Surface Pressure-Dependent Interactions of Secretory Phospholipase

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Surface Pressure-Dependent Interactions of Secretory Phospholipase A2 with Zwitterionic Phospholipid Membranes Wei-Ning Huang,*,† Yu-He Chen,‡ Chia-Lu Chen,§ and Wenguey Wu*,‡ †

Department of Biotechnology, Yuanpei University, Hsinchu, Taiwan Department of Life Science, National Tsing Hua University, Hsinchu, Tawian § Department of Medical Laboratory Science and Biotechnology, Yuanpei University, Hsinchu, 300, Taiwan ‡

ABSTRACT: The hydrolytic activity of secretory phospholipase A2 (PLA2) is regulated by many factors, including the physical state of substrate aggregates and the chemical nature of phospholipid molecules. In order to achieve strong binding of PLA2 on its substrates, many previous works have used anionic lipid dispersion to characterize the orientation and penetration depth of PLA2 molecules on membrane surfaces. In this study, we applied monolayer technique with controllable surface area to investigate the PLA2s of Taiwan cobra venom and bee venom on zwitterionic phophatidylcholine monolayers and demonstrated an optimum hydrolytic activity at a surface pressure of 18 and 24 mN/m, respectively. By combining polarized attenuated total reflection Fourier-transform infrared spectroscopy and monolayer-binding experiments, we found that the amount of membrane-bound PLA2 decreased markedly as the surface pressure of the monolayer was increased. Interestingly, the insertion area of the PLA2s decreased to near zero as the surface pressure increased to the optimum pressure for hydrolytic activity. On the basis of the measured infrared dichroic ratio, the orientation of the PLA2s bound to zwitterionic membranes was similar to that observed on a negatively charged membrane and was independent of the surface pressure. Our findings suggest that both PLA2s were located on the membrane surface rather than penetrating the membrane bilayer and that the deeply inserted mode is not a favorable condition for the hydrolysis of phospholipids in zwitterionic phospholipid membranes. The results are discussed in terms of the easy access of catalytic water for the PLA2 activity and the mobilization of its substrate and product to facilitate the catalytic process.

’ INTRODUCTION Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 fatty acyl ester bond of membrane phospholipids, releasing fatty acids and lysophospholipids in the process. Aggregated substrates, such as micelles and vesicles, support 100-fold greater activity than that of the corresponding monomeric phospholipid substrates.1 In the conditions in which the phospholipid substrate is aggregated, the activity of PLA2 is still affected by many factors such as surface charge,2 vesicle curvature,3,4 lipid composition,5 membrane phase,6 monolayer surface pressure,7,8 and interfacial water activity.9,10 Two different explanations have been proposed for the hydrolytic activation of PLA2. According to the “substrate model”, the properties of the aggregated substrate form a preferred density, flexibility, or orientation at the interface,11 whereas the “enzyme model” suggests that the enzyme is activated via a conformational change following binding to the aggregated substrate. Although X-ray crystallography,12,13 nuclear magnetic resonance,14,15 and infrared spectroscopy16,17 have revealed small conformational changes in PLA2 following complexes with various inhibitors and aggregated substrates, the detailed activation mechanism of hydrolysis remains obscure. Despite many reports showing that PLA2s have active and inactive membrane-bound states depending on the physical and chemical properties of the substrates,7,17 these membrane-bound states have proven to be difficult to characterize.18 r 2011 American Chemical Society

The orientation and penetration depth of PLA2 in highly negatively charged phospholipid aggregates have been characterized by infrared,19,20 electron paramagnetic resonance,21,22 and fluorescence spectroscopy.20,23 On the basis of the results of electron paramagnetic resonance analysis of spin-labeled bee venom PLA2 (bvPLA2), it appears that bvPLA2 actually sits shallowly on the anionic phospholipid membrane surface rather than penetrating the membrane.21 Similar studies with human IIa PLA2 found that supramolecular aggregates were formed on negatively charged vesicles, and the IIa PLA2 molecule remained in a peripherally bound state on the micelles.22 In contrast, Tatulian and collaborators20 used polarized infrared spectroscopy and membrane depth-dependent fluorescence quenching to investigate the orientation and penetration depth of human pancreatic PLA2. They found that human pancreatic PLA2 penetrated deeply into negatively charged membranes, and the membrane structure disorder played a major role in PLA2 activity.24 Many studies have used negatively charged lipids to obtain a strong and extensive interaction between PLA2 and membrane Received: January 20, 2011 Revised: April 20, 2011 Published: May 10, 2011 7034

