J. Phys. Chem. B 2002, 106, 5793-5799
5793
Structural Reorganization of Phospholipid Headgroups upon Recrystallization of an S-Layer Lattice Markus Weygand,#,† Kristian Kjaer,‡ Paul B. Howes,§ Barbara Wetzer,| Dietmar Pum,| Uwe B. Sleytr,| and Mathias Lo1 sche*,† Institute of Experimental Physics I, Leipzig UniVersity, Linne´ str. 5, D-04103 Leipzig, Germany, Materials Research Department, Risø National Laboratory, DK-4000 Roskilde, Denmark, Department of Physics and Astronomy, UniVersity of Leicester, Leicester, LE1 7RH, U.K., and Center for Ultrastructure Research and Ludwig-Boltzmann-Institute for Molecular Nanotechnology, UniVersity for Agricultural Sciences, A-1180 Vienna, Austria ReceiVed: December 31, 2001; In Final Form: March 25, 2002
Structural details of the coupling of bacterial surface (S)-layers to the phospholipid, dipalmitoylphosphatidylethanolamine (DPPE), have been characterized using X-ray and neutron reflectometry. We studied the binding and recrystallization of S-protein isolated from B. sphaericus CCM2177 at DPPE monolayers on aqueous surfaces. Particular emphasis has been put on investigations of the lipid/protein interface in a joint refinement of X-ray and neutron data which reveals alterations of the molecular-level organization of the lipid headgroups upon protein binding and recrystallization: Peptide material interpenetrates the phospholipid headgroups almost in its entire depth but does not affect the hydrophobic lipid acyl chains. Consistent with FTIR results, we find that the headgroup hydration is reduced by ∼40% upon peptide interpenetration. On average, the equivalent of ∼65 electrons associated with the peptide, i.e., less than one peptide side group, interacts directly with one DPPE headgroup within the surface film. This suggests that the protein attaches to specific molecular moieties within the lipid monolayer which may form a lateral pattern within the film area that reflects the properties of the monomolecular protein crystal sheet.
Introduction Biological materials play an ever increasing role in modern research on advanced materials.1 In the field of biomimetic surface modification, crystalline bacterial S-layers,2-4 monomolecular protein crystal sheets that form the outermost cell envelope component on a large number of prokaryotic organisms, have already attracted a lot of attention and possess the potential to become a preeminent technology,5,6 as their application spectrum ranges from nanosieves to carrier structures for artificial vaccines.7 Surface functionalization by means of supramolecular construction kits6,8 is a particularly promising field of S-layer technology.6,7 Although S-layers on Grampositive bacteria are attached to the outer cell wall in vivo, i.e., to the peptidoglycan layer, purified S-layer protomers have been reported to recrystallize at a wide range of interfaces and surfaces, including phospholipid model membranes.9-11 Particularly, the capability of S-protomers to recrystallize at lipid surfaces has been thoroughly studied12-15 because it is technologically attractive if utilized for the formation of molecularscale scaffoldings11,16,17 envisaged to stabilize biomembrane models11,18-20 such as Newton Black Films,21 vesicles, or solidsupported bilayer membranes.22,23 Earlier studies focused on S-layer/lipid interactions characterized S-layer recrystallization * To whom correspondence should be addressed. Phone: +49 (341) 9732 488. Fax: +49 (341) 97-32 479. E-mail:
[email protected]. † Leipzig University. ‡ Risø National Laboratory. § University of Leicester. | University for Agricultural Sciences. # Current address: Niels Bohr Institute for Astronomy, Physics and Geophysics, Københavns Universitet, DK-2100 Copenhagen, Denmark.
