Langmuir 1999, 15, 1337-1347
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Lateral Diffusion of Lipids in Silane-, Dextran-, and S-Layer-Supported Mono- and Bilayers Erika Gyo¨rvary,† Barbara Wetzer, and Uwe B. Sleytr* Center for Ultrastructure Research and Ludwig Boltzmann Institute for Molecular Nanotechnology, University of Agricultural Sciences, Gregor-Mendel Strasse 33, A-1180 Vienna, Austria
Axel Sinner, Andreas Offenha¨usser,* and Wolfgang Knoll Max Planck Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany Received July 7, 1998. In Final Form: November 17, 1998 Supported lipid bilayers on planar silicon substrates have been formed using crystalline bacterial cell surface (S-layer) protein as support onto which DMPC (pure or mixture with 30 mol % cholesterol) or DPPC bilayers were deposited. Lateral diffusion of fluorescence lipid probes in these layers have been investigated with fluorescence recovery after photobleaching technique (FRAP). For comparison, hybrid lipid bilayers (lipid monolayer on alkylsilanes) and lipid bilayers on dextran composed of the same lipids as for S-layersupported systems were studied. The mobility of lipids was highest in the S-layer-supported bilayers. No significant difference in mobility was observed for supports of the two S-layer proteins from Bacillus coagulans E38-66 or Bacillus sphaericus CCM2177. DMPC/cholesterol-layers revealed mostly a homogeneous structure, whereas in planar DPPC layers defects could be observed. In S-layer-supported DPPC bilayers, clear cracks could be seen below Tm whereas above Tm inhomogeneous round structures were formed. In another set of experiments the supported bilayers have been covered by S-layer proteins using three different techniques for protein recrystallization (trough, vertical, and horizontal). The recrystallization of S-layers was visualized in large scale by electron microscopy (EM) and more specific on the different substrates by atomic force microscopy (AFM). The S-layer cover induced an enhanced mobility of the probe in the lipid layer. Furthermore it was noticed that the S-layer lattice cover could prevent the formation of cracks and other inhomogenities in the bilayers.
Introduction Stable supported lipid layers on solid substrates are of practical and scientific interest. Providing a natural environment with solid-supported lipid layers for protein immobilization and maintenance of protein function is an important option for the design of biosensors. Solidsupported bilayers may also be used as model systems to study natural biological membranes, since they are supposed to maintain the thermodynamic and structural properties of free bilayers.1 This should also enable the use of several surface sensitive techniques for a better understanding of native biological membrane systems. Hybrid bilayers2,3 are formed by a lipid monolayer on top of a hydrophobic layer (such as alkane silane or alkane thiol) on a solid substrate (e.g., silicon or gold). Bilayers may be separated by ultrathin layers of water4 or soft polymer cushions1 (such as dextran). An alternative strategy to separate lipid bilayers from a solid support is to use crystalline bacterial surface layer proteins (S-layer proteins). S-layers can now be considered as one of the most commonly observed prokaryotic cell surface structure (for review see refs 5 and 6). With few exceptions, the cell * To whom correspondence should be addressed. † Permanent address: Department of Physical Chemistry, Åbo Akademi University, Porthansgatan 3-5, FIN-20500 Åbo, Finland. (1) Sackmann, E. Science 1996, 271, 43. (2) Miller, C.; Cuendat, P.; Gra¨tzel, M. J. Electroanal. Chem. 1990, 278, 175. (3) Plant, A. L. Langmuir 1993, 9, 2764. (4) Groves, J. T.; Ulman, N.; Boxer, S. G. Science 1997, 275, 651. (5) Messner, P.; Sleytr, U. B. In Advances in Microbial Physiology; Rose, A. H., Ed.; Academic: London, 1992; Vol. 33, p 213.
walls of archaebacterial cells consist exclusively of crystalline surface layers. S-layers were also detected in hundreds of different species of nearly every taxonomic group of walled eubacteria. S-layers are composed of a single protein or glycoprotein with a molecular weight between 40 and 200 kD. The crystalline protein layer can exhibit either oblique, square, or hexagonal lattice symmetry with spacings between the individual units in the range of 3-30 nm. Most S-layers are 5-15 nm thick, and the lattice packing leads to pores of identical size (2-6 nm) and morphology. S-layer lattices are highly anisotropic as they have an inner and outer face with different topography and physicochemical properties. In general, the inner face is more corrugated than the outer face. Also the surface charge on the faces differs and depends on pH due to the varying amino- and carboxyl-group content. It has been reported that S-layer proteins have the capability to crystallize in suspension,7 at solid surfaces,8 at the air/water interface,9 at floating lipid monolayers,10 and on liposomes.11 The interaction of the lipid headgroups with the S-layer protein lattice in a lipid/S-layer composite film can significantly modulate the characteristics of the lipid film (6) Sleytr, U. B.; Messner, P.; Pum, D.; Sa´ra, M. Crystalline Bacterial Cell Surface Proteins; Academic Press: Austin, 1996. (7) Sleytr, U. B.; Messner, P. In Electron Microscopy of Subcellular Dynamics; Plattner, H., Ed.; CRC Press: Florida, 1989. (8) Pum, D.; Sleytr, U. B. Supramol. Sci. 1995, 2, 193. (9) Pum, D.; Sleytr, U. B. Colloids Surf. A: Physicochem. Eng. Aspects 1995, 102, 99. (10) Pum, D.; Weinhandl, M.; Ho¨dl, C.; Sleytr, U. B. J. Bacteriol. 1993, 171, 2762. (11) Ku¨pcu¨, S.; Sa´ra, M.; Sleytr, U. B. Biochim. Biophys. Acta 1995, 1235, 263.