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Langmuir surfaces in model systems. However, as mammalian cell membranes are rich in zwitterionic lipids and contain only a small percentage of negatively charged lipids, most biological experiments involving the modulation of PLA2 activity have been performed mainly involving zwitterionic lipids. It is therefore also important to know the binding state of PLA2 on zwitterionic membrane surface. Recently, neutron reflectivity and ellipsometry studies have indicated that PLA2 partially penetrates, to an extent of 5 Å, the outer-chain region of phosphatidylcholine membranes when the PLA2 activity switches from a lag phase to a burst phase.25 We have recently demonstrated that PLA2 bound strongly to the structural defects of a supported single dipalmitoylphosphatidylcholine bilayer but only exhibited weak catalytic hydrolysis activity.26 This result implies that lipid packing and membrane curvature are also modulating parameters for the catalytic hydrolysis of PLA2 in Taiwan cobra venom (Naja atra; aPLA2). In this study, we combine two techniques, Langmuir
Blodgett film and attenuated total reflectance (ATR) Fourier-transform infrared (FTIR) spectroscopy, to investigate the catalytic hydrolysis of acidic aPLA2 and basic bvPLA2 on zwitterionic lipid monolayers at different lateral pressures. We also used interaction-energy calculations, involving several interaction sources including hydrophobicity, electrostatic charge, and surface pressure, to determine and explain the PLA2s penetration depth into the membranes. Our results highlight that, at least for the acidic aPLA2 and basic bvPLA2, the deeply inserted mode is not a favorable condition for the hydrolysis of phospholipids in zwitterionic phospholipid membranes.

’ EXPERIMENTAL SECTION Chemicals and Reagents. aPLA2 (Naja atra) was provided by the late Dr. Yang Chen-Chung. Its purity was confirmed by high-performance liquid chromatography on a reverse-phase C18 column and by electrospray-ionization mass spectrometry. bvPLA2 (Apis mellifera) was purchased from Sigma Aldrich (St. Louis, MO). The phospholipids 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (diether-PC14) were purchased from Avanti Polar Lipids (Alabaster, AL). Monolayer Studies. Monolayer experiments were performed using a Langmuir minitrough (Joyce-Laebl, Ltd.) with a custom-built glass trough (16  11 2 cm, L  W  H) and a Petri dish (5 cm in diameter) for fixed surface-pressure experiments and PLA2 enzyme-activity assays, respectively. As described previously,27 the lipids were dissolved in a chloroform/methanol 1:1 (v/v) solution and spread gently onto the air
water interface in the trough to form a lipid monolayer. For surface area expansion experiments, the nonhydrolyzed lipid of diether-PC14 was used. PLA2 was injected into the subphase solution, and the monolayer surface area as a function of time in fixed surfacepressure conditions was monitored. In enzyme activity assay experiments, DMPC was spread on a Petri dish and the area of surface was fixed according to the size of the dish. Since different amounts of applied DMPC would spread and equilibrate as a monolayer with different surface pressure, we use the decreasing rate of surface pressure as an indicator of the enzyme activity when PLA2 hydrolyzes the phospholipid to release its product into the subphase.27,28 The composition of the subphase solution was 100 mM NaCl, 10 mM Tris (pH 7.4), and 2 mM CaCl2. For protein/lipid ratio determination, the 10 mM Tris buffer was changed to 1 mM phosphate buffer (pH 7.4) to reduce the interference in the FTIR spectra by Tris. The temperature was maintained at 25 °C. FTIR Spectroscopy Experiments. The attenuated internal total reflectance plate was a 50  5  2 mm germanium crystal (Harrick,

