Interplay of Phospholipase A2 with S-Layer ... - ACS Publications

Mar 4, 2003 - The results indicate that the isoporous S-layer protein lattice represents no barrier for the PLA2 diffusing from the bulk to the monola...
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Langmuir 2003, 19, 3393-3397

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Interplay of Phospholipase A2 with S-Layer-Supported Lipid Monolayers Bernhard Schuster,* Petra C. Gufler, Dietmar Pum, and Uwe B. Sleytr Center for Ultrastructure Research and Ludwig-Boltzmann-Institute for Molecular Nanotechnology, Universita¨ t fu¨ r Bodenkultur Wien, Gregor-Mendel-Strasse 33, 1180 Vienna, Austria, Europe Received October 30, 2002. In Final Form: January 20, 2003 The present study reports on the enzymatic interplay of porcine pancreatic phospholipase A2 (PLA2) on a monolayer composed of dimyristoylphosphatidylethanolamine (DMPE) in the absence and presence of a recrystallized monomolecular layer of the bacterial cell surface (S-layer) protein SbpA. The PLA2-induced hydrolysis of plain and SbpA-supported DMPE monolayers was monitored by the decay of the surface pressure Π. The same duration of the lag period was observed with SbpA-supported and the corresponding plain DMPE monolayer. The results indicate that the isoporous S-layer protein lattice represents no barrier for the PLA2 diffusing from the bulk to the monolayer surface. This supposition was also confirmed by size exclusion experiments. Most important, the recrystallization of SbpA does not induce defects in the DMPE monolayer from which the PLA2 can benefit. With an S-layer-supported DMPE monolayer, a drop in Π was also observed, but since there are two contributions to Π (SbpA and DMPE monolayer), this parameter decreased to the value for an SbpA monolayer on the air/water interface. The observed results suggest that the prevalent proportion of the DMPE molecules remains in a physicochemical state that allows recognition by PLA2. Thus, S-layer proteins are suitable structures to support lipid films and membranes, and they constitute promising building blocks for new nanotechnological and biomimetic applications for functional lipid membranes.

Introduction Biological self-assembly systems play an ever increasing role in modern research on advanced materials.1 In this context, monomolecular protein lattices (S-layers) that form the outermost cell envelope component on a large number of prokaryotic organisms have already attracted a lot of attention as patterning elements in molecular nanotechnology.2 Surface functionalization by means of supramolecular construction kits is a particularly promising field of S-layer technology.3 While, in Gram-positive bacteria, S-layer proteins are attached to the rigid peptidoglycan-containing cell wall, isolated S-layer protomers have been reported to recrystallize at a wide range of interfaces and surfaces.2,3 Particularly, the capability of S-layer proteins to recrystallize at lipid surfaces (Figure 1) has been thoroughly studied,4-6 since it is technologically attractive if utilized for stabilizing functional lipid membranes. Further on, S-layer lattices have been suggested as ultrathin, intermediate layers to separate lipid membranes from solid supports.4,7 Soft biofunctional interfaces on solids have been demonstrated to be essential * Corresponding author. E-mail: [email protected]. Phone: +43-1-47654-2200. Fax: +43-1-4789112. (1) Ratner, B.; Hoffman, A.; Schoen, F.; Lemons, J. In Biomaterials Science. An Introduction to Materials in Medicine; Academic Press: San Diego, CA, 1996. (2) Sleytr, U. B.; Sa´ra, M.; Pum, D.; Schuster, B. In Nano-Surface Chemistry; Rosoff, M., Ed.; Marcel Dekker: New York, 2001; pp 333389. (3) Sleytr, U. B.; Sa´ra, M.; Pum, D.; Schuster, B. Prog. Surf. Sci. 2001, 68, 231-278. (4) Schuster, B.; Sleytr, U. B. Rev. Mol. Biotechnol. 2000, 74, 233254. (5) Weygand, M.; Wetzer, B.; Pum, D.; Sleytr, U. B.; Cuvillier, N.; Kjaer, K.; Howes, P. B.; Lo¨sche, M. Biophys. J. 1999, 76, 458-468. (6) Diederich, A.; Sponer, C.; Pum, D.; Sleytr, U. B.; Lo¨sche, M. Colloid Surf., B: Biointerfaces 1996, 6, 335-346. (7) Schuster, B.; Pum, D.; Sa´ra, M.; Braha, O.; Bayley, H.; Sleytr, U. B. Langmuir 2001, 17, 499-503.

