Báo cáo khoa học: S-Layers as a basic building block in a molecular construction kit ppt

MINIREVIEW S-Layers as a basic building block in a molecular construction kit Uwe B. Sleytr, Eva M. Egelseer, Nicola Ilk, Dietmar Pum and Bernhard Schuster Center for NanoBiotechnology, University of Natural Resources and Applied Life Sciences Vienna, Austria Introduction Methods for organizing materials at the nanometer level are essential for the fabrication of supramolecular structures and devices. Thus, molecular self-assembly systems that exploit the molecular scale manufacturing precision of biological systems are prime candidates in nanobiotechnology. Crystalline bacterial cell surface layer (S-layer) pro- teins have been optimized during billions of years of biological evolution as building blocks of one of the simplest self-assembly systems (Fig. 1) [1–3]. S-Layers are now recognized as the most common outermost cell envelope components of prokaryotic organisms [3,4]. Most S-layers are composed of a single protein or glycoprotein species endowed with the ability to assemble into monomolecular arrays on the supporting envelope layer, representing the simplest biological membrane developed during evolution. The wealth of information accumulated on the structure, chemistry, morphogenesis, genetics, and function of S-layers has led to a broad spectrum of application in nanobiotech- nology and biomimetics [2,5]. Most importantly, S-layers represent very versatile self-assembly systems with unique features as the structural basis for a com- plete supramolecular construction kit, involving all major types of biological molecules: proteins, lipids, glycans, nucleic acids, and combinations of these. Keywords biomimetics; biosensors; nanobiotechnology; nanoparticles; S-layers Correspondence U. B. Sleytr, Center for NanoBiotechnology, University of Natural Resources and Applied Life Science Vienna, Gregor-Mendel-Strasse 33, 1180 Vienna, Austria Fax: +43 1 4789112 Tel: +43 1 47654 2201 E-mail: uwe.sleytr@boku.ac.at This paper is dedicated to the memory of Margit Sa ´ ra (Received 6 October 2006, accepted 14 November 2006) doi:10.1111/j.1742-4658.2006.05606.x Crystalline arrays of protein or glycoprotein subunits forming surface layers (S-layers) are the most common outermost envelope components of prokaryotic organisms (archaea and bacteria). The wealth of information on the structure, chemistry, genetics, morphogenesis, and function of S-layers has revealed a broad application potential. As S-layers are periodic structures, they exhibit identical physicochemical properties for each molecular unit down to the subnanometer level and possess pores of identi- cal size and morphology. Many applications of S-layers in nanobiotechnol- ogy depend on the ability of isolated subunits to recrystallize into monomolecular lattices in suspension or on suitable surfaces and interfaces. S-Layer lattices can be exploited as scaffolding and patterning elements for generating more complex supramolecular assemblies and structures, as required for life and nonlife science applications. Abbreviations Bet v1, major birch pollen allergen; EGFP, enhanced green fluorescent protein; PSA, prostate-specific antigen; SbpA, S-layer protein of Bacillus sphaericus CCM177; SbsB, S-layer protein of Geobacillus stearothermophilus PV72 ⁄ p2; SbsC, S-layer proteins of Geobacillus stearothermophilus ATCC 12980; SCWP, secondary cell wall polymer; SgsE, S-layer protein of Geobacillus stearothermophilus NRS 2004 ⁄ 3a variant 1; S-layer, crystalline bacterial cell surface layer; SLH, S-layer-homology; S-liposomes, S-layer coated liposomes; SPR, surface plasmon resonance; SUM, S-layer ultrafiltration membrane; ZZ, two copies of the synthetic analog of the IgG-binding domain of protein A from Staphylococcus aureus. FEBS Journal 274 (2007) 323–334 ª 2006 The Authors Journal compilation ª 2006 FEBS 323 S-Layers, as periodic structures, exhibit identical physicochemical properties on each molecular unit down to the subnanometer level and possess pores of identical size and morphology. Moreover, functional groups are aligned on the surface and within the pores of the lattice in well-defined positions and orientation. The possibility to change the natural properties of S-layer proteins by genetic engineering and incorporate single-functional or multifunctional domains into S-layer lattices has opened up new strategies for the fine- tuning of their structural and functional features [6–8]. Major areas of application of S-layers include: (a) production of isoporous ultrafiltration membranes; (b) supporting structures for defined immobilization or incorporation of functional molecules (e.g. antigens, antibodies, ligands, enzymes); (c) matrix for the devel- opment of biosensors including solid-phase immuno- assays and label-free detection systems; (d) support and stabilizing matrices for functional lipid mem- branes, liposomes, and emulsomes; (e) adjuvants for weakly immunogenic antigens and haptens; (f) matrix for controlled biomineralization and structure for for- mation of ordered arrays of metal clusters or nano- particles as required for molecular electronics and nonlinear optics or catalysts [2,3,5,6,9–11]. General aspects of S-layers S-Layer proteins are widely distributed in the major lineages of archaea and in Gram-positive and Gram- negative bacteria [2–4,7]. In S-layer-carrying organ- isms, up to 20% of the total protein synthesis effort may be devoted to the production of S-layer proteins. S-Layers represent a fascinating model system for studying the dynamic process of assembly of a supra- molecular structure during cell