underneath surface monolayers constituted of various lipids under a variety of subphase conditions.16,24 They revealed indirectly, via biochemical techniques, the electrostatic character of the lipid/protein interactions but were incapable of addressing structural issues in further detail: Does the protein interact with the membrane surface, the lipid headgroup, or the hydrophobic interior of the membrane? How may this interaction be quantitatively assessed? This work addresses such questions and deals with the characterization of the microscopic interactions within a S-protein/lipid compound system on a molecular scale. Recently, X-ray and neutron scattering techniques have been particularly useful for studies of structural aspects of peptidelipid interactions and the membrane association of proteins.25,26 Related techniques used here are reflectivity measurements, applied in situ at the air/water interface, and their evaluation in a coupled mode thus utilizing contrast variation in X-ray and (various) neutron scattering length density (SLD) profiles. This results in an appreciation of the S-layer/lipid interface in unprecedented structural detail. Materials and Methods Sample preparation and data collection have been described earlier.14 In brief, Bacillus sphaericus (B. sphaericus) strain CCM2177 from the Czech Collection of Microorganisms (Brno, Czech Republic) were grown in continuous culture and their S-layer proteins extracted using guanidine hydrochloride (GHCl, 5 M in 50 mM Tris-HCl buffer, pH 7.2, 20 °C) as described.27 After dialysis against H2O, self-assembly products were sedimented at 40.000 × g and 4 °C immediately before using the protein solution in experiments. The clear supernatant that contained the disassembled S-layer protein monomers (∼2 mg/
10.1021/jp0146418 CCC: $22.00 © 2002 American Chemical Society Published on Web 05/14/2002
5794 J. Phys. Chem. B, Vol. 106, No. 22, 2002 mL of the solution) was injected into the subphases of dipalmitoylphosphatidylethanolamine (DPPE or DPPE-d62, respectively, in X-ray or neutron experiments; both from Avanti Polar Lipids, Inc., Birmingham, AL) monolayers in the Langmuir film balance. The lipid was spread from CHCl3/CH3OH (3:1, Merck, Darmstadt, Germany, p.a. grade) on aqueous subphases made from either Milli-Q-filtered H2O with a resistivity R > 18.2 MΩcm (X-ray experiments; Millipore, Bedford, MA) or D2O triply distilled in an all-quartz/Teflon (PTFE) apparatus with R > 10 MΩcm28 (neutron experiments). These subphases were buffered with ∼1 mM boric acid (pH ) 9.0) and contained 10 mM CaCl2. After evaporation of the solvent, the films were compressed to a surface pressure, π, of typically 28 mN/m where the monolayer is in the hexatic TC phase. Phospholipid monolayer phases are denoted according to Kaganer et al.:29 G, gaseous; LE, liquid expanded; TC, tilted condensed; SC, solid condensed. X-ray reflectivity experiments were conducted at the undulator beamline BW130 (undulator gap: 15.6 mm) of HASYLAB (DESY, Hamburg, Germany) at a positron energy of ∼4.6 GeV. The experimental setup, in which a monochromator in Laue geometry deflects the impinging beam down to the sample at an adjustable angle,31 has been described in detail.32-34 X-ray wavelengths, chosen in different runs, were between λ ) 1.38 and 1.45 Å. The beam footprint on the sample was ∼5 × 50 mm2. The incident beam was attenuated using various calibrated Al absorbers in different regimes of Qz; at Qz > 0.4 Å-1, the full (nonattenuated) beam was used in the experiments. The sample chamber consists of a custom-built, computer-controlled Langmuir film balance (surface area 16 × 30 cm2) incorporated in a gastight, thermostated Al container with Kapton windows for the X-ray beam.35 Experiments were performed at room temperature (T ) 21 ( 1 °C) with a He atmosphere over the sample films. Neutron reflectivity experiments were performed at the liquid surface reflectometer TAS 9, the MARK-2 implementation of the MARK-1 reflectometer36 at TAS7, in the guide hall of the DR3 reactor at Risø National Laboratory. The cold neutrons, moderated using liquid hydrogen at 20 K, were monochromated by means of pyrolytic graphite (002) around λ ∼ 4.76 Å and deflected to the sample surface in a geometry similar to that of the X-ray experiments.36 The incident and reflected beams were shaped by means of two computer-controlled vertical Cd slits each, located at the entrance and exit, respectively, of an 80’ Soller collimator. A Be filter operating at 77 K removed higher harmonics from the incident beam. The detector was a high pressure (∼5 atm) 3He chamber. The incident neutron beam was continuously monitored by a low pressure (∼0.05 atm) 3He detector just in front of the sample. The background count rate at each Qz position was determined by missetting the detector arm of the instrument by ∼3° horizontally away from the specular position. The sample chamber was of a design similar to that described for the X-ray measurements. Reflectivity data were obtained at T ∼ 21 °C on H2O and on D2O. In both types of experiments, polished (λ/10) Pyrex (boron silicate) glass blocks, inserted into the subphase to diminish the depth under the beam footprints on the monolayer to ∼300 µm, were used to suppress surface waves in the film balance. After characterization of a phospholipid surface layer, typically 5-10 mL of the S-layer protein solution were injected through the compressed phospholipid monolayer into the aqueous subphase. The protein was allowed to incubate the monolayer for 10-12 h, and progress of the film formation was monitored by recording the reflectivity at constant momentum transfer, Qz.