10.1021/la980827v CCC: $18.00 © 1999 American Chemical Society Published on Web 01/27/1999
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S-Layer Proteins. Bacillus coagulans, strain E38-66, was kindly provided by the O ¨ sterreichische Zuckerforschungsinstitut, Tulln, Austria, and Bacillus sphaericus, strain CCM2177, was from the Czech Collection of Microorganisms, Brno, Czech Republic. Growth of the bacteria in continuous culture, preparation of the cell walls, extraction of the S-layer protein with guanidine hydrochloride, and subsequent dialyzation of the protein solution were performed as described previously.15 The S-layer self-assembly products formed during dialysis were sedimented for 15 min at 40 000 g at 4 °C. The clear supernatant containing S-layer protein subunits was used for recrystallization experiments. The used protein concentration was about 2-3 mg/ mL. As previously demonstrated the S-layer proteins from both bacteria show the capability to recrystallize into coherent layers at the air/water interface and at floating lipid monolayers. The S-layer proteins from these two strains have been previously characterized,16 and their properties are summarized in Table 1. Preparation of Silicon Substrates. Silicon wafers purchased from WackerChemie (Burghausen, Germany) were used as substrates for all measurements. To prevent destructive interference of fluorescence absorption and emission light at the Si-crystal surface,17 silicon wafers were oxidized at 1000 °C (Institut fu¨r Mikrotechnik, Mainz, Germany), yielding a 250 nm
thick oxide layer. Substrate slides were cut from wafers and precleaned in Milli-Q water (Millipore Corp.), then activated for 10 min in 25% HNO3 and finally rinsed several times in Milli-Q water. The substrates were then dried at 120 °C and cooled under argon. Silanization of Silicon Substrates. Substrates were silanized in a 1 vol % solution of decyldimethylsilane (DMS; ABCR, Karlsruhe, Germany) in dry toluene (p.a., Merck) at room temperature for 1 h. Silanized substrates were finally rinsed with toluene, methanol (p.a., Merck), and Milli-Q water. Fresh solutions were prepared for each experiment. Silanization with hexamethyldisilane (HMDS; ABCR, Karlsruhe, Germany) was performed in an airtight glass vessel. Substrate slides with some drops of HMDS were baked in the glass vessel at 60 °C for 2 h and finally rinsed with methanol. All these substrates were used immediately after preparation. Dextran Coating of Silicon Substrates. Silicon slides were first cleaned as described above and then dried at 70 °C. The warm slides were then treated with a 0.2 vol % solution of epoxysilane ((3-(2,3-epoxypropoxy)propyl)trimethoxysilane) (ABCR, Karlsruhe, Germany) in 2-propanol (p.a., Merck) for 5 min. The slides were then baked at 70 °C for 2 h, rinsed with 2-propanol, and finally incubated in 30 wt % aqueous dextran solution (Dextran T500, Pharmacia Biotech, Sweden) for 24 h. Dextran is known to bind to silane by opening the epoxy groups.18 The slides were rinsed with Milli-Q water for several days at room temperature to remove the noncovalently bound polymer. Recrystallization of S-Layer Proteins on Silanized Silicon Substrates. Recrystallization of S-layer proteins on the silanized silicon substrates was carried out at room temperature in 10 mL dishes filled with buffer. S-layer protein from B. coagulans E38-66 (referred to hereafter as E38-66) was recrystallized on HMDS-coated substrates from 1 mM citrate buffer (Fluka) pH 4 and the S-layer protein from B. sphaericus CCM2177 (referred to hereafter as CCM2177) was recrystallized on DMScoated substrates from 10 mM CaCl2 1 mM borate buffer (Fluka) pH 9. The pH for the buffer solutions was adjusted with NaOH and HCl (Merck). The substrates were placed on the air/buffer interface prior to injection of 300 µL protein solution into the subphase. After recrystallization overnight the substrates were removed from the interface, rinsed, and stored in the same buffer solution as used for recrystallization. Recrystallized S-layers could also be chemically cross-linked with glutaraldehyde (2.5% in 50 mM cacodylate buffer pH 7.2 for 15 min at room temperature). Lipids. 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), and cholesterol (Avanti Polar Lipids, Alabama) were used as supplied and dissolved in chloroform. The chain melting phase transition temperatures Tm for the used lipids are 24 °C and 41 °C, respectively. Cholesterol (30 mol %) was mixed with DMPC in chloroform (referred to hereafter as DMPC/cholesterol). It is known that cholesterol addition broadens the phase transition of DMPC,19 and at cholesterol concentrations above 25 mol % the main transition becomes undetectable.20 The fluorescence probe used for fluorescence microscopy and as an indicator for lipid diffusion was N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (NBD-PE; Molecular Probes, Eugene, OR). Lipid mixtures of 1 mol % fluorescent lipid probe were used. The molecular weight of the probe was 956.25 g/mol. Bilayer Deposition. The procedure to deposite a lipid monolayer on silanized silicon substrates, a bilayer on dextran, or S-layer protein supports is schematically illustrated in Figure 1. Preparation of lipid films was carried out in a Langmuir trough with a Wilhelmy balance (Nima Technology Ltd, Coventry, England). Lipid monolayers were obtained by spreading a lipid solution on a subphase (1 mM Na-acetate buffer pH 7 or 10 mM
(12) Pum, D.; Sleytr, U. B. Thin Solid Films 1994, 244, 882. (13) Schuster, B.; Pum, D.; Sleytr, U. B. Biochim. Biophys. Acta 1998, 1369, 51. (14) Schuster, B.; Pum, D.; Braha, O.; Bayley, H.; Sleytr, U. B. Biochim. Biophys. Acta 1998, 1370, 280. (15) Sleytr, U. B.; Sa´ra, M.; Ku¨pcu¨, Z.; Messner, P. Arch. Microbiol. 1986, 146, 19. (16) Pum, D.; Sa´ra, M.; Sleytr, U. B. J. Bacteriol. 1989, 171, 5296.
(17) Brandsta¨tter, M.; Fromherz, P.; Offenha¨usser, A. Thin Solid Film 1988, 160, 341. (18) Elender, G.; Ku¨hner, M.; Sackmann, E. Biosens. Bioelectron. 1996, 11, 565. (19) McMullen, T. P. W.; McElhaney, R. N. Curr. Opin. Colloid Interface Sci. 1996, 1, 83. (20) McMullen, T. P. W.; Lewis, R. N. A. H.; McElhaney, R. N. Biochemistry 1993, 32, 516.