growth. An intact closed S-layer on an average-sized, rod-shaped prok- aryotic cell consists of  500 000 monomers. Thus, to maintain a complete S-layer on the surface of a cell growing with a generation time of 20–30 min, at least 500 copies of a single polypeptide species have to be synthesized, translocated to the cell surface, and incor- porated into the existing lattice per second. Under laboratory cultivation conditions, the yield of S-layer protein is strain-specific, ranging between 0.5 and 2.0 g wet weight per litre growth medium. Most S-layers are composed of a single homogen- eous protein or glycoprotein species with a molecular mass of 40–200 kDa. The degree of glycosylation of S-layer proteins can vary between 2% and 10% (w ⁄ w) [2,4]. Bacterial S-layer lattices are generally 5–20 nm thick, whereas S-layer lattices of archaea are up to  70 nm thick. Transmission electron microscopic studies on the mass distribution of S-layers (Fig. 1A) and subsequent 2D and 3D analysis, including compu- ter image enhancement, have produced structural information down to 0.35–1.5 nm [2]. High-resolution images of the surface topography of S-layers under biological conditions have been obtained by scanning force microscopy [2,12]. A common feature of S-layers is, with respect to the orientation on the cell, their smooth outer surface and more corrugated inner A B Fig. 1. (A) Electron micrograph of a freeze-etched and Pt ⁄ C-shadowed preparation of a Gram-positive organism exhibiting a square (p4) S-layer lattice. The bar corresponds to 100 nm. (B) Schematic drawing illustrating the various S-layer lattice types. In the oblique lattice, one morphological unit (red) consists of one (p1) or two (p2) identical subunits. Four subunits constitute one morphological unit in the square (p4) lattice type, whereas the hexagonal lattice type is either composed of three (p3) or six (p6) subunits. Modified from Sleytr et al [2] with permission from Wiley-VCH. Molecular construction kit based on S-layers U. B. Sleytr et al. 324 FEBS Journal 274 (2007) 323–334 ª 2006 The Authors Journal compilation ª 2006 FEBS surface. The proteinaceous subunits of S-layers are aligned either in lattices with oblique (p1, p2), square (p4), or hexagonal (p3, p6) symmetry (Fig. 1B) with a center-to-center spacing of the morphological units of  3–35 nm [2,8]. Hexagonal lattice symmetry is pre- dominant among archaea [3]. S-Layers are highly por- ous protein lattices with a surface porosity of 30–70%. As S-layers are mostly composed of identical species of subunits, they exhibit pores of identical size and mor- phology [2,3,5]. However, in many protein lattices, two or more distinct classes of pores with diameters in the range 2–8 nm have been identified. Results of amino-acid analysis and estimation of the secondary structure by comparison of protein sequence data and CD measurement of various S-layer proteins can be summarized as follows. There are large amounts of glutamic acid and aspartic acid ( 15 mol%), a high lysine content ( 10 mol%), and large amounts of hydrophobic amino acids ( 40–60 mol%). The hydrophilic and hydrophobic amino acids do not form extended clusters, and, in most S-layer proteins,  20% of the amino acids are organized as a-helix and  40% occur as b-sheets. The aperiodic folding and b-turn content may vary between 5% and 45%. In general, most S-layer subunits are weakly acidic proteins with isoelectric points in the range 4–6, except the S-layer proteins of lactobacilli and that of Methanothermus fervidus. Post-translational modifica- tions include cleavage of N-terminal or C-terminal fragments, phosphorylation, and glycosylation of amino-acid residues [13]. The latter is a remarkable characteristic of many archaeal and some bacterial S-layer proteins. The glycan chains and linkages differ significantly from those of eukaryotes [14]. Self-assembly of S-layer proteins One of the most fascinating properties of isolated native and recombinant S-layer proteins is their ability to form free-floating self-assembly products in solution (flat sheets, cylinders or spheres), to recrystallize into extended monomolecular layers on solid supports, at the air ⁄ water interface and on lipid films, and to cover liposomes and nanocapsules completely (Fig. 2) [5,8]. The reassembly occurs after removal of the disrupting agent used in the dissolution and isolation procedure. In general, complete disintegration of S-layer lattices into their constituent protein subunits on bacterial cells can be achieved using high concentrations of cha- otropic agents (e.g. guanidine hydrochloride, urea), by lowering or raising the pH, or by applying metal- chelating agents or cation substitution [5]. The forma- tion of self-assembled arrays is only determined by the amino-acid sequence of the polypeptide chains and consequently the tertiary structure of the S-layer protein species. In various S-layer proteins from Bacil- lacaea it has been shown that significant portions of the C-terminal or N-terminal part can be deleted with- out loss of the capability of the subunits for lattice formation [15]. Further, for the S-layer protein of Bacillus sphaericus CCM 2177 (SbpA), truncation of the amino-acid sequence led to a change in the S-layer lattice type from square (p4) to oblique (p1) lattice symmetry [16]. Fig. 2. Schematic drawing of the isolation of native S-layer proteins from bacterial cells and the reassembly of native and recombin- ant S-layer proteins into crystalline arrays in suspension, on a solid support, at the air ⁄ water interface and on a planar lipid film, and on liposomes or nanocapsules. An example of