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Ossining, NY) with an incident angle of 45°. The ATR plates were washed with deionized water and cleaned using a plasma cleaner. The incident infrared light was polarized with a germanium single-diamond polarizer (Harrick, Ossining, NY). For the ATR experiments, the lipid
protein monolayer was transferred to an ATR plate at a rate of 0.5 cm/min and a feedback motor controlled the area to keep the surface pressure constant under the transferred pressure.27 For the transmission experiments, the protein/ lipid mixture was dissolved in 10 mM Tris buffer (pH 7.4) and sealed between two CaF2 windows in a temperature-controlled demountable liquid cell and separated by a 12 μm Teflon spacer. FTIR spectra were collected using a Thermo Nicolet Nexus 470 spectrometer with a liquidnitrogen-cooled Hg
Cd
Te detector. The ATR and transmission spectra were based on the accumulation of 500 and 100 scans, respectively. Fourier-transformed data were treated with triangular apodization at a spectral resolution of 2 cm
1 as previously described.29 Dichroic Ratio Map. The theoretical dichroic ratio map of membrane-bound PLA2 was generated using the three-dimensional (3D) structure of the PDB codes of 1POA30 and 1POC31 for aPLA2 and bvPLA2, respectively. The angle of vibration moment of amide I between the CdO and the N
C amide plane for the secondary structures of the R-helix, β-sheet, and undefined structures are known to be 38°, 0°, and 20°, respectively.16,32,33 The amide I vibration moments of each residue in the various secondary structures were summed under an assumed rotation angle of PLA2 bound to the membrane surface. The initial orientation of PLA2 was aligned along the major helices on the Z-axis and the β-wing on the of the Y-axis (Figure 6). Electric field amplitudes at the germanium surface were calculated using the equations given by Harrick,34 and the dichroic ratio maps of amide I were then calculated as previously described.27 The θ and j angles represent the rotations on the X-axis and Y-axis, respectively. Because the values of the dichroic ratio are the same where the protein faces the Z axis or membrane, the resultant maps have j-angle symmetry. Interaction-Energy Mapping. A mean-field approach, also referred to as the implicit solvent method, was used in the computer simulation studies to obtain the interaction energies of the PLA2 molecules at the membrane surface. The calculation of total interaction energy (Etotal) was similar to our previous works, but a new term for surface pressure energy, i.e., Epress, was added as shown in eq 1: Etotal ¼ Ehydro þ Eelect þ Epress

ð1Þ

where the electrostatic energy, Eelect, is the sum total of the individual energies of the charged residues interacting with the membrane headgroup potential. The hydrophobic energy, Ehydro, is the same as accessible surface multiplied by a transferring-energy coefficient. The area of accessible surfaces of individual atoms was calculated using the Surface 4.0 software35 and using the IMPALA parameters for the transferring energy coefficient of each atom.36 We estimate Epress according to eq 2 as, when a protein is inserted into a membrane at different surface pressures, the system needs extra energy to perform the work: Epress ¼ π  Ap

ð2Þ

where Epress, π, and Ap represent the energy associated with protein insertion into the membrane, the surface pressure of the monolayer, and the insertion area of the protein in the membrane, respectively. The notation (θ, j) for the interaction-energy map is the same as that for the dichroic ratio map.

’ RESULTS Surface-Pressure-Dependent Area Expansion. Figure 1 shows the binding traces of PLA2s acting on a diether-PC14 monolayer as 7035

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Figure 2. Representative polarized ATR infrared spectra of dietherPC14 monolayers transferred on a germanium crystal surface. Black and gray lines represent the ATR-IR spectra determined by parallel and perpendicular incident light, respectively. The polarized IR spectra for pure diether-PC14 and added aPLA2 were obtained at a surface pressure of 14 mN/m; IR spectra for added bvPLA2 were obtained at a surface pressure of 18 mN/m.

Figure 1. PLA2s binding-induced area increases as a function of time at different initial pressures. The PLA2 concentration was 50 nM: Taiwan cobra venom (A) and bee venom (B).

indicated by the area expansion after treatment with 50 nM PLA2s at different surface pressures. The aPLA2 and bvPLA2 exhibited similar binding behaviors, in which the surface pressure increase markedly decreased the extent of area expansion. However, the exact area of expansion was distinctive: the bvPLA2 expanded the area of the lipid monolayer by nearly 100% at 16 mN/m surface pressure, whereas the aPLA2 only generated a 4% surface expansion under the same conditions. These results imply that more bvPLA2 bound to the lipid monolayer than aPLA2; another possibility is that the penetrated area of aPLA2s is smaller. To distinguish between these two possibilities, the lipid/protein monolayer was transferred to a germanium ATR plate and analyzed using parallel and perpendicular FTIR spectroscopy (Figure 2). The diether-PC14 monolayer showed alkene vibration signals only in the 2800
3000 cm
1 range. The characteristic ester-bond signal of lecithin at 1730 cm
1 was absent because diether-PC14 substitutes an ether bond for an ester bond. When protein was bound to the monolayer, the peptide bond signals of amide I and amide II were evident.