Table 1. Summary of S-Layer Proteins and Their Properties Used in This Work MW of protein subunit pI lattice type lattice constants or spacing thickness pore diameter
E38-66
CCM2177
97 000 4.3 oblique (p2) a ) 9.4 nm, b ) 7.4 nm, γ ) 80° 4-5 nm 2-3 nm
120 000 4.2 square (p4) 12.8 nm 10-12 nm 4-5 nm
(particularly its fluidity and local order in the nanometer scale). Therefore the terminology “semifluid membrane” has been used to describe this type of supramolecular structure.12 Voltage clamp studies on Langmuir films13 and Black lipid membranes14 confirmed that the associated S-layer did not impede the functionality of the lipid membrane or incorporated molecules. A particularly stable composite structure could be obtained after intra- and intermolecular cross-linking of the S-layer protein alone or with molecules from the lipid layer.6 The aim of this work was to investigate the possibility of using S-layer-coated substrates as supports for lipid bilayers. In addition, we were interested in obtaining more information about the interaction between the recrystallized S-layers and lipid membranes and to study how those interactions influence the fluidity in a lipid layer. Therefore, lateral diffusion coefficients were measured with fluorescence recovery after photobleaching (FRAP) technique and the lipid mobility was compared with both silane-supported lipid monolayers and dextran-supported lipid bilayers. Further we have also studied the effect of an S-layer coating on silane-supported monolayers and on S-layer- or dextran-supported bilayers. The recrystallization of S-layer proteins was observed by atomic force microscopy (AFM), and the mesoscopic lateral structure of the lipid bilayers was observed by fluorescence microscopy. Materials and Methods
Lateral Diffusion of Lipids
Figure 1. Schematic illustration of the preparation steps to produce (a) a monolayer on a silanized silicon substrate, (b) a dextran-supported bilayer, and (c) a S-layer-supported bilayer. Top-to-bottom: Compression of a lipid film to a chosen surface pressure, the monolayer transferred onto a dextran- or a S-layercovered silicon substrate by LB method. The silanized silicon substrate, the dextran- or the S-layer-supported lipid monolayer was horizontally placed onto the floating lipid monolayer and pressed trough the lipid film into the subphase. CaCl2 1 mM borate buffer pH 9). First the DPPC or DMPC (pure or mixture) monolayer was compressed to 30 mN/m (T ) 22 °C) or 35 mN/m (T ) 15 °C), respectively, corresponding a liquid condensed or solid film. The bilayer was prepared on dextranor S-layer-supported substrates using a technique reported by Tamm and McConnell.21 The first monolayer was deposited by the Langmuir-Blodgett (LB) method22 where the layer was transferred vertically during an upstroke with a speed of 4 mm/ min. During this process, surface pressure of the monolayer was kept constant by an electronic feedback control. The second monolayer was deposited horizontally by the Langmuir-Schaefer technique (LS)23 where the substrate was pressed through the lipid film and placed in a specially made glass holder in the subphase. Also for the deposition of a lipid monolayer on a silanized silicon substrate the LS-technique was used. The monoor bilayer-coated substrates were kept and handled under subphase in the glass holder until further analysis. Only one of the layers was fluorescence labeled by NBD-PE. Generally the inner monolayer was labeled with the fluorescent probe when the effect of the supporting material was studied. When a lipid mono- or bilayer was coated with S-layer proteins, the adjacent lipid layer was labeled with the fluorescent probe and the effect of this S-layer cover on the lipid membrane could be observed. Recrystallization of S-Layer Proteins on Lipid Layers. Recrystallization of S-layer proteins on lipid layers was carried out with different techniques depending on the lipid layer. In the first technique the procedure of Wetzer et al.24 was used. The preparation of an S-layer protein-covered lipid monolayer on a silanized silicon substrate, a bilayer on a dextran-layer, or an S-layer protein support using this technique is schematically illustrated in Figure 2. Recrystallization of S-layer proteins on spread and compressed phospholipid monolayers was carried out on a Langmuir trough (homemade, 60 × 200 mm2 and V ) 120 mL) with a Wilhelmy balance (Nima Technology Ltd., Coventry, England). The Langmuir monolayer was compressed to the surface pressure of a liquid condensed or solid film, and the silanized silicon substrates were placed on the floating lipid monolayer. A S-layer protein solution (2 mL) was injected under the monolayer. Recrystallization experiments were generally (21) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105. (22) Blodgett, K. J. Am. Chem. Soc. 1935, 57, 62. (23) Langmuir, I.; Schaefer, V. J. J. Am. Chem. Soc. 1937, 59, 2400. (24) Wetzer, B.; Pum, D.; Sleytr, U. B. J. Struct. Biol. 1997, 119, 123.
Langmuir, Vol. 15, No. 4, 1999 1339
Figure 2. Schematic illustration of the recrystallization of S-layer proteins beneath a floating lipid layer. Preparation of a S-layer-covered (a) monolayer on a silanized silicon substrate, (b) a dextran-supported bilayer, and (c) a S-layer-supported bilayer with the trough technique (see Figure 1). Top-tobottom: The silanized silicon substrate, the dextran- or the S-layer-supported lipid monolayer was horizontally placed on the compressed lipid film. The S-layer protein was injected beneath this floating lipid film and the substrate was left in this position until the S-layer protein had formed a closed crystalline monolayer. Finally the substrate was pressed through this lipid/protein crystal assembly into the subphase.
Figure 3. Schematic illustration of (a) vertical and (b) horizontal recrystallization techniques. Preparation of S-layercovered S-layer-supported bilayers. Top-to-bottom: The S-layersupported lipid bilayer was placed vertically or horizontally in a glass dish filled with subphase and S-layer protein was injected into the subphase. The substrates were left in this position until the S-layer protein had formed a closed crystalline monolayer. (This schematic illustration can also be used for dextran-supported bilayers covered with S-layer.) carried out overnight. Finally, the substrates were pressed through the floating lipid/protein composite layer into the subphase. Also, supported lipid bilayers could be covered by an S-layer applying this technique. Substrates with a dextran-layer, or an S-layer-supported lipid monolayer were placed on the floating lipid monolayer/protein crystal assembly and then pressed through the layer. In this work this technique is referred to hereafter as trough. In the other techniques the recrystallization of S-layer proteins on a phospholipid bilayer was carried out in a glass dish as shown in Figure 3. The preparation of phospholipid bilayers on S-layer protein or dextran-supported substrates was carried out as described above. These substrates were then placed vertically or horizontally (referred to hereafter as vertical or horizontal respectively) in a glass dish filled with 10 mL buffer. A S-layer protein solution (300 µL) was then injected into the subphase and the recrystallization was carried out overnight. Fluorescence Recovery after Photobleaching (FRAP). For measurements of diffusion coefficients, FRAP was applied using the method of circular uniform spot photobleaching which was first reported by Axelrod.25 In this version a circular region (∼4 µm diameter) of the fluorescent molecules bearing lipid bilayer was uniformly illuminated by an attenuated observation
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Gyo¨ rvary et al.