for maintaining the thermodynamic and structural properties of lipid films,8-10 a prerequisite for the formation of functional lipid membranes.11,12 Structural details of the nonnatural coupling of the bacterial S-layer protein SbpA, isolated from Bacillus sphaericus CCM 2177, to phosphatidylethanolamine (PE) monolayers have been characterized. Electrostatic interaction of SbpA with PE headgroups has been observed, accompanied only by a slight reduction in the order of the hydrophobic lipid acyl chains.5,13 Structurally intact protein motifs interpenetrate the PE headgroups at least up to the plane in which the phosphates are located. The PE headgroups reorient upon SbpA binding, but it is not clear whether a few headgroups per protein monomer unit in the S-layer reorient by a large extent or all lipid headgroups become slightly modulated.5 A standing hypothesis in membrane biology implies that the collective physical properties of lipid membrane components can modulate the activity of membraneassociated proteins. In this context the functioning of porcine pancreatic phospholipase A2 (PLA2) at lipid/water interfaces of phospholipid monolayers lends itself, as a biophysical model, to the study of generic effects involved in protein-membrane interactions.14 This interfacially activated enzyme catalyzes regio- and stereospecific (8) Sackmann, E.; Tanaka, M. Trends Biotechnol. 2000, 18, 58-64. (9) Sackmann, E. Science 1996, 271, 43-48. (10) Marsh, D. Biochim. Biophys. Acta 1996, 1286, 183-223. (11) Heyse, S.; Stora, T.; Schmid, E.; Lakely, J. H.; Vogel, H. Biochim. Biophys. Acta 1998, 1376, 319-338. (12) Knoll, W.; Frank, C. W.; Heibel, C.; Naumann, R.; Offenha¨usser, A.; Ru¨he, J.; Schmidt, E. K.; Shen, W. W.; Sinner, A. Rev. Mol. Biotechnol. 2000, 74, 137-158. (13) Sleytr, U. B.; Sa´ra, M.; Pum, D.; Schuster, B.; Messner, P.; Scha¨ffer, C. In Biopolymers; Steinbu¨chel, A., Fahnestock, S., Eds.; WileyVCH: Weinheim, Germany, 2003; Vol. 7, pp 285-338. (14) Hønger, T.; Jørgensen, K.; Biltonen, R. L.; Mouristen, O. G. Biochemistry 1996, 35, 9003-9006.

10.1021/la026771t CCC: $25.00 © 2003 American Chemical Society Published on Web 03/04/2003

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substrate, also termed “interfacial quality”.17,20 Several studies have proposed that the presence of defects in the lipid structure may act as a starting point for the enzyme activity.21 The present study investigates indirectly, via the action of PLA2, whether the attached S-layer lattice induces biologically relevant defects in the lipid structure or microheterogeneities which in turn modulate the activity of this enzyme. The method used to follow the PLA2 activity was the monitoring of the surface pressure of a phospholipid monolayer at the air/water interface. Materials and Methods

Figure 1. (A) Electron micrograph of a negatively stained preparation of the S-layer protein SbpA isolated from Bacillus sphaericus CCM 2177, recrystallized on a monolayer consisting of 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE). The scale bar represents 100 nm. (B) Computer image reconstitution of the protein mass distribution in the projection of SbpA: protein is white, and staining material is dark. The scale bar represents 10 nm. (C) Schematic view of the architecture of the S-layer-supported lipid monolayer (not drawn to scale).

hydrolysis of the sn-2 acyl ester linkage of sn-3-glycerophospholipids.15-17 The reaction products are free fatty acids and 1-acyllysophospholipids. PLA2 shows at the interface of aggregated substrates, such as phospholipid monolayers, a 10.000-fold greater activity compared with that of the corresponding monomeric substrate.18,19 The activity of the PLA2 and the hydrolysis kinetics depend on the morphology and the physicochemical state of the (15) Scott, S. P.; White, D. L.; Otwinowski, Z.; Yuan, W.; Gelb, M. H.; Sliger, P. B. Science 1990, 250, 1541-1546. (16) Verger, R. Trends Biotechnol. 1997, 15, 32-38. (17) Nielsen, L. K.; Risbo, J.; Callisen, T. H.; Bjørnholm, T. Biochim. Biophys. Acta 1999, 1420, 266-271. (18) Verger, R.; Mieras, M. C. E.; de Haas, G. H. J. Biol. Chem. 1973, 248, 4023-4034. (19) Beisson, F.; Tiss, A.; Rivie´re, C.; Verger, R. Eur. J. Lipid Sci. Technol. 2000, 102, 133-153.

