Effect of the Concentration of Cytolytic Protein Cyt2Aa2 on the Binding

Sep 9, 2015 - Bacillus thuringiensis is known by its insecticidal property. The insecticidal proteins are produced at different growth stages, includi...
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Effect of the Concentration of Cytolytic Protein Cyt2Aa2 on the Binding Mechanism on Lipid Bilayers Studied by QCM‑D and AFM Sudarat Tharad,† Jagoba Iturri,§ Alberto Moreno-Cencerrado,§ Margareta Mittendorfer,§ Boonhiang Promdonkoy,‡ Chartchai Krittanai,*,† and José L. Toca-Herrera*,§ †

Institute of Molecular Biosciences, Mahidol University, 25/25 Phuttamonthon 4 Road, Salaya Campus, Nakhon Pathom 73170, Thailand ‡ National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand § Institute for Biophysics, Department of Nanobiotechnology, University of Natural Resources and Life Sciences Vienna (BOKU), Muthgasse 11, Vienna 1190, Austria S Supporting Information *

ABSTRACT: Bacillus thuringiensis is known by its insecticidal property. The insecticidal proteins are produced at different growth stages, including the cytolytic protein (Cyt2Aa2), which is a bioinsecticide and an antimicrobial protein. However, the binding mechanism (and the interaction) of Cyt2Aa2 on lipid bilayers is still unclear. In this work, we have used quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM) to investigate the interaction between Cyt2Aa2 protein and (cholesterol-)lipid bilayers. We have found that the binding mechanism is concentration dependent. While at 10 μg/mL, Cyt2Aa2 binds slowly on the lipid bilayer forming a compliance protein/lipid layer with aggregates, at higher protein concentrations (100 μg/mL), the binding is fast, and the protein/lipid layer is more rigid including holes (of about a lipid bilayer thickness) in its structure. Our study suggests that the protein/lipid bilayer binding mechanism seems to be carpet-like at low protein concentrations and pore forming-like at high protein concentrations.

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INTRODUCTION

to an increase in the resistance of the insect larvae to the receptor requiring Cry protein.4 Cyt proteins are produced in an inactive dimeric form. To exert their cytolytic activity, these proteins require a proteolytic activation in order to remove the N- and C-terminal amino acids of the molecule.5 An active Cyt protein can directly bind to unsaturated phospholipids in the membrane such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin.6 The 3D structure of both inactive and active Cyt proteins is formed by single α−β domains, in the shape of a β-core sandwiched by α-helices.7 Such structure would resemble that of fungal volvatoxin A2 (VVA2)8 and Erwinia virulence factor (Evf),9 both lipid binding proteins. Cyt protein was shown to exhibit in vitro cytolytic activity in various cell types including insect cells, mammalian cells, and bacteria.10,11 Alternatively, specific in vivo larvacidal activity to Dipteran insect larvae such as mosquitoes and black flies was also reported.12 Besides the cytolytic activity, Cyt proteins can also synergize their activity with Cry proteins to overcome the

Bacillus thuringiensis (Bt) is a naturally occurring Gram-positive soil bacterium known by its insecticide properties. The insecticidal proteins are produced in two different stages of the Bt growth. At the early vegetative stage, vegetative insecticidal proteins (Vip) are produced, whereas the two families of delta-endotoxin1Crystal (Cry) and Cytolytic (Cyt) proteinsare expressed later as a parasporal crystal protein during the sporulation stage.2 Delta-endotoxin has already demonstrated insecticidal activity against susceptible insect larvae including the larvae of Lepidopteran and Coleopteran, a plant pest, as well as of Dipteran, a disease vector (e.g., dengue and malaria). In addition, there are also less well characterized Bt subspecies active on other insect orders as well as nematodes. According to insecticide properties against diverse insect species, Bacillus thuringiensis has been developed to be used as a commercial bioinsecticide beside chemical insecticides. Not only toxic proteins, but also insecticidal protein coding-genes can be applied to a transgenic plant field in order to produce pest-resistant crops.3 Nevertheless, the prolonged application of Bacillus thuringiensis in the fields leads © XXXX American Chemical Society