Figure 3. ATR infrared spectra of aPLA2 mixed with different amounts of diether-PC14. The amount of aPLA2 was 5 μg, and the amount of the diether-PC14 was 0, 10, 20, 30, 40, or 50 times that of the aPLA2. Inset: Correlation of the IR signal intensity with the lipid/protein ratio of bvPLA2 (squares) and aPLA2 (circles).

To obtain a standard calibration curve for the protein/lipid ratio of the monolayer, a different molar ratio lipid/PLA2 was prepared and air-dried on an ATR germanium crystal. Quantitative analysis of the FTIR spectra on the intensity of the lipid alkene and PLA2 amide I at different molar ratios correlated well with the protein/lipid ratio (Figure 3). The amide I intensity was nearly constant when different amounts of lipid were added to 5 μg samples of PLA2. This means that the sample’s thickness is much thinner than the penetration depth of the evanescent field of infrared37 and that the standard curve for the lipid/protein ratio determination was appropriate for the monolayer samples. The slope of the standard curve of aPLA2 was slightly greater than that for bvPLA2 because the molecular weight of aPLA2 is less than that of bvPLA2 (13k versus 15k Da, respectively). Correlation of Insertion Area and Hydrolysis Activity. Figure 4A shows that the proportion of enzyme bound to the monolayer decreased rapidly as the surface pressure of the monolayer increased. To determine the ratio of PLA2 and lipid, we compare the IR spectra of the nonpolarized standard curves of Figure 3 with a monolayer of polarized parallel spectra subtracted 7036

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Figure 5. Infrared dichroic ratio of the methylene stretch of the dietherPC14 monolayer (upper) and the amide I stretch of aPLA2 (middle) and bvPLA2 (lower) at different surface pressures.

Figure 6. Definition of the orientation for PLA2 molecules laying on the zy interface and the major helices aligned on the Z-axis and the β-wing on the of the Y-axis. The original point of coordinate is defined by the center of the protein molecule. The θ and j angles represent the rotations on the X-axis and Y-axis, respectively. The aPLA2 and bvPLA2 are beside the left and right sides of Y-axis, respectively.

Figure 4. Enzyme activity (A; black), bound amounts (A; gray) and insertion area (B) of PLA2s interacting with lipid monolayers as a function of surface pressure. Enzyme activity assay by the initial slope of surface pressure decrease of DMPC monolayer after adding bvPLA2 (squares) or aPLA2 (circles) 4 nM. (C) Wavenumber shift of pure diether-PC14 (triangles) and that mixed with bvPLA2 (squares) and aPLA2 (circles) at different temperatures.

0.43 times from the perpendicular one.38 Although the amount of interfacial PLA2 was decreased, the hydrolysis rate of aPLA2 and bvPLA2 on the DMPC monolayer are optimal at surface pressures of 18 and 24 mN/m, respectively. Differences in optimum pressures at different PLA2 and lipid compositions have been reported previously.7,39 It is not clear why changing the surface pressure of the monolayer alters enzyme activity, as several other molecular parameters can contribute to the surface pressure. Patterns observed in hydrolysis rate versus surface pressure profile could be explained by two counterbalancing factors. As

the surface pressure increases, the amount of enzyme present in the interface decreases, whereas the specific activity of the enzyme increases. One can estimate the penetration area per PLA2 molecule on the lipid monolayer by the amount of bound PLA2 and the total expansion area of monolayer. Figure 4B shows that the insertion areas of aPLA2 and bvPLA2 were decreased when the monolayer surface pressure increased, and that the insertion areas were near zero at 18 and 24 mN/m, respectively. This observation suggests that the fastest hydrolysis velocity was achieved when PLA2 does not enter the membrane. Because the amount of bound PLA2 decreased significantly within a narrow range of surface pressure increase, we supposed the pressure of maximum activity of individual PLA2 was close to the apparent optimal pressure. The lipid packing of the monolayer was equal to the bilayer membrane packing when the surface pressure was approximately 30 mN/m,40 suggesting that PLA2 bound on the bilayer membrane in a peripheral mode. Differential scanning calorimetry (DSC) and infrared spectroscopy further support this conclusion. Figure 4C shows the alkene frequency profile of dietherPC14 mixed with PLA2 as a function of temperature. The phase transition of diether-PC14 occurred at 26 °C and was not influenced by the addition of aPLA2 or bvPLA2. DSC was also used to ensure that the enthalpy and transition temperature of 7037