beam of an argon ion laser. Fluorescence intensity was detected as the prebleach intensity value (Fpre). For bleaching, which implies photoexcitation of the fluorescent dyes and irreversible chemical reaction with oxidation reactants (e.g., solved oxygen), the laser beam was switched for an interval of 5-40 ms to the full power level about a factor 105-106 higher than observation level. Immediately after switching back to the observation beam, the recovery of the fluorescence intensity from the bleached area was recorded. The recovery is caused by the diffusion of unbleached dye labeled lipids from outside the bleached area into it. The shape of the recorded recovery curve is dependent on instrumental parameters (spot radius w) and system properties (diffusion coefficient D). The diffusion coefficients of the lipids were analyzed by the model of Soumpasis:26
{
F(t) ) F(∞) - {F(∞) - F(0)} 1 - exp
-2τ/t
[ ( ) ( )]} 2τ 2τ I0 + I1 t t
and D ) w2/4τ with F(t) as fluorescence recovery at time t after bleaching, F(∞) final recovery, F(0) fluorescence intensity at the end of the bleach pulse, and τ the characteristic recovery time. I0 and I1 are spherical Bessel functions of zero and first order. The relative recovery Rrel is indicating the fraction of mobile lipids and is defined as
Rrel )
F(∞) - F(0) Fpre - F(0)
D and Rrel were evaluated by fitting the theoretical curve to the data using a Marquart-Levenberg nonlinear least-square-fitting routine. The samples were measured right after film deposition. The chosen amount of NBD-PE probe (1 mol %) was higher than used by some other groups (Vaz,27 Almeida,28 Derzko29), although these groups studied multilayers rather than bilayers. However, the lower amount of dyes would have serious disadvantages for our experiments in regard of fluorescence microscopy and noise level of the FRAP measurement. Therefore, we decided to use the 1 mol % approach. All the samples were first analyzed at a temperature below Tm and then above Tm. At least eight photobleaching measurements were performed and results of good fitting quality were averaged. Large errors were rather caused by inhomogenities in the samples than by method, instrument or statistic. Atomic Force Microscopy (AFM). A Nanoscope III (Digital Instruments, Inc., Santa Barbara, CA) was used for structural studies of the S-layer protein recrystallization on the various substrates. The measurements were carried out in contact mode under fluid with a 12 µm scanner. Standard silicon nitride tips (NanoProbes, Digital Instruments, Inc.) with a nominal spring constant of 0.06 N/m were used. To prevent the tip from modifying the sample surface the applied force was first minimized. Electron Microscopy (EM). The S-layer recrystallization was controlled also in a large scale with electron microscopy. The protein or protein/lipid (produced by the trough technique) films were transferred onto carbon coated EM grids. The grids were horizontally placed on the film surface and removed by hand with forceps. The protein films were chemically cross-linked with 2.5% glutaraldehyde solution (in 0.1 M sodium cacodylat buffer pH 7.2) for 20 min and negatively stained with 2% uranyl acetate in water for 20 min as previously described.10 Transmission electron micrographs were taken with a Philips CM12 instrument (Philips, Eindhoven, The Netherlands) operated at 80 kV.
Results The investigated systems are schematically illustrated in Figure 4. In system I a lipid monolayer is deposited on (25) Axelrod, D.; Koppel, D. E.; Schlessinger, J.; Elson, E.; Webb, W. W. Biophys. J. 1976, 16, 1055. (26) Soumpasis, D. M. Biophys. J. 1983, 41, 95. (27) Vaz, W. L.; Clegg, R. M.; Hallmann, D. Biochemistry 1985, 24, 781. (28) Almeida, P. F. F.; Vaz, W. L. C.; Thompson, T. E. Biochemistry 1992, 31, 6739. (29) Derzko, Z.; Jacobson, K. Biochemistry 1980, 19, 6050.
Figure 4. Schematic illustration of the different solidsupported lipid layer systems studied with fluorescence recovery after photobleaching (FRAP) method. System I: lipid monolayer on silanized silicon substrate. System II: dextran-supported lipid bilayer. System III: S-layer-supported lipid bilayer. Columns A and B are systems without and with S-layer cover, respectively. Table 2. Diffusion Coefficients (D) and Fluorescence Recoveries (R) of NBD-PE in DMPC/Cholesterol- and DPPC-Monolayers on Decyldimethylsilane (DMS) or Hexamethyldisilane (HMDS) Supports. Monolayers Were Also Coated by S-Layer (E38-66trough or CCM2177trough) monolayera DMS-1‚DMPC/CHOL HMDS-1‚DMPC/CHOL HMDS-1‚DMPC-CCM2177trough DMS-1‚DPPC HMDS-1‚DPPC DMS-1‚DPPC-E38-66trough HMDS-1‚DPPC-CCM2177trough a
T [°C]
D [µm2 s-1]
R [%]
15 30 15 30 30 45 45 45 45
0.038 ( 0.006 0.144 ( 0.006 0.026 ( 0.005 0.071( 0.003 0.190 ( 0.028 0.935 ( 0.159 0.095 ( 0.026 0.821 ( 0.157 0.113 ( 0.006
80 ( 3 83 ( 1 34 ( 3 83 ( 1 94 ( 1 95 ( 1 75 ( 3 83 ( 5 79 ( 3
1‚: monolayer.
a silane layer covalently coupled to a silicon substrate, whereas in the other systems a lipid bilayer is supported by a soft polymer cushion composed of dextran-layer (system II) or by an S-layer (system III). In all three systems an S-layer lattice could be deposited as an outermost layer. The pressure-area isotherms of the spread phospholipid monolayers showed the characteristic features observed by other groups30 (data not shown). The DMPC isotherm measured at 15 °C revealed a liquid expanded phase at all measured surface pressures. Cholesterol was used in DMPC-layers because of its known influence on the phase behavior of lipid bilayers.19 This addition of cholesterol had a condensing effect on the isotherm. The DPPC isotherm at 22 °C showed four different regions: a homogeneous liquid expanded state, a region of coexisting liquid expanded and liquid condensed domains (periodic patterns), a liquid condensed state, and a solid state. In this study, DPPC-monolayers were transferred at a solid state where the lateral compressibility is low and the aliphatic chains are expected to be densely packed. Hybrid Lipid Bilayers on Silicon Substrates. (System I). DMPC/Cholesterol. Table 2 presents the results from fluorescence lipid probe diffusion in a DMPC/ cholesterol-monolayer deposited on a silanized silicon substrate. The probe diffusion could be measured at temperatures below and above the phase transition temperature Tm. The NBD-PE molecular probe in the outer DMPC/cholesterol-monolayer showed low probe mobility at 15 °C, but the probe diffusion increased with temperature. However, there were some differences in the (30) Mingotaud, A.-F.; Mingotaud, C.; Patterson, L. K. Handbook of Monolayers; Academic Press Inc.: San Diego, 1993; Vol 1.