Received: February 12, 2015

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DOI: 10.1021/acs.langmuir.5b02849 Langmuir XXXX, XXX, XXX−XXX

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Langmuir resistance of larvae stains, acting as Cry protein receptors.13 Despite their common larvicidal activity, Cry and Cyt proteins have entirely different amino acid sequences.1,7,14 Cytolytic proteins can be produced by several Bacillus thuringiensis subspecies with partial variations in their amino acid sequences. Hence, attending to their amino acid sequence identity, cytolytic proteins are classified into three classes: Cyt1, Cyt2, and Cyt3.15 The cytolytic protein Cyt2Aa2 of our particular interest is produced by B. thuringiensis subsp. darmstadiensis. The full length of the Cyt2Aa2 gene is highly expressed in Escherichia coli as an inclusion protein. After proteolytical processing, Cyt2Aa2 adapts to an ∼23 kDa active form. Its toxicity is found against Aedes aegypti and Culex quinquefasciatus mosquito larvae and also includes a hemolytic activity.16 Furthermore, it shows the capability to bind and form a protein complex with a synthetic lipid membrane without the requirement of a receptor.17 The precise mechanism of action for Cyt protein is still unclear, although two possible models have been proposed. First, according to a pore forming model, Cyt protein would oligomerize on the lipid membranes to form a well define pore.7,18−20 On the contrary, by following a detergent-like model, Cyt protein aggregates on the lipid membranes surface and dissolves the lipid membrane at a critical concentration.21,22 In this work, we present the influence of the Cyt2Aa2 protein concentration on the protein−lipid binding mechanism and the final protein−lipid (nano)structure by means of complementary quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM) measurements. QCM-D provided information in real time about the Cyt2Aa2 protein mass bound to mixed-lipid bilayers, as well as the variation of the viscoelastic properties of the protein−lipid layer. In turn, AFM helped to elucidate the topographical changes produced due the protein−lipid complex formation and the stiffness of the final protein−lipid layer.

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quartz sensors (QSX 303, 4.95 MHz, Q-Sense AB) were sonicated in 2% (w/w) SDS solution for 20 min and then rinsed with Milli-Q water. The crystals were dried under N2 stream, treated with UV/ Ozone (Bioforce Nanosciences, USA) for 30 min and mounted into the QCM-D chamber. Then, previously degassed PBS buffer (pH 7.4) was injected to the chamber by means of a peristaltic pump (SM935C, Ismatec, Zürich, Switzerland) in a flow rate of 50 μL/min for at least 1 hour, to ensure stability of the baseline. Lipid bilayers were formed by a liposome fusion method. Thus, 0.1 mg/mL LUVs in PBS were pumped into the chamber (flow rate 50 μL/min) for 10 min, followed by a thorough PBS rinse (50 μL/min, 1 hour) that allowed removal of intact LUVs. Subsequently, the corresponding protein solutions were flushed into the chamber at a flow rate of 50 μL/min for 7 min. After injection, the flow was either stopped or decreased down to 4 μL/min, depending on the followed method. In all cases, the system was left to evolve until a plateau was observed, indicating full completion of the process. Experiments were performed at 25 °C. Real time variations of frequency (Δf) and dissipation (ΔD) parameters were observed at several overtones (n = 3, 5, 7,...13) throughout the QCM-D experiment. The mass adsorption (Δm) on the crystal surface can be proportionally related to changes in frequency through the Sauerbrey equation: Δm = −(C/n)Δf, where C corresponds to the mass sensitivity constant (−17.7 ng cm−2 Hz−1), and n is the overtone number. Simultaneously, viscoelasticity of the film on the crystal surface is related to an energy dissipation value following an equation: D = Edissipated/2πEstored. Low dissipation values indicate the presence of a rigid layer, whereas high values relate to softer layers. Applicability of Sauerbrey’s model is limited to rigid layers, while for soft and dissipative films more complex models would be required. In our case, (ΔD/Δf)abs ratio ≤ 0.2 × 10−6 all along the measurement, and the system under analysis is considered to fulfill the requirements for a Sauerbrey-like calculation.23 Additionally, the ΔD versus Δf plot can be also used to analyze QCM-D data. Each point of ΔD/Δf plot represents a dissipation and frequency data at a certain time, which provides a more detailed view on the viscoelastic evolution of films per mass unit (Δm) change. The shape of the ΔD/Δf plot, as well as the slopes derived, can specifically characterize one type of process and make possible the differentiation between them. Atomic Force Microscopy. The protein−lipid structures were visualized by AFM technique with a J-scanner controlled by NanoScope V multimode (Bruker, USA) software. Silicon-nitride probes (DNP-S10, Bruker, USA) with a nominal spring constant of 0.24 N/m were used in the experiments. Prior to its use in the AFM fluid cell, the cantilever was cleaned with UV/Ozone for 20 min. Once mounted, the system was kept until stabilization of the deflection signal. The AFM images were obtained in tapping mode, at low forces to prevent sample damage, at a scan rate lower than 2 Hz. AFM measurements were performed on silicon wafers (IMEC, Leuven, Belgium) of area 1 cm2. The substrate was sonicated in 2% SDS for 20 min, rinsed with Milli-Q water, dried under N2 stream and treated with UV/ozone for 30 min and then mounted into the liquid cell of the AFM. The solutions containing the liposomes were injected into a chamber sealed by a silicone O-ring with a syringe, following the same sequence employed as for QCM-D experiments. All the images were processed with the Nanoscope program and SPIP (Image Metrology, Denmark).