Weygand et al.
Figure 1. Normalized X-ray reflectivity, R/RF, of a DPPE surface monolayer (π ) 28 mN/m) and a DPPE/CCM2177 multilayer film, prepared as described in the Material and Methods section, on an aqueous (H2O) subphase with 10 mM CaCl2 buffered at pH ) 9. T ) 21 ( 1 °C. Continuous lines show the modeled reflectivities of the DPPE monolayer (cf. Figure 4) and the complex S-layer/lipid layer. The latter was derived from a joint refinement of X-ray and neutron scattering data (cf. Figures 3 and 5).
Protein film formation was assumed to be completed when no change in the reflectivity signal was observed for more than 30 min. To protect against beam damage during the X-ray measurements, the sample chamber was continuously horizontally translated such that new sample film was moved into the beam footprint.14 This ensured that at any time of the experiments the illuminated film was largely undamaged. After completing the scattering experiments, the crystallinity and completeness of the reconstituted protein lattice was routinely checked by transferring the lipid/protein sample films to EM grids coated with carbon films.13,14 Typically, 15-20 EM grids were placed at different locations on the film balance in the region of the beam footprint. They were inspected in the EM (Philips CM12, Eindhoven, NL) after fixation with glutaraldehyde and negative staining with uranyl acetate. To estimate the (X-ray or neutron) SLD of the protein, the amino acid composition of the S-layer protein from B. sphaericus CCM2177 has been determined14 using a standard procedure.37 Volumetric information for the individual amino acids, folded into the peptide chain within the protein, has been derived by starting from the molecular volumes from crystal data38 and adding an extra 3% for packing deficiencies of the peptide.39 In similar measurements of streptavidin binding to biotinylated lipid monolayers,35,40,41 this procedure yielded a realistic estimate of the solvent-inaccessible volume if proton-deuteron exchange on the protein in D2O was taken into account.35,39 An estimate of the SLDs was finally derived by weighting the electron numbers or neutron scattering lengths of the amino acids with their relative abundance and dividing the result by their, analogously computed, associated volume, yielding Fpe ) 0.412 Å-3 and Fpn ) 1.93 × 10-6 Å-2, respectively. This served as a starting point for further data refinement (see below). Results and Data Interpretation Two X-ray reflectivity data sets, obtained at π ∼ 28 mN/m for a pure DPPE monolayer and at π ∼ 34 mN/m for a compound DPPE/CCM2177 surface film on subphases with 10 mM CaCl2, are compared in Figure 1. The abbreviation “CCM2177” is from here on used to denote the S-layer protein from B. sphaericus CCM2177. The adsorbed protein consists of a recrystallized S-layer, i.e., a molecularly thin protein sheet crystal, as demonstrated in situ in similar preparations that were
Reorganization of Phospholipid Headgroups
J. Phys. Chem. B, Vol. 106, No. 22, 2002 5795
Figure 2. Normalized neutron reflectivity, R/RF, of a DPPE-d62/ CCM2177 multilayer film on an aqueous (D2O) subphase with 10 mM CaCl2 buffered at pH ) 9. T ) 21 ( 1 °C. The continuous line shows the modeled reflectivity of the complex S-layer/lipid layer as derived from a joint refinement of X-ray and neutron scattering data (cf. Figures 3 and 5).