Lateral Diffusion of Lipids
mobility of the probe depending on the silane used as a support. The lipid monolayer on DMS showed larger diffusivity than on HMDS. The fluorescence recoveries were around 80-84% with an exception, HMDS-DMPC/ cholesterol (34%). We think this low value is due to the fact that the lipid monolayer interacts with the HMDS monolayer which leads to a higher friction due to epitaxial coupling. Especially for the very short and imperfect ordered HMDS-layer, this effects the portion of the mobile lipids as well. From fluorescence micrographs (data not shown) it could be observed that the DMPC/cholesterol layer was homogeneous on both silane layers at both temperatures. S-layer protein CCM2177 was allowed to recrystallize on a floating pure DMPC monolayer and transferred onto a solid substrate with the trough technique as described earlier (see Figure 1). The recrystallization degree of S-layer proteins on lipid layers was determined with atomic force microscopy (AFM). From the AFM images it could be seen that the recrystallization was not successful and that probably the S-layer proteins were only adsorbed on the lipid monolayer (data not shown). Also the electron micrographs of negatively stained preparations support this assumption (data not shown). DPPC. The results for DPPC monolayers on a silanized silicon are also shown in Table 2. At temperatures below the phase transition temperature of DPPC, the lipid mobility was almost negligible therefore the values are not tabulated. However, it could be seen that the rate of diffusion increased around 42 °C. The fluorescence images of a DPPC layer on a silanized substrate showed a clear pattern and dark spots of submicron size (data not shown). The pattern formation was even more pronounced on the HMDS substrate. The inhomogenities stayed also visible at temperatures above Tm. The difference between the two silanes used as a support could also be seen in the diffusion data; DMS-DPPC showed higher probe mobility than HMDS-DPPC. The fluorescence recovery was 95% for DMS-DPPC but only 75% for HMDS-DPPC. The aim was to recrystallize both S-layer proteins onto DPPC hybrid bilayers with the trough technique. The AFM and EM images showed that E38-66 was probably only adsorbed on the lipid monolayer (data not shown), whereas CCM2177 showed recrystallization (see Figure 5a) as observed previously.24 From the fluorescence micrographs in Figure 5, part b, a clear pattern is visible in the DPPC monolayer covered by S-layer proteins; however, by heating these samples to 45 °C the lipid monolayer became more homogeneous. The CCM2177 cover slightly increased the probe mobility in the HMDS-supported DPPC-monolayer. One reason for this promoted S-layer protein recrystallization on the floating lipid film might be the reduced lipid mobility in the DPPC film compared to that in the DMPC film. Lipid Bilayers on Dextran. (System II). Recently various groups have used polymers as a lubricating layer to reduce the interaction energy between the solid substrate and the lipid bilayer.31,32 In our study dextran is used as lubricating polymer layer. Dextran swells under water and forms a flexible and smooth polymer cushion which seems to be suitable as a support for lipid bilayers.18 DMPC/Cholesterol. The diffusion coefficient for a supported symmetric bilayer of DMPC/cholesterol on dextran was measured (see Table 3). The fluorescence (31) Ku¨hner, M.; Tampe´, R.; Sackmann, E. Biophys. J. 1994, 67, 217. (32) Spinke, J.; Yang, J.; Wolf, H.; Liley, M.; Ringsdorf, H.; Knoll, W. Biophys. J. 1992, 63, 1667.
Langmuir, Vol. 15, No. 4, 1999 1341
(a)
(b)
Figure 5. (a) Deflection mode AFM image (left) of a DMSsupported DPPC monolayer covered by CCM2177trough. The Z scale is 10 nm. EM micrograph (right) of the recrystallized CCM2177trough on a DPPC monolayer. The scale bar represents 100 nm. (b) Fluorescence micrographs of a DMS- or a HMDSsupported DPPC monolayer covered by E38-66trough or CCM2177trough, respectively. The scale bar represents 10 µm in all micrographs. Table 3. Diffusion Coefficients (D) and Fluorescence Recoveries (R) of NBD-PE in Dextran-Supported DMPC/ Cholesterol- and DPPC-Bilayers. Bilayers Were Also Coated by S-Layer (E38-66 or CCM2177) with Different Techniques (Trough, Vertical or Horizontal) bilayera Dex-2‚DMPC/CHOLb Dex-2‚DPPCb Dex-2‚DPPCt Dex-2‚DPPCt-E38-66trough Dex-2‚DPPCt-E38-66vertical Dex-2‚DPPCt-E38-66horizontal Dex-2‚DPPCt-CCM2177trough Dex-2‚DPPCt-CCM2177vertical Dex-2‚DPPCt-CCM2177horizontal t:
T [°C]
D [µm2 s-1]
R [%]
15 30 45 45 45 45 45 45 45 45
0.066 ( 0.005 0.511 ( 0.023 1.365 ( 0.171 2.204 ( 0.185 2.667 ( 0.356 1.543 ( 0.370 1.318 ( 0.188 1.580 ( 0.148 0.953 ( 0.166 1.621 ( 0.119
95 ( 1 98 ( 1 100 ( 4 98 ( 1 100 ( 2 75 ( 9 104 ( 3 102 ( 2 113 ( 5 100 ( 2
a b: inner monolayer mixed with NBD-PE probe. 2‚: bilayer. outer monolayer mixed with NBD-PE probe.
probe was in the inner monolayer, and the fluorescence images showed that this layer was homogeneous. The fluorescence recoveries were high and the diffusion coefficient was around 0.07 µm2 s-1 at 15 °C and around 0.5 µm2 s-1 at 30 °C.
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Figure 6. Fluorescence micrographs of a dextran-supported DPPC bilayer where the inner (left) or outer (right) monolayer is labeled with the fluorescence probe. The scale bar represents10 µm in all micrographs.
DPPC. The diffusion coefficient of a symmetric DPPCbilayer on dextran was measured and the measurement of probe mobility was performed separately in both layers (see Table 3). The diffusion coefficient could be determined only at temperatures above Tm (at lower temperatures the lipid diffusion was insignificant). At 45 °C the diffusion coefficient was about 1.4 µm2 s-1 in the inner monolayer and about 2.2 µm2 s-1 in the outer monolayer. In both samples the fluorescence recoveries were high. However, the fluorescence micrographs showed that both monolayers had at 25 °C some round spots where fluorescence was missing, but at 45 °C the micrographs were homogeneous (see Figure 6). Further experiments were made with dextran-supported bilayers where the outer DPPC monolayer was mixed with the NBD-PE probe. Three different techniques: trough, vertical, and horizontal (referred hereafter with a lower index), were used to recrystallize S-layer proteins onto this bilayer. The coating techniques were described in materials and methods (see Figures 2 and 3). Both S-layer protein strains, E38-66 and CCM2177, were used for recrystallization. From the AFM images it could be observed that the S-layer protein self-assemblies were sedimented on the horizontally resting bilayer (data not shown) and therefore this technique was decided to be unsuitable for recrystallization. However, with the vertical technique CCM2177 could smoothly be recrystallized on the DPPC bilayer (see Figure 7, part a). From the Figure 7, part b, it can be seen that at both temperatures E3866trough-covered bilayers had areas were fluorescence was lacking, whereas E38-66vertical fluorescence images were homogeneous but relatively dark. The fluorescence recovery values (see Table 3) show that the smallest recovery rate was measured in DPPC bilayers covered with E3866vertical. The lipid layer covered by CCM2177 showed some modulations in the fluorescence images at 25 °C (see Figure 7, part c). At 45 °C the fluorescence image of CCM2177trough became homogeneous, while CCM2177vertical showed still inhomogenities in the fluorescence distribution. The high fluorescence recovery rate in the DPPC bilayer covered by CCM2177vertical cannot be explained yet; however, we think one reason might be the inhomogeneities which were very bright indicating an enrichment of fluorophores. The bilayer covered with S-layer proteins by the vertical technique showed lower probe diffusion than the sample prepared with the trough technique (see Table 3).