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Figure 7. (A) Theoretical contour plot of infrared dichroic ratio of amide I at the designated rotation angle of the aPLA2 molecule. Shaded areas represent the theoretically determined infrared dichroic ratios for those rotation angles falling within the experimentally determined values. (B) Equipotential contour plot of aPLA2/PC monolayer interaction energy at a surface pressure of 30 mN/m. The energy interval of the contour plot is 2 kcal/mol. (C) Separated interaction energy profile of the aPLA2/PC system (θ = 90 o, j = 100 o) at a surface pressure of 30 mN/m at different penetration depths. (D) Total energy profiles of the C orientation at different surface pressures and penetration depths. The surface pressures of these curves were 30, 25, 18, 15, 10, 5, and 0 mN/m (from top to bottom).

diether-PC14 were also not perturbed by the addition of PLA2s (data not shown). The phase-transition temperature was two degrees higher than that of DMPC. It has been previously shown that the alkyl ether analogues of phosphatidylcholine have a higher phase-transition temperature than their diester counterparts.41 Many membrane insertion proteins, such as cardiotoxins or δ-lysin, reduce the endothermic enthalpy and change the frequency profile of methylene vibration.42,43 In conclusion, the phase behaviors of lipid/PLA2s samples suggest that lipid packings of the membrane are not perturbed significantly by the binding of PLA2s; therefore, PLA2 adopts a peripheral lipid binding mode, rather than insertion into the zwitterionic bilayer under the experimental conditions. Calculation of the Orientation and Interaction Energy. Linear dichroism infrared spectroscopy is a useful tool for determining the orientation of biomolecules in membranes.16,37 Figure 5 shows the dichroic ratio of the methylene group of diether-PC14 and the amide I of PLA2s at different surface pressures. The methylene group of diether-PC14 had a nearconstant value of a dichroic ratio of 1.1. This value was higher than that for ester bond lipids, such as dipalmitoylphosphatidylcholine (DPPC), DMPC, and dimyristoylphosphatidylglycerol (DMPG), whose dichroic ratios were all less than 1.0.27,44,45 Under low hydration state conditions when drying on an ATR plate, the ether-linked phospholipid could exist as a mixture of an interdigitated and noninterdigitated form to allow the tilting of

the bilayer.46 When PLA2 was added to the subphase of the diether-PC14 monolayer, the infrared intensity of methylene decreased because proteins were occupying the monolayer surface. The dichroic ratios of the amide I group of aPLA2 and bvPLA2 were in the range of 1.3
1.2 and 1.2
1.1, respectively, and the values were independent of surface pressure (Figure 5). This finding suggests that the orientations of PLA2s were not influenced by surface pressure, but that the insertion area decreased sharply at higher surface pressures. To determine the orientation of PLA2s based on their interaction with membranes, we first aligned a PLA2 molecule along its major helices on the Z-axis and the β-wing of the Y-axis (Figure 6). The dichroic ratio of amide I and the interaction energy of PLA2s were then calculated at different rotation angles (Figures 7A,B and 8A,B). The shadow areas of the dichroic ratio map represent the possible orientations that fit the experimental data. The molar absorptivity of the β sheet structure has been reported to be 1.4
1.6 times larger than that of the R-helix structure at the amide I region.47 We tried to weight the vibration moment of the β sheet for the dichroic ratio calculation and observed no significant changes for the dichroic ratio map because the content of the β sheet structure is small, with only about 7
12% PLA2s. By taking account of both the experimentally allowed dichroic ratio and the interaction energy minima of PLA2 in membrane monolayer, one could determine (90o, 100o) in Figure 7B and 7038

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Figure 8. (A) Theoretical contour plot of infrared dichroic ratio of amide I at the designated rotation angle of the bvPLA2 molecule. Shaded areas represent the theoretically determined infrared dichroic ratios for those rotation angles falling within the experimentally determined values. (B) Equipotential contour plot of the bvPLA2/PC monolayer interaction energy at a surface pressure of 30 mN/m. The energy interval of the contour plot shown in the Figure is 2 kcal/mol. (C) Separated interaction energy profiles of the bvPLA2/PC system (θ = 100 o, j = 140 o) at a surface pressure of 30 mN/m at different penetration depths. (D) Total energy profiles of the C orientation at different surface pressures and penetration depths. The surface pressures of these curves were 30, 24, 20, 15, 10, 5, and 0 mN/m (from top to bottom).