characterized with grazing-incidence small-angle X-ray diffraction33 (GISAXD) at the BW1 undulator beamline of HASYLAB (DESY, Hamburg).14 The crystallinity of the samples characterized in this work was checked ex situ with electron microscopy13 after every individual experiment. During protein incubation, the monolayer was maintained at constant area and the surface pressure raised gradually from π ) 28 to 34 mN/m. Obviously, the lipid/protein data set represents a much more complex structure than the pure lipid data set. However, even simple lipid data at high resolution, extending out in Qz to ∼0.8 Å-1, have recently turned out to provide a challenging task for data modeling at the molecular level because the conventional slab or “box” models fall short at a realistic data description at high Qz.42,43 In particular, the application of the box model to phospholipid monolayers, such as PEs of various chain lengths,44 yielded structures in which the headgroup hydration appears rather unrealistic,43 apparently dropping upon compression from 20+ H2O molecules associated with the PE headgroup at low π (LE phase) to -50 Å) cf. Figure 5. The inset in part (a) shows a magnified view of the electron density (normalized to the value of the water subphase, F0e ) 0.3336 Å-3) in the region where the primary S-protein layer faces the secondary layer (see text). The dashed trace in this inset shows that the electron density profile of the primary layer after reflection around the line, z ) -145 Å, and a multiplication by × 0.3 matches the original profile almost perfectly.
sets under the assumption that the underlying molecular structure is equivalent in both experimental situations. A rather complete description of the free-form electron density profile derived from the experimental data shown in Figure 1 has already been presented.14 Figures 1 and 2 of ref 14 give a good appreciation of subsequent states of model refinement. The final result of the data refinement achieved in that work showed that the reconstituted S-layer does actually not correspond to a neat monomolecular protein layer. Rather, the protein interface structure consisted of a monolayer, presumably densely packed and crystalline as revealed from GISAXD, to which a partially filled (volume fraction, ∼30%), secondary monolayer is coupled in a back-to-back mode with respect to the uniaxial protein orientation.14 Figure 3 shows the corresponding electron density profile in full detail. It extends from the alkane/air interface (which defines z ) 0) to about z ) -250 Å. Along z, ∼30 Å correspond to the region occupied by the lipid monolayer. As already discussed earlier,14 this section of the electron density profile is largely unchanged upon S-layer reconstitution, indicating that the overall structure of the lipid monolayer remains intact.50 In fact, (wide-angle) grazing incidence X-ray diffraction from the lipid acyl chains indicated that the hydrophobic part of the monolayer is virtually unaffected
5796 J. Phys. Chem. B, Vol. 106, No. 22, 2002
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by the bound and recrystallized S-layer protein.15 A similar conclusion was more indirectly drawn from a fluorescence microscopic characterization of S-layer reconstitution.12 The lower part of Figure 3 shows a coarse decomposition of the overall electron density profile into four components: The lipid acyl chains, the lipid headgroup, protein matter organized in a structured film underneath the lipid headgroup, and water that fills the space within the protein film which is not occupied by peptide material. Thus, the latter contribution reproduces negatively that of the protein, i.e., Vw(z) ) 1 - Vp(z), where the Vi denote the volume fractions of water and protein. These components have been derived as follows. The lipid components have been determined from refinement of the model with neutron data (see below). The relative proportions of peptide and water have been determined under the assumption that the volume occupied by the protein may be described by means of a uniform electron density, Fpe ) 0.412 Å-3, computed as described in the Materials and Methods section. An increase of the experimentally determined Fe value at a particular distance z from the surface, Fe ) Fe(z), above the value for water (assumed to be equivalent to bulk water, F0e ) 0.3336 Å-3) is thus attributed to a volume fraction of protein, Vp(z), respectively, of water, V0(z), that obey νw(z) + νp(z) ) 1. Then, clearly, Fe(z) ) νp(z)Fpe + ν0(z)F0e , whence
νp(z) )
Fe(z)/F0e - 1 Fpe /F0e - 1
(1)
Within the protein monolayer, Vp varies roughly between 0.2 and 0.7.14 This protein monolayer extends roughly from z ) -30 to -145 Å. Beyond z ) -145 Å, the electron density profile suggests that a secondary monolayer with a low area occupancy is attached to the primary monolayer in an inside-out orientation. An expanded section of the electron density profile, normalized to F0e , is shown in the inset in Figure 3. The corresponding region of Fe/F0e is located around the interface between the primary and the secondary protein layers. This region is characterized by a large electron density, peaked at z ∼ -110 Å. A second, minor peak in Fe (and, thus, in Vp) is observed around z ) -180 Å. Its relative magnitude (i.e., its deviation from Fe/F0e ) 1) amounts to ∼30% of that of the larger peak and is most likely