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Lipid Bilayers on S-Layer Proteins. (System III). The silanized silica substrates were treated with S-layer proteins as described previously in materials and methods. Pum et al.8 showed with AFM studies that silanized substrates are suitable for S-layer protein crystallization. However, S-layer proteins isolated from different bacterie prefer to crystallize on silanes with different hydrophobicity (unpublished observations). In our study, recrystallization of E38-66 on HMDS was successful but did not cover the whole substrate (see Figure 8, part a). DMS was found to be more suitable for the recrystallization of CCM2177 than HMDS. In this case the recrystallization was successful and small recrystallized patches with different lattice orientation could be observed all over the substrate (see Figure 8, part b). However, these patches did not form large flat areas as E38-66 did. To increase the mechanical stability of the S-layer it was cross-linked with glutaraldehyde which introduces intermolecular covalent linkages between the individual S-layer protein subunits.33 DMPC/Cholesterol. Table 4 shows the lateral mobility data of S-layer-supported lipid bilayers. It can be seen that the mobility in a DMPC/cholesterol bilayer on an S-layer protein support was comparable to the other systems (I and II), and when the thicker S-layer lattice (CCM2177) was used, the mobility seemed to be even slightly higher than with E38-66. It can be observed from the fluorescence images in Figure 8, part c, that the S-layer as a supporting layer seems to cause some inhomogenities to the inner lipid monolayer. The samples were full of small spots where fluorescence was lacking and these spots were also present when the samples were heated above Tm. Small differences could be observed between the two S-layer proteins studied. The density of these spots was higher at the E38-66-supported bilayer than at the CCM2177-supported bilayer and this might explain the slightly smaller diffusion coefficient values for bilayers on E38-66. If the S-layer was not cross-linked with glutaraldehyde, the mobility of lipids in the inner monolayer was reduced and the fluorescence data revealed that the inner layer contained even more holes or quenched areas than on cross-linked S-layer supports (data not shown). DPPC. The same trend could be seen in the DPPCbilayers with NBD-PE probe in the outer monolayer. The fluorescence recovery was high in all cases (see Table 4), but the probe mobility was almost 10 times higher when the supporting protein layer was cross-linked with glutaraldehyde. From the fluorescence micrographs in Figure 8, part d, it can be observed that the supporting protein layer caused some cracks in the inner monolayer at 25 °C. At higher temperatures the fluorescence probe was squeezed out from the monolayer and inhomogenities were formed. A DPPC-bilayer on supporting S-layer protein lattice was also coated with S-layer proteins with the trough technique. The probe mobility in such bilayers could be measured to be as high as 2.5-3.1 µm2 s-1 (see Table 4). AFM images showed a clear lattice structure in CCM2177supported DPPC-bilayers covered with recrystallized CCM2177trough (see Figure 9a). The fluorescence images of this kind of bilayers revealed again cracks in the bilayer at temperatures below Tm (see Figure 9, part b). Surprisingly, instead of the formation of inhomogenities at 45 °C, only some differences in the fluorescence intensity were observed. (33) Sleytr, U. B.; Sa´ra, M. Trends Biotechnol. 1997,15, 20.
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(a)
(b)
(c)
Figure 7. (a) Deflection mode AFM image of a dextran-supported DPPC bilayer covered by CCM2177vertical. The Z-scale is 7 nm. (b) Fluorescence micrographs of a dextran-supported DPPC bilayer covered by E38-66 with different techniques. The scale bar represents10 µm in all micrographs. (c) Fluorescence micrographs of a dextran-supported DPPC bilayer covered by CCM2177 with different techniques. The scale bar represents 10 µm in all micrographs.
Discussion Comparison of the lateral diffusion coefficients in DMPC/cholesterol- and DPPC-bilayers. It is generally known that diffusion in DMPC-bilayers is slower than in DPPC membranes29 and that an isothermal addition of cholesterol to fluid phase phospholipid bilayers (that is at T > Tm) produces a more ordered bilayer structure thus reducing diffusion coefficients.34 Although cholesterol leads to the formation of more ordered mixed DMPC/ cholesterol-layers, the fluid-like state of the lipid layer
remained. Therefore, the fluorescence micrographs were homogeneous indicating a stable and continuous layer (data not shown). The diffusion coefficients measured in all DMPC/ cholesterol samples at temperatures above and below Tm showed a clear dependency on the supporting system, which will be discussed later. Our values of diffusion coefficients were ca. 30-60% compared with data obtained (34) Mu¨ller, H.-J.; Galla, H. J. Biochim. Biophys. Acta 1983, 733, 291.
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(c)
(a)
(b)
(d)
Figure 8. (a) Deflection mode AFM image (left) of a HMDS support covered by E38-66. The Z scale is 6 nm. EM-micrograph (right) of the recrystallized E38-66 at the air/subphase interface. The scale bar represents 100 nm. (b) Deflection mode AFM image (left) of a DMS support covered by CCM2177. The Z scale is 4 nm. EM micrograph (right) of the recrystallized CCM2177 at the air/ subphase interface. The scale bar represents 100 nm. (c) Fluorescence micrographs of a S-layer-supported (glutaraldehyde crosslinked) DMPC/cholesterol-bilayer. The scale bar represents 10 µm in all micrographs. (d) Fluorescence micrographs of S-layersupported (glutaraldehyde cross-linked) DPPC bilayer. The scale bar represents 10 µm in all micrographs.
with various systems: D ) 2.8 µm2 s-1 (Elender et al.18 for DMPC/cholesterol (20 mol %, T ) 30 °C) bilayer on dextran), D ) 2.2 µm2 s-1 (Almeida et al.28 for DMPC/ cholesterol (30 mol %, T ) 30 °C) multilayers on a glass support), D ) 1.62 µm2 s-1 (Johnson et al.35 for DMPC/ cholesterol (40 mol %, T ) 27 °C) multilayers on a glass support). However, it is known that in binary lipid layers containing only 20 mol % cholesterol exist two thermodynamically distinct domains (a cholesterol-poor and a cholesterol-rich domain), whereas in binary mixtures with high cholesterol concentration (such as 30 mol %) only one domain exists (a cholesterol rich domain, known as the liquid-ordered phase).20 (35) Johnson, M. E.; Berk, D. A.; Blankschtein, D.; Golan, D. E.; Jain, R. K.; Langer, R. S. Biophys. J. 1996, 71, 2656.