(100°, 140°) in Figure 8B as a possible orientation of aPLA2 and bvPLA2, respectively, on the membrane surface. The rotation angle (90°, 100°) was a generally accepted orientation of group I PLA2,19 and it was located in a local energy minimum position. In contrast, other orientations with the lowest interaction energy of aPLA2 at rotation angle (120°, 0°) does not fall into the experimentally allowed value of the dichroic ratio. Similarly, the bvPLA2 orientation with a rotation angle of (100°, 140°) also matched well with the experimentally determined value of the dichroic ratio of amide I. This orientation is also consistent with a previous report of bvPLA2 bound to negatively charged membranes21 (Figure 8A,B). We should emphasize that other possible orientations of PLA2 also exisit based on this approach, but the apparent consistency of our results with previous work suggests strongly that both aPLA2 and bvPLA2 could adopt a peripheral binding mode without penetrating deeply into zwitterionic membrane monolayers.

’ DISCUSSION The binding mode for efficient enzyme activity on a membrane can be described by two important factors: the orientation and the penetration depth. In this study, we show that the individual enzyme activity of secretory PLA2s from cobra and bee venom reaches a maximum on zwitterionic phospholipid membranes when the binding mode is peripheral, at which condition the insertion area approaches zero. In addition, the binding mode is more significant than the binding amount as far as enzyme activity is concerned. The defected area of DPPC on a supported single bilayer26 may reflect the low surface pressure condition of DMPC, which was strong binding but low catalytic activity. Since the orientations of PLA2s on zwitterionic membranes are similar to the previous reports of PLA2s on negatively charged membranes, it implies that the orientation of PLA2 is not primarily driven by electrostatic interaction.48 This conclusion is also consistent with the observation that the total net charge of aPLA2 and bvPLA2 7039

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2 and þ3 at neutral pH conditions, respectively, but aPLA2 and bvPLA2 exhibit similar perepheral binding modes. The major phophospholid substrates of PLA2 on the mammalian membrane surface are zwitterionic phosphatidylcholine. Our results suggest that aPLA2 exhibits the highest activity without significantly penetrating the membrane monolayer surface. It has been proposed that the accessibility of catalytic water at the hydrolytic site plays an important role in the enzymatic activity of PLA2. The higher enzymatic activity of PLA2 in the peripheral binding mode than in the deeply penetrated mode is consistent with this interpretation. Substrate and product mobilization also play an important role in the hydrolytic activity of interfacial enzymes. However, there has been very few studies on PLA2 catalytic activity along this line. One could argue that higher enzymatic activity with less membrane penetration could reflect the optimal condition of substrate and product mobilization for PLA2. Since the detected optimal membrane pressure for its enzymatic activity is still lower than the membrane bilayers, our results imply that there may be other components in the venom to facilitate the process. For instance, cardiotoxins (CTXs) in cobra venom and melittin in bee venom have been shown for some time to be able to exhibit synergistic effects with PLA2. Although the detailed mechanism remains unclear, it is tempting to suggest that both components could function to facilitate the substrate mobilization in biological membranes. The suggested mechanism implied that toxin PLA2 acts mainly on specific locations on a cell surface, rather than just a random hydrolytic enzyme. Finally, according to the interaction energy profile of PLA2s as a function of penetration depth at reported rotation angles (Figures 7C and 8C), the major factor contributing to the binding energy was hydrophobic interaction. In addition, the repulsion energy of surface pressure increased rapidly as the penetration depth increased. On the basis of the depth-dependent interaction energy curves at different surface pressures as shown in Figures 7D and 8D, both results indicate that the hydrophobic force drives PLA2s to insert into the membrane at low surface pressure. The mobilizing of substrates and the delivering of products are important processes if cellular reactions involve both hydrophobic and hydrophilic molecules that reside within the chemically distinct environments.49 The peripheral binding mode of PLA2s on a membrane may well represent the balance to adopt its orientation for substrate and product mobilization. It should be pointed out that, without atomic resolution structures of membrane-bound PLA2, it remains difficult to propose a detailed mechanism about the hydrolytic activation of PLA2. However, the methods we employed in this study serve as a quick and intuitive way to calculate the interaction energy between membranes and proteins without requiring chemical labeling of the studied materials. These experimental strategies could provide an effective tool for the analysis of the membrane/protein interactions.