MATERIALS AND METHODS

Protein Preparation. The inclusion protein of Bacillus thuringiensis cytolytic protein (Cyt2Aa2) was expressed and purified by following the method described by Promdonkoy and Ellar.19 The purified inclusion protein was solubilized in 50 mM carbonate buffer (pH 10.0) at 30 °C for 1 h. The soluble protein was then separated by centrifugation at 10 000g for 10 min. In order to obtain active Cyt2Aa2, the soluble protein was incubated with 2%(w/w) Chymotrypsin (Sigma, Germany) at 30 °C for 2 h, and proteins were subsequently kept at 0 °C. Purity and molecular weight of Cyt2Aa2 were determined by SDS-PAGE (Invitrogen, USA), and the final protein concentration was determined by UV280 absorption (NanoDrop, USA). All the protein solutions employed were freshly prepared and filtered before each experiment. Preparation of Lipid Vesicles. Large unilamellar vesicles (LUVs) were prepared by extrusion. The solutions of 1-palmitoyl,2-oleoyl-snglycero-3-phosphocholine (POPC) and cholesterol (Sigma, Germany) in chloroform were mixed in a 13:1 molar ratio. The lipid mixture was dried under N2 stream to form the lipid film and incubated under a continuous N2 stream for 1 h to remove the residual solvent. Then, the lipid film was incubated in a phosphate buffered saline (PBS) solution of pH 7.4 (10 mM phosphate buffer, 2.7 mM potassium chloride, and 137 mM sodium chloride) (Sigma, Germany) at room temperature for 1 h and then vortexed until complete resuspension to form a final concentration of 1 mg/mL. The vesicle mixture in PBS was pressed repeatedly through a 50 nm polycarbonate membrane by using a MiniExtruder (Avanti, USA) and then stored at 4 °C. Quartz Crystal Microbalance with Dissipation. QCM-D experiments were performed in a Q-Sense E4 instrument (Q-Sense AB, Sweden). Prior to their use in the experiments, silicon-coated

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RESULTS Cytolytic Protein Cyt2A Binding on Supported Lipid Bilayers. Protein adsorption was determined by quartz crystal microbalance with dissipation technique. First, supported lipid bilayers were formed on top of the SiO2 sensors by means of a liposome fusion method. Liposomes (POPC:cholesterol, 13:1) were injected into the QCM-D chamber, and the lipid bilayer formation could be followed in situ by monitoring the changes in both frequency (Δf) and dissipation (ΔD) values B