The diffusion coefficients for DPPC-layers were higher that for DMPC/cholesterol-layers. Compared with the data measured for DPPC layers by other groups some differences could be seen: D < 0.01 µm2 s-1 (McConnell et al.36 for a DPPC-monolayer (T ) 22 °C) on a OTS-treated glass support), D ) 5.5 µm2 s-1 (Tamm et al.21 for a DPPCbilayer (T ) 45 °C) on an oxidized silicon). Our values for diffusion coefficients for the DPPC-monolayers were in the same range; however, the values for the DPPC-bilayers were again lower compared with the data from Tamm. The finding of Johnson et al.,35 that smaller fluorescence probes diffuse faster than the larger ones, can neither be the explanation for this slow probe diffusion in our study. We think that these differences might be due to the fact that the other groups mainly used multilayers on glass where all the layers were labeled; whereas we used mono-
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Table 4. Diffusion Coefficients (D) and Fluorescence Recoveries (R) of NBD-PE in S-Layer-Supported (Cross-Linked or Un-Cross-Linked with Glutaraldehyde) DMPC/Cholesterol- and DPPC-Bilayers. DPPC Bilayers Were Also Coated by S-Layer (E38-66trough or CCM2177trough) bilayera HMDS-E38-66-2‚DMPC/CHOLb HMDS-E38-66*-2‚DMPC/CHOLb DMS-CCM2177-2‚DMPC/CHOLb DMS-CCM2177*-2‚DMPC/CHOLb HMDS-E38-66-2‚DPPCb HMDS-E38-66*-2‚DPPCb DMS-E38-66-2‚DPPCt-E38-66trough DMS-CCM2177-2‚DPPCb DMS-CCM2177-2‚DPPCt-CCM2177trough
T [°C]
D [µm2 s-1]
R [%]
15 30 15 30 15 30 15 30 45 45 45 45 45
0.093 ( 0.009 0.701 ( 0.036 0.289 ( 0.028 0.595 ( 0.059 0.122 ( 0.010 0.800 ( 0.082 0.090 ( 0.008 0.227 ( 0.031 2.853 ( 0.280 0.303 ( 0.011 3.071 ( 0.904 3.016 ( 0.224 2.490 ( 0.284
92 ( 1 94 ( 0 96 ( 2 98 ( 1 92 ( 1 86 ( 4 83 ( 2 91 ( 3 96 ( 2 91 ( 1 89 ( 9 94 ( 1 92 ( 2
a 2‚: bilayer. b: inner monolayer mixed with NBD-PE probe. *: un-cross-linked with glutaraldehyde. t: outer monolayer mixed with NBD-PE probe.
(a)
(b)
Figure 9. (a) Deflection mode AFM image of a CCM2177supported DPPC bilayer covered by CCM2177trough. The Z scale is 8 nm. (b) Fluorescence micrographs of a S-layer-supported DPPC bilayer covered by S-layer. The scale bar represents 10 µm in all micrographs.
and bilayers where only one layer was labeled. However, the main reason for these differences are probably due to the interaction of the bilayer with a polymer or a S-layer. In contrast to the system used in our study the bilayer in Tamm’s work is floating on a thin water layer on oxidized silicon. In principle can lipid molecules also exchange between the two monolayers of the bilayer; however, this transverse diffusion (also called flip-flop) is a slow process with halftimes of hours or even days.37,38 Due to the measurement
of the samples right after the bilayer preparation, we restricted our attention to the lateral diffusion. Inhomogeneous Lipid Structures or Bilayer Defects. Inhomogeneous lipid structures or bilayer defects was seen in this study. The scale of the inhomogeneities was of similar or smaller size than the bleach spot (4 µm). To avoid large systematic errors, we checked the bilayer before every FRAP measurement by fluorescence microscopy and tried to omit areas of unusually high inhomogeneity. Large errors in the diffusion coefficients might be a result of such inhomogeneities. This kind of inhomogeneities can be observed under some conditions, e.g., because of temperature variations or rapid coating or inhomogenities in the substrate properties. Monolayers transferred from solid or liquid condensed state appear to form more stable and close to equilibrium bilayers. However, Tamm et al.21 noticed that a bilayer supported by a fixed geometry is stable only in a certain temperature-pressure region. It is well-known that DPPC-bilayers expand laterally at the chain melting phase transition by about 20% in area.39 This lateral expansion creates an excess lateral pressure in the membrane that is sufficient to squeeze out lipids into inhomogenities of different shapes. Therefore DMPC was mainly used in a mixture of 30 mol % cholesterol because the phase transitions are impeded with cholesterol addition40 and also the bending modulus of the mixture is higher than in pure DMPC.41 These factors inhibit the DMPC/cholesterol-bilayers to decouple from the substrate and to form inhomogenities.18 It seems that the mobility of lipids in a monolayer on a substrate coupled silane is dominated by the frictional effects with silane which inhibits formation of inhomogeneous round structures. Also the energetic barrier is higher because of the asymmetry of the system due to only one mobile layer. In a silane-supported DPPC monolayer (transferred at solid state), a clear pattern and holes could be seen in the fluorescence images below and above Tm (data not shown). These inhomogenities are probably due to the supporting silane layer and the fact that the holes do not disappear (36) McConnell, H. M.; Tamm, L. K.; Weis, R. M. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 3249. (37) Marsh, D. CRC Handbook of Lipid Bilayers; CRC Press: Florida, 1990. (38) Homan, R.; Pownall, H. J. Biochim. Biophys. Acta 1988, 938, 155. (39) Tra¨uble H.; Sackmann, E. J. Am. Chem. Soc. 1972, 94, 4499. (40) Needham, D.; Mcintosh, T. J.; Evans, E. Biochemistry 1988, 27, 4668. (41) Lipowsky, R.; Sackmann, E. Structure and Dynamic Membranes; North-Holland/Elsvier: Amsterdam, 1995.