Formation of Phospholipid Monolayer. Phospholipid monolayers were obtained by spreading 10 µL of a solution of 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE; Sigma, St. Louis, MO) dissolved in chloroform/methanol (9/1) at a concentration of 1 mg/mL. Twenty minutes after spreading, the monolayer in the gaseous state was compressed at a rate of 10 cm2 per minute by a Teflon barrier. The Langmuir trough (Type 611, NIMA, Coventry, England) was rinsed with methanol, ethanol, and water before use. Doubly distilled and deionized water (Milli-Q system, Millipore Corp.) was used for making the subphase and for washing the trough. The Wilhelmy method was used to measure the surface pressure (sensor PS4, NIMA). The drift of the surface pressure was smaller than 0.2 mN/m per hour. The subphase contained 10 mM CaCl2 and 0.8 mg/mL β-cyclodextrin (Sigma) in 0.5 mM Tris buffer adjusted to pH 9.0. β-cyclodextrin, a cyclic oligosaccharide, forms inclusion complexes with smaller molecules which fit into its hydrophobic cavity. In the present study, β-cyclodextrin was used to remove the waterinsoluble lipolytic products (fatty acids and 1-acyllysophospholipids) from the air/water interface. All experiments were performed at the temperature 21 ( 0.5 °C. Isolation and Recrystallization of S-Layer Protein. Growth, cell wall preparations, and extraction of the S-layer protein SbpA from B. sphaericus CCM 2177 (Czech Collection of Microorganisms) were performed as described elsewhere.22 For the recrystallization of SbpA, the lipid monolayer was generated as described before and the SbpA solution was injected underneath the lipid film to a final SbpA concentration of 0.1 mg/mL. The same procedure but without a preformed lipid monolayer was used to recrystallize SbpA at the air/water interface. The recrystallization of SbpA was finished after 3.5 h on both interfaces, as determined by transmission electron microscopy (see below). Transmission Electron Microscopy (TEM). The recrystallization of the S-layer protein was monitored after 0.5, 1.5, 2.5, 3.5, and 4.5 h by transmission electron microscopical studies (Philips CM12, Eindhoven, The Netherlands) on negatively stained preparations. The electron microscope grids were carefully placed onto the composite DMPE/S-layer film and removed after several seconds by hand with forceps. Image contrast was enhanced by negative staining of the protein-lipid film with uranyl acetate (2% in distilled water) after fixation with glutaraldehyde (2.5% in 0.1 M cacodylate buffer, pH 7.2). Porcine Pancreatic Phospholipase A2. Porcine pancreatic phospholipase A2 (PLA2) was obtained from Sigma and used with no further purification. The final PLA2 concentration was 0.2 µg/mL in all experiments. The global kinetic constant of the hydrolysis, Qm, also called “interfacial quality”, takes into account the influence of the various physicochemical parameters of the substrate on the enzyme activity and was estimated by the equation Qm ≡ νm/ΓSCE0, as previously described.18,23,24 CE0 corresponds to the PLA2 concentration in the subphase and was calculated to be 8.57 × 1012 molecules cm-3. The surface substrate (20) Verger, R. Methods Enzymol. 1980, 64, 340-392. (21) Grandbois, M.; Clausen-Schaumann, H.; Gaub, H. Biophys. J. 1998, 74, 2398-2404. (22) Sleytr, U B.; Sa´ra, M.; Ku¨pcu¨, Z.; Messner, P. Arch. Microbiol. 1986, 146, 19-24. (23) Panaitov, I.; Verger, R. In Physical chemistry of biological interfaces; Baszin, A., Norde, W., Eds.; Marcel Dekker: New York, 2000; pp 359-400. (24) Panaiotov, I.; Ivanova, M.; Verger, R. Curr. Opin. Colloid Interface Sci. 1997, 2, 517-525.