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Langmuir (Supporting Information, Figure SI1). The resulting Δf and ΔD values after the bilayer rinse in PBS were established as the zero/baseline value for the second part of the experiment, which involved the protein−bilayer interaction. The 10-fold difference in protein concentrations (10 μg/mL and 100 μg/ mL) was chosen in order to study the effect of Cyt2Aa2 concentration on the lipid bilayer binding mechanism. Control experiments concerning the adsorption of Cyt2Aa2 on SiO2 and the binding capability of protoxin and the mutant N145A onto the formed lipid bilayer are shown in the Supporting Information (Figure SI2).17 The experiments delivered a negative result: Cyt2Aa2 did not adsorb on SiO2, while protoxin and mutant N145A did not bind on the lipid bilayer. The Cyt2Aa2/lipid bilayer binding experiments followed two different protocols, in either continuous or stopped flow conditions (Figure 1).

revealed saturation of the lipid bilayer surface for high protein concentrations already after the first injection, since Δf5 and ΔD5 values did not change when an additional protein supply took place. However, for low protein concentrations, Cyt2Aa2 could still continuously bind before saturation was reached at Δf5 = −39.1 Hz and ΔD5 = 7.6 × 10−6. Moreover, binding kinetics after the first injection was faster than after the second injection, as determined from the colored time evolution observed from the ΔD/Δf plots of each individual injection (see Figure SI3). Hence, while the total mass of Cyt2Aa2 bound in both high and low protein concentrations brought no significant differences, the dissipation values showed a very interesting behavior. The low protein concentration showed a dissipation value larger than the high protein concentration, although the amounts of protein binding on the lipid bilayer are similar. This data suggest that the protein concentration employed affects the protein−lipid structure formation. Experiments in continuous flow conditions were also performed to determine the dynamic binding of Cyt2Aa2 on the lipid bilayer. The protein solutions were continuously fed into QCM-D chambers until stabilization of Δf and ΔD values. The results revealed that protein binding curves of the high concentration solutions almost overlapped for continuous and stopped flow measurements. A sudden decrease/increase in the frequency and dissipation parameters, respectively, was followed by a stable plateau (Δf5 = −36.1 Hz and ΔD5 = 1.3 × 10−6) only few minutes after injection. However, for low protein concentrations, Cyt2A gradually bound onto the lipid bilayer surface until a saturation state was reached in Δf5 = −52.5 Hz and ΔD5 = 12.5 × 10−6. Interestingly, for low concentrations, both the total protein bound and the dissipation value were significantly higher than in the high concentration case. The continuous flow curve for low Cyt2Aa2 concentrations is also featured by a shoulder-like behavior as the binding process takes place, which could be considered a consequence of a two-state binding process. This binding trend was also observed in stopped flow experiments (Figure 1). The ΔD5 was plotted against Δf5 (ΔD/Δf plot) in order to correlate the protein binding to the rigidity of the film formed on the crystal surface (Figure 2). For the high protein concentration, ΔD/Δf plots in stopped and continuous flow conditions were similar (Figure 2A). ΔD/Δf ratio is very low, and the rate of mass increasing is faster than the dissipation value, indicating the formation of a rigid layer. Before the system reaches a saturation state after 20 min (cluster of timeindicating color), the protein−lipid complex seems to rearrange to a more rigid structure, as deduced from the slight drop observed in ΔD (Figure 2B). The ΔD/Δf plot highlights once again the absence of interaction after a second protein injection. The very low dissipation of the high protein concentration (ΔD ≤ 2 × 10−6) could suggest a crystal-like structure formation induced by Cyt2Aa2, similar to those formed by other proteins on the same substrate.24 In comparison, the slope of the curve for low protein concentrations is higher than for high concentrations, even from the very initial moments after protein injection (Figure 2A). Interestingly, the protein−lipid layer of the low protein concentration did not reach to a stable state (Figure 2B). The dissipation value still increases, even though the protein saturated on the lipid bilayer. This suggests that the protein−lipid structure in the low concentration regime shows more compliance than the protein/lipid structure formed at high protein concentration (not being able to reach the equilibrium state during the measuring time).

Figure 1. Cytolytic protein binding on lipid bilayers determined by QCM-D. The protein solutions were exposed to the lipid bilayer by either stopped flow or continuous flow. Employed flow conditions are shown on top of the upper image. Represented data correspond to the fifth overtone. The frequency curves of 100 μg/mL protein concentration overlap each other (in flow and stopped-flow conditions).