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upon heating over Tm suggests that a lipid layer is missing in these areas. Also in dextran-supported DPPC-bilayers, some holes were visible but contrary to silane-supported monolayers these holes healed at T > Tm. This suggests that these holes were formed in course of lipid layer transfer. Tamm et al.21 explained the origin of such holes with tension variations. If the bilayer on the solid support is under tension (i.e., the actual lateral bilayer pressure is smaller than its two-dimensional equilibrium pressure) and if the tension becomes high enough (lateral pressure low) the bilayer will finally rupture. In our work, the first layer was transferred vertically during an upstroke with a standard LB-transfer speed of 4 mm/min. For example, the holes in S-layer-supported DMPC/cholesterol bilayers (see Figure 8, part c) appear to be coupled to the solid support (did not heal when heated), which suggests that there might be some interaction of the hole perimeter with the substrate. These interactions did not reduce the lipid diffusion in the surrounding lipid layer though the fluorescence recoveries maintained high (e.g., 80%) and the bilayer could still considered to be in a fluidlike state. The S-layer-supported DPPC-bilayer transferred at a solid state showed boundaries which resembled ruptures (see Figure 8, part d). The bilayer appeared not to be coupled to the support, because lipids from the planar bilayer squeezed out to form inhomogenities at temperatures above Tm. Although lipid diffusion to these inhomogenities occurred to a large extent, the bilayer still showed fluorescence recovery in a range of >94%. Comparison of Diffusion Coefficients in Hybrid and Supported Bilayers. When a bilayer is in frictional contact with a solid substrate, the mobility of molecules is reduced. This reduction is a consequence of the frictional contact between the fluid bilayer and the solid support, which generates two additional friction forces between the molecule and the solid support: frictional shear stress and direct frictional force.1 The diffusion depends on the support used,42 and therefore, silane-supported monolayers show lower diffusion values than the dextran and S-layer-supported systems. In our study, we used HMDS and DMS for silanization. For HMDS, only three methyl groups are responsible for the hydrophobization of the silicon substrate and the formed coating is therefore rather inhomogeneous. Therefore the monolayer was probably in frictional contact with the support showing low diffusion and fluorescence recovery values. Because silanization with DMS formed a better organized layer, the interaction between the silane and lipid layers was small. These factors might be mainly responsible for the higher diffusion coefficients of the DMS-supported lipid monolayers. The results from the dextran-supported lipid bilayers indicate that lipids could move more freely on dextran supports than on silane layers. In dextran-supported bilayers, some differences in the diffusion coefficients could be detected depending on which lipid layer (inner or outer layer) was labeled with NBD-PE. The inner layer showed lower values than the outer layer and this difference suggests a weak frictional contact between the inner lipid layer and the dextran support. In this work it could be shown that S-layer proteins as a supporting layer could keep lipid bilayers in a fluidlike state and therefore a high lipid mobility was measured. S-layer coated supports could be made mechanically and chemically more resistant by introducing inter- and intramolecular covalent linkages (e.g., with glutaraldehyde) between the individual S-layer subunits.33 The lower (42) Merkel, R.; Sackmann, E.; Evans, E. J. Phys. France 1989, 50, 1535.
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probe mobility in bilayers on un-cross-linked S-layer supports may be due to some interactions between the S-layer lattice and lipids. In some bilayers this un-crosslinked S-layer support caused some structural changes in the lipid bilayer. However, in AFM images, no structural differences between native and cross-linked S-layers could be observed.12 Therefore all (if not otherwise mentioned) S-layer proteins on solid substrates were first cross-linked with glutaraldehyde. S-layer Protein Lattice as a Cover for Lipid Monoor Bilayers. By using S-layer as a cover for lipid layers, the formation of many mono- or bilayer defects can be inhibited. For example, pattern or inhomogeneous round structure formation in silane-supported lipid monolayers or in dextran-layer or S-layer-supported bilayers, respectively, could be inhibited by covering the sample with S-layer proteins (see Figures 5, part b and 7, parts b and c). In some cases, although the formation of inhomogenities could largely be prevented by a S-layer coating, some partitioning of the fluorescence probe to different areas occurred at T > Tm (see Figure 9b). All three covering techniques differ clearly and therefore also the interactions between lipid layers and S-layer proteins are dissimilar. When S-layer proteins are recrystallized onto a bilayer using the trough technique, the phase state of the floating monolayer plays a critical role in the recrystallization process.43 If the lipid layer is in a liquid expanded state, the lipids are mobile and may easily interact with the adsorbed S-layer proteins. However, this interaction might be so disturbing that the recrystallization process of the proteins in a large scale is impeded and the proteins might only be adsorbed on the lipid monolayer. If the lipid monolayer was in a liquid condensed or in a solid state, closer interaction between the lipids and proteins occurred where the recrystallization was not disturbed. The interaction is primarily electrostatic and the contact between the lipid monolayer and the adsorbed protein occurs through the “primary” binding sites on the protein surface.44 The detected fluctuations of the fluorescence distribution (see Figure 7, part c) in the transferred lipid layers is suggested to originate from this specific interaction during the recrystallization process. These contact points of the interaction seemed to decrease the probe mobility, whereas S-layer proteins adsorbed on lipid monolayers enhanced the fluidity of the lipid bilayer. In the vertical technique, the bilayer is seen as a solid layer and the lipids were not able to the same extent to interact with the S-layer proteins as in the trough technique. Therefore also the fluorescence images were more homogeneous than the from trough technique (see Figure 7, part c). In the horizontal technique the sedimentation of S-layer self-assembly products disturbs the crystallization system and was therefore considered not to be a suitable covering technique. As a conclusion the lipid layers beneath the S-layer protein lattice could maintain their fluidity and in almost all cases lipid mobility was even higher under a S-layer lattice. Between the two S-layer proteins used, only some minor differences could be seen in diffusion coefficients. The lipid layers showed higher diffusion values under the S-layer lattice of proteins with smaller pore diameter and thinner structure (E38-66). This difference may be due to the different protein properties or degree of recrystallization. (43) Diederich, A.; Sponer, C.; Pum, D.; Sleytr, U. B.; Lo¨sche, M. Colloids Surf. B: Biointerfaces 1996, 6, 335. (44) Wetzer, B.; Pfandler, A.; Gyo¨rvary, E.; Pum, D.; Lo¨sche, M.; Sleytr, U. B. Langmuir 1998, 14, 6899.
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Acknowledgment. Volker Scheumann is acknowledged for his help performing the AFM studies and Jacqueline Friedmann for the EM measurements. One part of this work was supported by the Austrian Science Foundation (Projects S7204 and S7205) and the Austrian Ministry of Science and Transportation. Another part was supported by the Deutsche Forschungsgesellschaft
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(Grant Of 22/2-1) and the Ministerium fu¨r Bildung, Wissenschaft, Forschung und Technologie (Project 0310852). Erika Gyo¨rvary was financed by Jenny and Antti Wihuri Foundation and Åbo Akademi Research Institute Foundation. LA980827V