associated with a partial secondary protein monolayer. This is quantitatively demonstrated by the dotted line which represents a segment of the electron density profile of the primary layer, mirrored around z ) -145 Å and multiplied with a factor of 0.3: Apparently, the dotted line retraces the secondary maximum almost perfectly. The detailed structure and structural rearrangements of the lipid headgroups are the focus of this work. We start with an analysis of the pure lipid structure. At π ) 28 mN/m, the roomtemperature isotherm of DPPE suggests an area per molecule, A ∼ 42 Å2. This is in line with twice the unit cell area of the ) 38.6 Å2, derived from GIXD.15 The chain lattice, 2 × AGIXD 0 discrepancy in the two results derives presumably from grain boundaries within the surface monolayers where the molecules do not contribute to diffraction, such that the value estimated from GIXD constitutes a lower limit. Figure 4 shows the Fe profile of the DPPE monolayer at 28 mN/m, derived from the data shown in Figure 1 (upper trace) in more detail. The overall profile has been determined with the model-free data inversion approach. Its decomposition into components (lower traces) that represent the acyl chains, hydrated lipid headgroups, and
Figure 4. Chemical interpretation of the electron density profile of a pure DPPE monolayer at π ) 28 mN/m and its decomposition into submolecular fragments. For details, see text.
subphase water is inspired by the VRDF approach and complies with the following rationale. The acyl chains form a homogeneous layer interfacing with the air compartment. It is assumed to obtain a symmetric envelope whose lower boundary, near z ) -25 Å, is fixed by the number of electrons on the two DPPE chains and the value of A and is thus unambiguously determined. The half-width of the acyl chain distribution is ∼19 Å. In contrast, the extension of the PE headgroup remains ambiguous, because the electron density of the ethanolamine is rather similar to that of (bulk) water. Consequently, if one decomposes the overall Fe profile, the lower end of the resulting headgroup distribution function cannot be determined. This is indicated in Figure 4 which shows three alternate traces that correspond to PE groups hydrated with nw ) 3, 5, and 7 water molecules, respectively.51 Correspondingly, the apparent onset of the bulk subphase shifts to progressively lower z values as nw is increased in the model. If we assume that five water molecules are thus laterally associated with each headgroup, as suggested by recent investigations of other phospholipid monolayers,43,45,47 the apparent half-width of the PE distribution functions is ∼10 Å, such that the entire DPPE molecule at the surface extends along ∼29 Å. Model Refinement on the DPPE/CCM2177 Layer System with Neutron Data. Although model-independent data inversion methods, such as the one used in this work, are instrumental in identifying SLD profiles that might possibly describe the physical situation well, neither a cross-check on the chemical plausibility of the resulting profile nor a cross-reference between various (e.g., X-ray and neutron) data sets is tractable. Thus, for a co-refinement with neutron data, the electron density profile shown in Figure 3a has to be further decomposed and parametrized. Of particular interest in the context of this work was the interface between the protein layer and the lipid surface monolayer. Because it has been earlier determined that peptide material does not insert into the acyl chain region,14,15 we only need to consider short-range interpenetration of the peptide into the lipid. We have described this interaction as a analytical contribution to the electron density profile that decays monotonically toward zero and allows for a “smooth landing” of the corresponding distribution function. Because the analysis of the pure lipid SLD profile has suggested that the DPPE is confined to a range that extends about 30 Å across the interface, it may be safely assumed that below z ) -40 Å only protein determines the SLD profile of the DPPE/CCM2177 system. For
Reorganization of Phospholipid Headgroups
J. Phys. Chem. B, Vol. 106, No. 22, 2002 5797 in a loss of about two water molecules associated with the phosphate, and concurrently gains slightly in distribution width. Although the peptide interpenetrates deeply into the PE headgroup, there is minimal interaction, if any, with the lipid’s acyl chains. The electron density associated with the inserted peptide amounts to ∼61 electrons (on average) per lipid molecule in the surface film, equivalent to about one amino acid or two sidegroups (of average complexity). It should be emphasized that the explicit form of the analytical function used to characterize peptide insertion is not relevant for the details of the results: The amount of scattering length introduced into the headgroup region is rather independent of the technical details. Discussion
Figure 5. Chemical interpretation of the electron density profile of the DPPE/CCM2177 multilayer structure in the region of the lipid surface monolayer and comparison with the structure of the pure lipid monolayer. The overall profiles (continuous lines) have been decomposed into submolecular fragments (dashed lines, DPPE/CCM2177 system; dotted lines, pure lipid monolayer) in a refinement of the data shown in Figures 1 and 2. For details, see text.