Interplay of PLA2 with Lipid Monolayers

Figure 2. Monolayer compression isotherm of DMPE. The compression rate is 10 cm2/min. Subphase conditions: 10 mM CaCl2, 0.8 mg/mL β-cyclodextrin, 0.5 mM Tris, pH ) 9.0; T ) 21 °C. concentration (ΓS, DMPE molecules cm-2) was deduced using the area per DMPE molecule (Figure 2) at the adjusted initial surface pressure. The hydrolysis rate (νm, DMPE molecules cm-2 min-1) was obtained from a semilogarithmic plot of the change in Π versus time. For the plain DMPE monolayers, the data points after the lag phase up to the inflection point have been considered. Size Exclusion Experiments. S-layer ultrafiltration membranes (SUMs, Nanosearch GmbH, Vienna, Austria) were produced from cell wall fragments of B. sphaericus CCM 2120 as previously described.25 In brief, cell wall fragments were deposited on nylon microfiltration membranes with an average pore size of 0.4 µm (SM 25058, Sartorius AG, Goettingen, Germany) and cross-linked with glutaraldehyde, and finally, Schiff bases were reduced. The integrity of the active ultrafiltration layer was checked by filtration of a ferritin solution. Ferritin (L ∼ 12 nm) must be completely retained by defect-less SUMs. The rejection property of the SUM was determined by filtration of 0.5 mg/mL PLA2 dissolved in 0.5 mM Tris buffer, 10 mM CaCl2, pH 9.0. The SUM was clamped in a filtration chamber (MPS-1, Amicon, Danvers, MA) and rotated at 3000 rpm. After filtration, the protein absorbances of the feed solution, the filtrate, and the retentate were determined with a spectrophotometer (Hitachi U-2000, Tokyo, Japan) at 280 nm. The rejection coefficient, R, was calculated by the equation R ) ln(Cr/C0)/ln(V0/Vr), where Cr and Vr represent the final protein concentration and the final volume of the retentate, respectively, and C0 and V0 are the initial protein concentration and initial volume, respectively.25,26

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Figure 3. Change in surface pressure (Π) as a function of time. PLA2 was injected (final concentration 0.2 µg/mL) into the subphase underneath the DMPE monolayers at different initial pressures. Subphase conditions as indicated in Figure 2.

Hydrolysis of DMPE Monolayers by PLA2. The phospholipid DMPE was chosen because the S-layer protein SbpA assembles to large closed lattices on this lipid monolayer6 (Figure 1A) and zwitterionic phospholipids such as phosphatidylcholines (PCs)27,28 or PEs29 are frequently used as substrates for various PLA2. Isotherms of DMPE showed the well-known transition from the liquid-expanded to the liquid-condensed phase at an area per molecule of ∼0.67 nm2 (Figure 2). Injection of PLA2 underneath DMPE monolayers led to a decrease in surface

pressure Π after a relatively long lag period. This reflects also the intrinsic features of the used PLA2, as pancreatic PLA2 is known to penetrate slowly into lipid monolayers.18,23 To monitor only the DMPE molecules via measuring Π, and, thus, the substrate concentration, it was mandatory to remove the reaction products from the air/ liquid interface. This is an allowed procedure, as the reaction products are known to be not essential for the activation of porcine pancreatic PLA2.30 To solubilize the water-insoluble, lipolytic reaction products, the nonsurface-active β-cyclodextrin was added as a selective product acceptor.31,32 In accordance with other studies, β-cyclodextrin had no influence on the isotherms of DMPE (data not shown). The PLA2-induced drop in Π was observed for DMPE monolayers with an initial surface pressure in the range 12.0-28.4 mN/m. After the lag period, Π did not decrease continuously but flattened to show an inflection point (Figure 3). The inflection points were (0.7 mN/m (numbers of experiments (n) ) 8) close to the Π value at which the phase transition has been observed (Figure 2). This behavior reflects most probably, due to the hydrolysis of DMPE molecules by PLA2, an increase in area per DMPE molecule, and thus, the DMPE monolayer passed through the phase transition from an initially liquid-condensed to a liquid-expanded phase. In addition, an impact of the phase transition on the PLA2 activity has been observed with dipalmitoyl-PC small and large unilamellar vesicles.30 The PLA2-induced decrease in Π per minute after the lag period showed a maximum at an initial Π of 24.9 mN/m and decreased at higher and lower initial Π values. The global kinetic constant of hydrolysis, Qm, was estimated, and as expected, an influence of the initial Π on Qm has been found (Table 1). The bell-shaped correlation is also in accordance with previously published studies, for example, for pancreatic PLA2 hydrolyzing a dinonanoyl-PC33 or a didodecanoylglycerol monolayer.32 Two nonmutually exclusive interpretations for this bell-shaped correlation, taking into