In stopped-flow experiments, QCM-D chambers were initially filled with protein solutions at a certain flow rate (see Materials and Methods) and then the system was left to evolve while the pump was off. As observed in Figure 1, a high protein concentration (100 μg/mL) showed a change in the frequency of Δf5 = −36.4 Hz after 1 h incubation, almost twice as much as the variation measured for low protein concentration (10 μg/ mL), Δf5 = −20.8 Hz. Such difference between values correlates to the different amounts of protein in solution. The Cyt2Aa2 concentration employed also impacts the kinetics of the process, since the high concentration shows an initial curve of frequency and dissipation values steeper than the low concentration. The dissipation value measured for low protein concentrations (3.7 × 10−6) appeared to be twice that of high protein concentrations (1.7 × 10−6), indicating that Cyt2Aa2 at the low protein concentration (10 μg/mL) induces the formation of a softer protein−lipid layer than at high protein concentration (100 μg/mL). The completion of the protein binding process could then be determined by a second Cyt2Aa2 injection step, in the same conditions as those explained above. Results C

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Figure 2. ΔD/Δf plot of cytolytic protein binding on the lipid bilayer. (A) The point of the second protein injection is indicated; (0,0) position corresponds to the first injection. (B) The time evolution of the experiment is indicated by the color bar (0−120 min).

Figure 3. Time sequence of the Cyt2Aa2 absorption. The AFM image shows the height micrographs of lipid−cholesterol bilayers (deposited on silica surfaces) exposed to both 10 (A−C) and 100 μg/mL (D−F) protein solutions. The time sequence starts after equilibration of the measuring system (t0 = 20 min), which corresponds to images A and D. Images B and E were taken at (t0 + 60 min), while C and F were measured at (t0 + 120 min).

Cytolytic Protein−Lipid Bilayer Structures. To confirm the results shown by QCM-D data, the protein−lipid structures were investigated by AFM. Since the comparison between height profiles turned out to be highly difficult on rough surfaces as that of the silicon-coated QCM-D sensor, the topography of the protein−lipid interaction was limited to samples from stopped flow condition measurements, which were performed on smooth silicon wafers.25 AFM images of the substrate and the lipid-cholesterol bilayer can be found in the Supporting Information (Figure SI4). In addition, Figure SI5 shows a representative force−distance curve taken on the bilayer; the histogram depicts the estimated values of the

bilayer thickness (without taking into account the elastic regime of the force−distance curve). Figure 3 shows the time sequence of Cyt2Aa2 film formation and the influence the injected protein (toxin) concentration has on the final structure of the protein−lipid layer complex. These experiments followed the same steps and injection/incubation times as those applied for QCM-D for a proper comparison. As mentioned in the Materials and Methods section, any eventual disturbance of the structures formed was minimized by operation in tapping mode. The AFM (height) images depict two different structures. On one hand, the exposure of the original lipid bilayer to low concentrations (10 μg/mL) of the D

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Figure 4. (A,C) Height micrographs of 10 μg/mL and 100 μg/mL Cyt2Aa2 protein films, respectively, measured after 120 min in tapping mode and in the presence of PBS buffer (pH 7.4). Vertical scale: 5 nm. (B,D) Height profiles, as obtained from the blue straight lines drawn in panels A and C, respectively.

cytolytic protein induced an island-like structure (aggregates) of visible roughness (Rq = 0.6 nm), which shows a continuous coating growth evolution in time as seen from Figure 3A−C. On the other hand, as it can be seen from Figure 3D−F, the 100 μg/mL protein produced a rather homogeneous film featured by the presence of holes (of approximately 4 nm depth). However, and due to the AFM operational mechanism, the first minutes after protein injection could not be monitored. The injection of the protein solution into the liquid chamber generated turbulences in the system that affected the correct functioning of the cantilever. Thus, a certain equilibration time was required prior to the first measurement. Duration of such process was optimized to 20 min after protein injection (t0). This explains the presence of an already formed film in the case of high Cyt2Aa2 concentrations for the first micrograph obtained (Figure 3D). Such images fully correlate to QCM-D results, where the whole binding process is shown to be over only after 5 min for 100 μg/mL Cyt2Aa2. A deeper analysis of the formed protein−lipid structures can be seen in Figure 4. The figure depicts height images after exposing the lipid bilayer to both concentrations of Cyt2Aa2 protein. Figure 4A shows that low toxin concentrations led to the formation of aggregates of similar size. The obtained values for such aggregates are about 100 nm diameter, and heights of ∼3.5 nm. A different trend can be observed for higher concentrations. The height (Figure 4C) images show that holes are formed when the lipid bilayer is exposed to a protein concentration of 100 mg/mL.