z > -40 Å, the volume fraction distributions of peptide and of water, Vp and Vw, have thus been parametrized as
( (z + 40l Å) )(ν(z ) -40 Å) -
ν(z) ) 1 -
b
(z + 40 Å) tan R);
-40 Å < z < -40 Å + l (2)
where l represents free parameters for peptide or water, lp or lw, that determine the maximum length of insertion into the lipid monolayer and a ) tan R and b are constants. R is determined by the slope of the electron density contribution of peptide or of water at z ) - 40 Å - δ. To obtain a “smooth landing” of the distribution function at z ) lp - 40 Å, b ) 0.5 has been used for the peptide in the model. For the description of the peptide-associated water, b ) 1 was used. The parametrization described above allows us to separate for z > -40 Å the electron density contributions of the acyl chains, the inserted peptide, and the hydration water it introduces into the lipid monolayer. The contribution of the lipid headgroup itself derives from the difference between the overall SLD profile, as derived from the model-independent fit, and the three contributions discussed above. Figure 5, which shows the corresponding decomposition of the overall SLD profile into components after data refinement with the X-ray and neutron reflectivity results of Figures 1 and 2, shows that this leads to an electron distribution in the lipid headgroup region that resembles very closely those deduced for pure lipid monolayers with established modeling concepts.43,52 This in turn validates the chosen approach of model construction. It is obvious that the chosen model would be underdetermined in the lipid headgroup region if only X-ray reflectivity data on the hybrid lipid/protein layer structure were available. However, a simultaneous evaluation of the X-ray and neutron results measured from samples of nominally identical preparation enables a unique determination of the model parameters. The result is visualized in Figure 5. The formally independent interpenetration lengths for peptide and its associated water turn out to be rather similar under the stated choices of the model: lp ) 16.0 Å and lw ) 14.9 Å.53 As visualized in the decomposed SLD profile, the lipid headgroup loses electron density upon S-layer recrystallization, because of dehydration which results
The (nonnatural) coupling of S-protomers to (phospho)lipid membranes is believed to have application potential for the stabilization of biomembrane models and the design of molecularly well-defined supramolecular architectures that permit control of membrane permeation. It has recently been shown that biomembrane models, such as planar bilayer membranes21 or liposomes,19,20 may be stabilized against thermal, mechanical, or electrical stress. It has also been demonstrated that membrane proteins incorporate readily into such stabilized membranes18 and may thus functionalize them. Such functionalized membranes have in turn been successfully transferred to macroscopic, porous supports, polyamide microfiltration membranes,54 and have thus for the first time been prepared in a stable format that might in the future be turned into real-life technical applications. Although the 3D atomic-level structure of S-layer proteins is still an unresolved problem, electron microscopy and atomicforce microscopy have revealed some general structural properties of the surfaces of S-layer lattices.6,7,9,10,55,56 In addition, the relative location of individual amino acids on specific S-proteins and their assembly products is being mapped out using molecular biology techniques.57,58 Still, the coupling of Sprotomers to lipid surfaces and their subsequent recrystallization into coherent S-layer lattices12 has not been fully understood. Because the exposure of cationic groups at the interface has been reported to be a requirement for S-layer recrystallization, electrostatic interactions play certainly a prominent role in the coupling.13 In addition, specific steric requirements, such as a maximum size of the lipid headgroups, are a prerequisite for S-layer reconstitution at a lipid surface.13 This is consistent with the finding that anionic groups on the protein are located in shallow pockets at those protein surfaces facing the membrane,59,60 such that these charges are only accessible to moieties which protrude from the interface to some extent. Vice versa, this suggests that the binding of the S-protein to the lipid interface occurs via the insertion of a structurally intact protein motif that forms a pocket capable of sterically specific recognition. Thus, it