(25) Sa´ra, M.; Sleytr, U. B. J. Membr. Sci. 1987, 33, 27-49. (26) Weigert, S.; Sa´ra, M. J. Membr. Sci. 1995, 106, 147-159. (27) Li, J.; Chen, Z.; Wang, X.; Brezesinski, G.; Mo¨hwald, H. Angew. Chem., Int. Ed. 2000, 39, 3059-3062. (28) Maloney, K. M.; Grandbois, M.; Graninger, D. W.; Salesse, C.; Lewis, A. K.; Roberts, M. F. Biochim. Biophys. Acta 1995, 1235, 395405. (29) Zhou, F.; Schulten, K. Proteins 1996, 25, 12-27.

(30) Lichtenberg, D.; Romero, G.; Menashe, M.; Biltonen, R. L. J. Biol. Chem. 1986, 261, 5334-5340. (31) Ivanova, M.; Ivanova, T.; Verger, R.; Panaiotov, I. Colloids Surf., B: Biointerfaces 1996, 6, 9-17. (32) Ivanova, M.; Verger, R.; Panaiotov, I. Colloids Surf., B: Biointerfaces 1997, 10, 1-12. (33) Verger, R.; de Haas, G. H. Annu. Rev. Biophys. Bioeng. 1976, 5, 77-117.

Results and Discussion

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Table 1. Global Kinetic Constant of the Hydrolysis (Interfacial Quality Qm) of DMPE Monolayers at Various Surface Pressures and DMPE/SbpA Layers by Porcine Pancreatic PLA2 film at the air/liquid interface DMPE DMPE DMPE DMPE DMPE + SbpA a

surface pressure Π (mN m-1)

1015Qm (cm3 s-1 molecule-1)

17.7 22.7 24.9 28.4 22.7

1.0a 1.1 ( 0.03 (n ) 4) 1.3a 1.0a 0.2 ( 0.05 (n ) 4)

Mean value of two independent measurements.

account the packing of the substrate monolayer34 or (and) conformational changes of the enzyme at the interface,33,35 have been proposed. The calculated Qm values showed the same magnitude as dipalmitoyl-PC and dioleoyl-PC monolayers hydrolyzed by Viperus berus phospholipase A2 in the presence of β-cyclodextrin,31 although Qm values 2 orders of magnitude higher have also been observed under the same conditions.23 Recrystallization of SbpA on DMPE Monolayers. Recrystallization of the S-layer protein SbpA was investigated by TEM after 0.5, 1.5, 2.5, 3.5, and 4.5 h. Negatively stained preparations showed large-scale, monomolecular crystalline arrays after a recrystallization period of 3.5 h (Figure 1A). The randomly oriented crystallites reached an average size of several micrometers and formed a closed monolayer of SbpA on the DMPE monolayer. As the area of the covered lipid monolayer was very large (∼60-80 cm2), it cannot be completely excluded that cracks may exist at certain locations in the S-layer lattice. The surface pressure of a DMPE monolayer increased rather rapidly after the injection of SbpA into the subphase from initially 10.5 to 22.7 ( 0.6 mN/m (n ) 4) (Figure 4A). The increase of Π at constant area was generally due to protein adsorption on the lipid interface. Crystalline patches could not be detected by TEM before a recrystallization period of 2.5 h. Thus, the subsequent recrystallization process had no significant influence on Π. SbpA was also selfassembled on the air/water interface at a final protein concentration of 0.1 mg/mL. Again, Π increased rapidly from zero to 18.4 ( 0.4 mN/m (n ) 4) (Figure 4A). In accordance with the results of the recrystallization of SbpA on lipid monolayers, it was not possible to conclude from the increase of Π whether SbpA has been only adsorbed to the air/water interface or assembled to a closed, crystalline S-layer lattice. The only way to determine the recrystallization of SbpA was to inspect the negatively stained samples taken at several locations of the composite layered film by TEM (data not shown). If after 3.5 h recrystallization time small patches (TEM-grids) or large areas (glass slides, 22 × 22 mm2) of the S-layer at the air/water interface (Figure 4B) or the composite S-layer/ lipid monolayer were removed by the Langmuir-Schaefer technique, Π dropped to a value depending on the transferred film area. In any case, no further change in Π could be observed for another 2 h (Figure 4B). Thus, one can conclude that at the end of recrystallization no surface-active monomeric or oligomeric proteinaceous precursors were present in the subphase. Hydrolysis of S-Layer-Supported DMPE Monolayers by PLA2. PLA2 was injected underneath DMPE monolayers with and without an attached monomolecular SbpA lattice. Both films were adjusted to an initial Π of (34) Muderhwa, J. M.; Brockmann, L. J. Biol. Chem. 1992, 267, 24184-24192. (35) Pattus, F.; Slotboom, A. J.; de Haas, G. H. Biochemistry 1979, 13, 2691-2697.