From the profile analysis (Figure 4D), a thickness of about 4 nm can be estimated (of the order of a lipid bilayer). Although both topography and structural features from the above-shown high concentration-derived films seem not to change much during the recorded time sequence, a closer analysis over the depth of the appearing pores brings the idea of a “dynamic” assembly (see Supporting Information, Figure SI6). Three hundred force−distance curves were measured on both types of samples, revealing differences in the approaching curves. The comparison between the slopes when the tip is in contact with the sample provides direct information about the stiffness of the sample as can be observed in the diagram show in Figure 5. Hence, the final protein/lipid layer is slightly more rigid when the initial lipid bilayer is exposed to a protein concentration of 100 μg/mL. This result is in agreement with the QCM-D observations. It was also observed that the curvature of the force−distance measurement, before the tip reaches the sample, is larger at low protein concentration. This suggests a stronger repulsive interaction (Supporting Information, Figure SI7). This result can be easily confirmed by converting such a force versus tip−sample separation plot into its semilogarithmic version (Ln F vs separation). Thus, the lower the value of the obtained slope, the higher the tip− sample repulsion. This occurs for 10 μg/mL of Cyt2Aa2. E

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surface. Furthermore, Cyt2Aa2 did not remove the lipid bilayer from the surface, even though the protein was incubated with the lipid bilayer overnight (data not shown). Thus, the binding mechanism of Cyt2Aa2 at low concentration resembles that of carpet-like or aggregation models rather than a detergent-like action (protein aggregation seems to increase with time, achieving width values of dozens of nanometers). The thickness of the more crystal-like structure and the protein aggregation complex were approximately 4 and 3.5 nm, respectively. Both values are comparable to the core β-sheets of Cyt2A (β5 = 3.6 nm, β6 = 4.3 nm, and β7 = 5.3 nm), which have the appropriate size to contribute to the formation of both type of structures.7 In addition, the planar lipid bilayer experiment might suggest that Cyt2A forms a cation-selective channel.28 This could partially explain the repulsion force between the silicon nitride tip and the protein−lipid film, if the cations (K+ and Na+) in the buffer solution are entrapped inside the pore or the protein complexes, leaving a high anion density on the film surface. The UV/ozone-treated silicon nitride tip normally carries a negative charge, which causes the repulsion from the protein−lipid layer (this is reflected in the curvature of the approaching force− distance curves). Since the protein is active along the experiments, it can be considered to be well inserted in the lipid layer, and therefore cations might be pumped toward the Si substrate. On the contrary, in case the protein would not have been active, cations would be collected and immobilized while being in solution, in a way that the ion concentration cannot be modified when forming the lipid-bilayer structure. These hypotheses are currently being investigated in detail using force-volume measurements, which will be the subject of another manuscript. In another study, the hemolysis time course of Cyt2Aa1 revealed a different kinetic activity between two ranges of protein concentration, 0.25−2.0 μg/mL and 4.0−64.0 μg/mL. The first range (0.25−2.0 μg/mL) showed a gradual hemolysis compared to a rapid up hemolysis (steep curve) of the second one.6 The hemolysis curve tends to resemble the Cyt2Aa2/lipid binding curve. The similarity of these results indicates that the lipid membrane can be disrupted by two different mechanisms (forming different complexes) depending on the Cyt2Aa2 protein concentration.

Figure 5. Slope values obtained from force−distance curves taken on a silicon substrate (reference), and films formed under high and low concentrations of Cyt2Aa2 protein. The table shows the mean value and the standard deviation (of three hundred measurements).