is not individual amino acid side chains interpenetrate the lipid layer but, rather, intact and well-defined motifs formed by the peptide. The observation of one amino acid interpenetrating the lipid layer on aVerage per headgroup has consequently to be interpreted in terms of specific contacts between the protein and lipid that occur locally at the interface. This conclusion is consistent with the observation that the acyl chain order of the lipid monolayer does not decrease by S-layer recrystallization.12,14,15 There thus emerges a molecular model for the coupled S-layer/phospholipid film structure in which the protein interacts with the lipid surface at specific, locally confined sites at their
5798 J. Phys. Chem. B, Vol. 106, No. 22, 2002 mutual interface. In view of the paracrystalline structure of the reconstituted S-layer, it is likely that these interactions take place at periodically recurring sites that match the protein lattice. It is quite conceivable that the interstitial lipid molecules, i.e., those that do not interact with the protein, are largely unaffected in their physicochemical properties. This is in turn consistent with the observation that nonconstitutive membrane proteins, such as the heptameric pore, R-hemolysin, insert readily into S-layersupported bilayer membranes.18 It would be surprising if such an insertion process should occur into a lipid layer affected by interactions with the S-layer that are laterally homogeneous on the molecular scale. From this notion, the view arises that some of the lipids bound to the paracrystalline S-layer are immobilized, whereas others, presumably the largest fraction, are more mobile since their headgroups are not specifically attached to the S-layer. We expect thus that some of the lipids bound to the S-lattice show a lateral mobility significantly reduced below that of the remaining fraction61 and have coined the phrase “semifluid membrane” to describe this property of the attached lipid layer.10 Conclusions The reconstitution of S-layers at phospholipid membranes is an exciting example of supramolecular self-organization at interfaces.11 The vast variety of natural S-layer proteins and the possibilities of chemical and genetic modification of the protein building blocks makes this a unique construction set for targeted surface modifications and molecular system design. Electrostatic interaction has been inferred from studies of S-protein adsorption and crystallization at various lipid monolayers13 and from a partial reorganization of the phospholipid headgroups14 upon S-layer formation under lipid monolayers and is also a likely coupling mechanism of S-layer proteins to lipid vesicles. It has been consistently observed in FTIR spectroscopy12 and in GIXD14,15 that S-layer coupling does not reduce the chain order in the adjacent lipid layer. In fact, the interaction of the protein is essentially confined to the lipid headgroup region: For the S-layer system from B. sphaericus CCM2177 attached to DPPE monolayers, we demonstrated in this study that the protein interacts strongly with the lipid headgroups. Upon protein attachment, the lipid headgroups dehydrate slightly, whereas the distribution width of the phosphate moiety (which is the headgroup component with the largest electron density) increases thus indicating that the protein infers on the headgroup at least a slight reorganization. From a quantitative evaluation of the SLD profiles it appears unlikely that isolated peptide side-chains intercalate the lipid headgroup region because this would exert a more massive, and more visible, impact on the monolayer. Instead, we propose a model in which structurally intact protein motifs interpenetrate the PE headgroups at least up to the level where the phosphates are located. Such a model in turn speaks in favor of a physicochemical state of the attached lipid membrane in which many, or even most, of the lipids in the proximal leaflet retain their lateral mobility. Consequently, only a limited proportion is impeded by the attachment of the repetitive domains of the S-layer lattice. Acknowledgment. This work has been supported by the German Science Foundation (SFB 294, TP F3), the Austrian Science Foundation (Projects S7204/S7205), the Fonds der Chemischen Industrie, Frankfurt, the Danish DanSync program, and the IHP Contract HPRI-CT-1999-00040 and the TMR contract ERBFMGECT950059 of the European Commission. We gratefully acknowledge time at beamline BW1 in
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