Figure 4. (A) Increase of Π as a function of time in the course of SbpA recrystallization (1) on a DMPE monolayer (initial Π was 10 mN/m) and (2) at the air/water interface. The final SbpA concentration was 0.1 mg/mL; subphase conditions were as in Figure 2. (B) Decrease of Π due to the Langmuir-Schaefer transfer of a patch of the crystalline S-layer on a glass slide (22 × 22 mm2).

Figure 5. PLA2-initiated decrease of Π for S-layer-supported (A) and plain (B) DMPE monolayers. Subphase conditions are given in Figure 2. The final PLA2 concentration was 0.2 µg/mL.

22.7 ( 0.6 mN/m. The lag period was determined to be 88 ( 12 min for the plain (n ) 4) and 91 ( 10 min for the S-layer-supported DMPE monolayer (n ) 4), respectively (Figure 5). The long duration of the lag period may be explained by the intrinsic features of the pancreatic PLA2, as this enzyme is known to penetrate slowly into the

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interface,35 especially with zwitterionic phospholipids as substrates.18 In addition, the lag period is known to depend on, besides the establishment of an equilibrium between the penetrated PLA2 and the PLA2 in solution,20 the number of lipid-packing defects.17,30 The latter is correlated indirectly with the duration of the lag period.36 From size exclusion measurements it is known that myoglobin (molecular size 4.4 × 4.4 × 2.5 nm3) passes through the pores of the S-layer lattice from B. sphaericus CCM 2120 with zero percentage rejection.26 This S-layer protein is known to be very similar to SbpA, as corresponding permeability measurements did indeed yield very similar results (M. Sa´ra, personal communication). It is conceivable that PLA2 (molecular size 4 × 3 × 2 nm3)37 may pass through the pores of the S-layer lattice and interact freely at least with the lipid molecules of the DMPE monolayer spanning the pore region. Indeed, size exclusion experiments revealed only 3 ( 1% rejection (n ) 5), or in other words, 97% of the PLA2 could pass through the S-layer pores. As the lag period revealed no significant difference in the duration for plain and S-layer-supported DMPE monolayers, one can conclude that (1) the isoporous S-layer lattice constitutes no barrier for the PLA2 diffusing from the bulk to the monolayer surface and (2) alterations of the molecular-level organization of the lipid headgroups upon protein recrystallization do not induce defects in the DMPE monolayer or at least the PLA2 cannot gain by the compositional heterogeneity. In contrast to the case of the plain DMPE monolayer, only a minor decrease of Π was observed with the SbpA/ DMPE layer due to the action of PLA2, and thus, no inflection point in the Π versus time plot was observed (Figure 5). In the latter case, both the crystalline SbpA and the DMPE monolayer may contribute to the measured Π. To get an approximate estimate on the influence of the crystalline SbpA layer on the hydrolysis of the adjacent DMPE monolayer by PLA2, Qm was calculated and found to be significantly lower than that for plain DMPE monolayers (Table 1). In general, the Qm value reflects altered physicochemical properties of the interface relevant to the hydrolysis activity of the PLA2. Π dropped slowly from initially 22.7 ( 0.6 mN/m (n ) 4) to 18.2 ( 0.8 mN/m (n ) 4), a value very close to 18.4 ( 0.4 mN/m, which has been measured for SbpA recrystallized at the air/water interface. To hydrolyze the DMPE molecules in the monolayer, PLA2 or at least a protein domain of the enzyme has to penetrate beyond the phosphate group to cleave the sn-2-acyl ester bond.15 S-layer proteins interact also with the headgroups of the DMPE molecules. Domains of SbpA interpenetrated the phospholipid headgroups almost in its entire depth and modulated the adjacent lipid monolayer in terms of altered hydration, orientation, and fluidity.5,6,13,38 As both proteins, SbpA and PLA2, target almost the same region near the phosphate group of the DMPE molecules, one can assume that the DMPE

