease when bound to lipids. A marked change in the aggregation behavior of alamethicin occurs at lipid-water interfaces, with the appearance of an intermediate phase. The fluidity of this phase is confirmed by both fluorescence measurements and surface rheology. The fluorescence micrographs of the lipid/peptide monolayers consisting of gray regions indicate the partial miscibility of the fluorescent dye, and hence a phase with lower intrinsic order than the solid domains present in alamethicin monolayers. The insertion of the peptide into the zwitterionic phospholipid monolayers is likely to arise from the dipole-dipole interaction since the peptide is not charged under the present subphase conditions (pH around 7). It is likely that the interaction of alamethicin with the zwitterionic phospholipids disrupts the close packing of peptides to form solid domains. This is also consistent with the observation of a disordered liquid expanded phase in lipid/peptide mixed monolayers at L/P > 1 where possibly the lipid bound peptide fails to aggregate at air-water interfaces. The weakly viscoelastic or viscous behavior observed for the intermediate phase in the linear viscoelastic measurements indicates the emergence of a distinct phase with high plasticity, in DOPC (DOPE)/alamethicin monolayers. This makes the monolayer more amenable to deformation and rearrangement under an external stress. A similar decrease in the monolayer rigidity has been reported recently for apolipoproteins adsorbed at lipid-water interfaces.26 The observation of the intermediate phase with meshlike filamentous aggregates has not been reported earlier in alamethicin/lipid monolayers, though a similar morphology has been seen in other lipid-peptide systems.22-24,27,28 From fluorescence microscopy studies, it was proposed that the formation of these structures increases the fluidity of the monolayer. This was inferred due to the appearance of the gray regions which implied partial miscibility of fluorescent probe in this phase. In the present study, linear viscoelastic measurements on alamethicin/lipid mixed monolayers with a homogeneous intermediate phase where a purely viscous behavior is observed, provide a direct evidence of the fluidity of this phase. It is also likely that the fluidity of this phase determines the permeability of lipid monolayer for ions in its function as ion channels in lipid bilayers. We would also like to point out that both DOPE and DOPC exhibit a similar behavior which indicate that the (25) Smith, E. C.; Crane, J. M.; Laderas, T. G.; Hall, S. B. Biophys. J. 2003, 85, 3048. (26) Bolanos-Garcia, V. M.; Renault, A.; Beaufils, S. Biophys. J. 2008, 94, 1735. (27) Gidalevitz, D.; Ishitsuka, Y.; Muresan, A. S.; Konovalov, O.; Waring, A. J.; Lehrer, R. I.; Lee, K. Y. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6302. (28) Ege, C.; Lee, K. Y Biophys. J. 2004, 87, 1732.

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headgroup does not significantly influence the lipid-peptide interactions. This is however consistent with the earlier studies where it was seen that the pressure-area isotherms are similar for DOPC/alamethicin and DOPE/alamethicin mixed monolayers.6 An interesting observation in these monolayers is also the homogeneous region of intermediate phase which was possibly missed out in P-A isotherms of mixed alamethicin/lipid monolayers. The transition was not recognized possibly because it interferes with the well characterized transition of LE/solid phases seen in alamethicin monolayers. The homogeneity of this phase also allows for the determination of their viscoelastic properties by microrheology technique, which will a subject of our future investigations.

4. Conclusions The aggregation behavior of the antimicrobial peptide alamethicin has been investigated at air-water and lipid-water interfaces using fluorescence microscopy and surface rheology techniques. Fluorescence micrographs of the monolayers reveal the coexistence of LE and solid phases with the area fraction of solid domains increasing with surface concentration of alamethicin. Linear viscoelastic measurements of the monolayer indicate an asymptotic increase of the storage modulus above a critical area fraction of solid domains. Under steady shear, the monolayers exhibit a finite yield stress which increases with surface concentration. The influence of zwitterionic phospholipids DOPC and DOPE on the viscoelastic propeties of alamethicin monolayers have also been examined. For alamethicin adsorbed on the lipid monolayers formed at the air-water interface at different surface concentrations, a steady decrease in the storage modulus is observed on increasing the lipid surface concentration. This is accompanied by a rheological transition from a viscoelastic to a viscous behavior of the monolayers. Surface rheology of mixed alamethicin/lipid monolayers also confirm the increase in the fluidity of monolayers with increase in lipid/peptide molar ratio. Fluorescence micrographs of lipid/alamethicin monolayers reveal, in addition to bright region of LE phase and dark solid phase seen in alamethicin monolayers, gray regions of filamentous network of aggregates consisting possibly of both alamethicin and lipids. The viscous behavior of the alamethicin/lipid monolayers formed at equimolar ratios consisting of homogeneous gray regions (Figure 7B), termed as the intermediate phase confirms the lower intrinsic order of this phase. In conclusion, our studies bring out clearly the relevance of surface shear rheology in conjunction with fluorescence microscopy in characterizing lipid/peptide monolayers. A systematic investigation of monolayer characteristics of antimicrobial peptides in the presence of lipids have important implications in the treatment of pulmonary lung disorders as well as in the design of novel antibiotics. Acknowledgment. A.K.S. acknowledges a CSIR Bhatnagar fellowship for financial support. R.K. thanks the Indian Institute of Science for the Centenary Postdoctoral Fellowship. The authors are grateful to Dr. V. A. Raghunathan for his help with fluorescence microscopy. The assistance of Deepthi B. K., Central Facility, Department of Physics, Indian Institute of Science, in carrying out the rheological measurements is acknowledged. LA8019765