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DISCUSSION Up to date, the binding mechanism of Cyt2Aa2 proteins to lipid bilayers is still unclear and therefore an open question. Our AFM results reveal that exposure of this bilayer to Cyt2Aa2 at either high or low protein concentrations leads to the formation of different protein−lipid complexes (Figure 3). A soft layer with aggregates is formed after exposing the lipid bilayer to low protein concentration (10 μg/mL), which matches with the dissipation values obtained by QCM-D data and the AFM images. For all the cases, the protein−lipid films seem to carry a negative surface charge as indicated from the repulsion force between the silicon-nitride tip and the layer at force spectroscopy measurements. At high protein concentration (100 μg/mL), Cyt2Aa2 seems to induce a protein−lipid rigid layer (crystal-like), which is in agreement with QCM-D results, with the presence of randomly distributed holes all over the coated area. Time lapse-AFM images show that the depth of the open pores (holes) increases in time (Figure SI6). Profile analysis gives a maximum pore depth value of ∼4 nm. Previous results have suggested either a pore-forming or a detergent-like mechanism as the most suitable one. In this study, the use of two protein concentrations differing in an order of magnitude, 10 μg/mL and 100 μg/mL, confirms that the protein concentration seems to be relevant for its binding (interaction) mechanism to the (cholesterol−)lipid bilayer. QCM-D results indicated that the protein concentration affects not only the total mass binding but also the rigidity of the layer formed. A high protein concentration (100 μg/mL) leads to formation of a rigid protein−lipid layer, reaching the saturation state very fast in comparison with the low protein concentration. Such findings point in the direction that the protein−lipid structure formed in high Cyt2Aa2 concentrations might correspond to a pore-forming model, where Cyt2Aa2 protein inserts into the lipid bilayer building a defined pore.19,26 On the contrary, the soft layer resulting from low protein concentrations could suggest a detergent-like action of Cyt2Aa2 taking place. Thus, proteins would bind (or aggregate) on the lipid bilayer until a critical point when the lipid bilayer would be dissolved.22,27 The excess of total mass measured (with QCMD) for protein−lipid films at low protein concentration includes the entrapped-water molecules in the vicinity of the

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CONCLUSION This work represents a step forward in understanding the Cyt2Aa2/lipid bilayer binding mechanism. The results show that such mechanism depends on the Cyt2Aa2 protein concentration. Furthermore, the protein concentration also changes the structure and the mechanical properties of the initial protein/bilayer. At high protein concentration, Cyt2Aa2 binds quickly on the lipid bilayer and forms a rigid protein− lipid layer with inserted holes. This structure might support the putative pore-forming model. On the contrary, when the bilayer is exposed to the lowest protein concentration, the binding process is slower, and aggregates are formed in the lipid bilayer. The final layer is more compliant than the layer formed at high protein concentration, possibly because aggregation induces the entrapment of water molecules. Cyt2Aa2 aggregation could correlate to a proposed carpet mechanism model. The thickness of both the crystal-like and the aggregation-derived structures correlate with the size of the core β-sheet of Cyt2Aa2. This investigation has led to three new main questions that are currently being investigated in our laboratory. The first one refers to the possible existence of a F

DOI: 10.1021/acs.langmuir.5b02849 Langmuir XXXX, XXX, XXX−XXX

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protein threshold concentration above which the hole mechanism dominates. The second one concerns the (negative) repulsive force between protein/lipid layer and the AFM-tip; we aim to elucidate the nature of such interaction, especially if it is driven by anions surrounding the protein/lipid layer. Finally, it is important to investigate the protein aggregation (at low toxin concentrations) as a function of time, since it should be of importance to understand protein− protein interactions and therefore its biological relevance.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b02849. Additional figures as described in the text (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Jacqueline Friedmann for artistic and technical support. This work was supported by the Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. Program (RGJ) (Grant No. PHD/0116/2551), and the National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Thailand (NSTDA). Financial support from RGJ to S.T. and B.P. is gratefully acknowledged. This work was partially supported by the International Graduate School BioNanoTech (IGS) Program of the Federal Ministry for Science and Research, Austria.

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REFERENCES

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DOI: 10.1021/acs.langmuir.5b02849 Langmuir XXXX, XXX, XXX−XXX