molecules interacting with or sterically shielded by the S-layer lattice cannot be hydrolyzed. In X-ray reflectivity measurements horizontal sections through the S-layer structure showed that about 75% of the area is occupied by protein.5,38 Thus, the predominant amount of DMPE molecules are at least shielded by SbpA. Upon PLA2induced hydrolysis, Π of an SbpA-supported DMPE monolayer decreased to the value observed for an SbpA lattice without a DMPE monolayer. Thus, the present results speak in favor of a physicochemical state of the attached lipid membrane in which the predominant part of the lipids in the proximal leaflet retained their lateral mobility and were accessible to the PLA2. This can just be achieved if not only the DMPE molecules located in the pore region but also significant parts of the SbpAassociated DMPE molecules were accessible to PLA2. Consequently, as also strengthened by recent X-ray and neutron data, only a limited proportion of the DMPE molecules are impeded by the attachment of SbpA resembling the repetitive domains of the proteinaceous lattice.38

(36) Hyvo¨nen, M. T.; O ¨ o¨rni, K.; Kovanen, P. T.; Ala-Korpela, M. Biophys. J. 2001, 80, 565-578. (37) Pritchard, L. Ph.D. Thesis, University of Strathclyde, England, 2000. (38) Weygand, M.; Kjaer, K.; Howes, P. B.; Wetzer, B.; Pum, D.; Sleytr, U. B.; Lo¨sche, M. J. Phys. Chem. B 2002, 106, 5793-5799.

LA026771T

Conclusion The S-layer lattice of SbpA neither constitutes a significant barrier for the PLA2 nor induces lipid packing defects which would result in shorter lag periods. The alterations of the molecular-level organization of the lipid monolayer upon S-layer protein binding and recrystallization observed by various biophysical methods do not cause serious inhibition of the PLA2. The present results are also supported by single channel measurements, as the intrinsic properties of R-hemolysin, like the unitary pore conductance, are approximately identical when reconstituted in folded or S-layer-supported lipid membranes.7,39 The presented composite model membrane has no biological relevance, as the used S-layer proteins are derived from bacteria with cell envelopes with an S-layer attached to a rigid peptidoglycan-containing layer and not to a lipid membrane.2 However, the present study demonstrated that the recrystallized S-layer lattice did not modulate a large proportion of the headgroup region of the phospholipid monolayer to an extent that could dramatically impede the recognition of the DMPE molecules by the biological interplay of the PLA2. On the other hand, S-layer proteins are suitable structures to stabilize lipid films and membranes4,40 and, thus, are promising building blocks for new nanotechnological and biomimetic applications for functional lipid membranes. Acknowledgment. This work was supported by the Ludwig Boltzmann Society, by grants from the Austrian Science Foundation, Projects 14419-MOB and 16295-B07, and by the Volkswagen Foundation, Germany, Project I/77 710.

(39) Schuster, B.; Sleytr, U. B. Bioelectrochemistry 2002, 55, 5-7. (40) Schuster, B.; Sleytr, U. B. Biochim. Biophys. Acta Biomembr. 2002, 1563, 29-35.