’ AUTHOR INFORMATION Corresponding Author

*(W.-N.H.) Address: Department of Biotechnology, Yuanpei University, No. 306, Yuanpei Street, Hsinchu 30015, Taiwan. E-mail: [email protected]. Tel: þ886-03-5381183 #8160. Fax: þ886-03-6102312. (W.W.) Address: Department of Life Science, National Tsing Hua University 101, Section 2, Kuang-Fu Road, Hsinchu, 30013, Taiwan. E-mail: wgwu@life. nthu.edu.tw. Tel: þ886-3-5731040. Fax: þ886-03-5717237.

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’ ACKNOWLEDGMENT This work was supported by a grant from the NSC. We thank Dr. Robert H. Glew and Dr. Da-Shin Wang for reading the manuscript and editorial suggestions. ’ REFERENCES (1) Hazlett, T. L.; Deems, R. A.; Dennis, E. A. Activation, aggregation, inhibition and the mechanism of phospholipase A2. Adv. Exp. Med. Biol. 1990, 279, 49–64. (2) Leidy, C.; Linderoth, L.; Andresen, T. L.; Mouritsen, O. G.; Jorgensen, K.; Peters, G. H. Domain-induced activation of human phospholipase A2 type IIA: local versus global lipid composition. Biophys. J. 2006, 90 (9), 3165–75. (3) Kensil, C. R.; Dennis, E. A. Action of cobra venom phospholipase A2 on large unilamellar vesicles: Comparison with small unilamellar vesicles and multibilayers. Lipids 1985, 20 (2), 80–3. (4) Burke, J. R.; Witmer, M. R.; Tredup, J. A. The size and curvature of anionic covesicle substrate affects the catalytic action of cytosolic phospholipase A2. Arch. Biochem. Biophys. 1999, 365 (2), 239–47. (5) Bell, J. D.; Biltonen, R. L. Molecular details of the activation of soluble phospholipase A2 on lipid bilayers. Comparison of computer simulations with experimental results. J. Biol. Chem. 1992, 267 (16), 11046–56. (6) Honger, T.; Jorgensen, K.; Biltonen, R. L.; Mouritsen, O. G. Systematic relationship between phospholipase A2 activity and dynamic lipid bilayer microheterogeneity. Biochemistry 1996, 35 (28), 9003–6. (7) Verger, R.; Rietsch, J.; Van Dam-Mieras, M. C.; de Haas, G. H. Comparative studies of lipase and phospholipase A2 acting on substrate monolayers. J. Biol. Chem. 1976, 251 (10), 3128–33. (8) Yokoyama, S.; Kezdy, F. J. Monolayers of long chain lecithins at the air/water interface and their hydrolysis by phospholipase A2. J. Biol. Chem. 1991, 266 (7), 4303–8. (9) Rao, C. S.; Damodaran, S. Is interfacial activation of lipases in lipid monolayers related to thermodynamic activity of interfacial water? Langmuir 2002, 18 (16), 6294–306. (10) Peters, G. H.; Moller, M. S.; Jorgensen, K.; Ronnholm, P.; Mikkelsen, M.; Andresen, T. L. Secretory phospholipase A2 hydrolysis of phospholipid analogues is dependent on water accessibility to the active site. J. Am. Chem. Soc. 2007, 129 (17), 5451–61. (11) Berg, O. G.; Rogers, J.; Yu, B. Z.; Yao, J.; Romsted, L. S.; Jain, M. K. Thermodynamic and kinetic basis of interfacial activation: Resolution of binding and allosteric effects on pancreatic phospholipase A2 at zwitterionic interfaces. Biochemistry 1997, 36 (47), 14512–30. (12) Chandra, V.; Jasti, J.; Kaur, P.; Dey, S.; Perbandt, M.; Srinivasan, A.; Betzel, C.; Singh, T. P. Crystal structure of a complex formed between a snake venom phospholipase A2 and a potent peptide inhibitor Phe-Leu-Ser-Tyr-Lys at 1.8 A resolution. J. Biol. Chem. 2002, 277 (43), 41079–85. (13) Ambrosio, A. L.; Nonato, M. C.; de Araujo, H. S.; Arni, R.; Ward, R. J.; Ownby, C. L.; de Souza, D. H.; Garratt, R. C. A molecular mechanism for Lys49
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