S-layer proteins reassembling with hexagonal (p6) lattice symmetry is shown here. Modified from Pum et al. [8], with permission from Springer. U. B. Sleytr et al. Molecular construction kit based on S-layers FEBS Journal 274 (2007) 323–334 ª 2006 The Authors Journal compilation ª 2006 FEBS 325 In suspension, the formation of S-layer self-assembly products starts by rapid nucleation of small oligomeric precursors, which subsequently form extended aggregates by a much slower self-assembly process. Depending on the S-layer protein species used and on the environmental conditions, double layers in back-to- back orientation may be formed. On solid supports, lattice formation is started after the attachment of nucleation sites, which may be oligomeric S-layer pro- tein or small self-assembly products from solution, and is continued by lateral growth in all directions until the proceeding front lines of the growing domains meet [12]. In this way, a closed mosaic of monocrystalline domains is formed. Depending on the S-layer proteins species used, on the environmental conditions and, in particular, on the surface properties of the support (e.g. hydrophobicity, surface charge), the size of the individual domains may be up to 20 lm in diameter. Genetics of S-layer proteins and their interaction with secondary cell wall polymers Several nanobiotechnological applications and functio- nalization of S-layer lattices depend on the recrystalli- zation of S-layer lattices in defined orientation with respect to their inner and outer surface. In this con- text, detailed information on the genetics of S-layer proteins and mechanisms determining in vivo S-layer assembly is important. As S-layer proteins represent an important class of secreted proteins, numerous S-layer genes from bac- teria and archaea have been sequenced and cloned [4,13]. For S-layer proteins of Gram-positive bacteria at least, common structural organization principles have been identified. A cell-wall-targeting domain was found at the N-terminal region of many S-layer pro- teins, which mediates binding to a specific hetero- polysaccharide, termed secondary cell wall polymer (SCWP), by a lectin-type binding. For Gram-positive bacteria, at least two types of binding mechanism between the N-terminal region of S-layer proteins and SCWPs have been described [16–18]. With respect to the first binding mechanism, so-called S-layer-homol- ogy (SLH) motifs, each comprising about 55 amino acids, recognize a distinct type of pyruvylated SCWP as the correct anchoring structure. The second binding mechanism has been described for Geobacillus stearo- thermophilus wild-type strains and is characterized by the interaction of a nonpyruvylated SCWP containing the negatively charged 2,3-dideoxydiacetamidomanno- samine uronic acid with a highly conserved N-terminal region lacking an SLH domain. However, the cell- wall-targeting domain is not necessarily located in the N-terminal region of the S-layer protein. Well-docu- mented examples of C-terminal anchoring are the S-layer proteins of Lactobacillus acidophilus ATCC 4556 and Lactobacillus crispatus [7]. To elucidate the structure–function relationship of distinct segments of S-layer proteins and to determine which amino-acid positions are surface-located and accessible, different strategies were pursued. In a first approach, N-terminally and C-terminally truncated forms were produced, and their self-assembly and recrystallization properties investigated [16,19,20]. The S-layer protein of Geoacillus stearothermophilus PV72 ⁄ p2 (SbsB) could be characterized by its two functionally and structurally separated parts, namely the specific SCWP-binding domain defined by the three consecutive SLH motifs and the larger C-terminal part responsible for formation of the crystalline array [21]. Interestingly, the deletion of even fewer than 15 C-ter- minal amino acids resulted in completely water-soluble forms of SbsB [15,19]. In contrast with SbsB, the S-layer proteins, SbsC of Geobacillus stearothermophi- lus ATCC 12980 and SbpA, turned out to be highly tolerant to deletions, as up to 179 and 237 amino acids, respectively, could be deleted at the C-terminus without interfering with lattice formation [16,20]. Another attempt to find which amino-acid positions in the primary sequence are located on the outer surface of the subunits, inside the pores, or at the subunit to subunit interface was seen in a cysteine scanning muta- genesis study with screening of the accessibility of the introduced cysteine residue in soluble, self-assembled and recrystallized S-layer proteins [19]. As, to date, no structural model at atomic resolution of any S-layer protein is available, elucidation of the 3D structure of S-layer proteins by X-ray crystallogra- phy is also being pursued. This can be explained by (a) the molecular mass of the S-layer subunits being too large for NMR analysis, (b) their high tendency to form 2D lattices preventing the formation of isotropic 3D crystals required for X-ray crystallography, and (c) the very low solubility of isolated subunits. First, 3D crystallization studies were carried out with water- soluble N-terminally or C-terminally truncated forms of SbsC. For the C-terminally truncated form, recom- binant SbsC 31-844 , crystals that diffracted to a resolu- tion of 3 A ˚ using synchrotron radiation could be obtained [22]. Native and heavy atom derivative data confirmed the results of the secondary-structure predic- tion, which indicated that the N-terminal region com- prising the first 257 amino acids is mainly organized as a-helices, whereas the middle and C-terminal parts of SbsC consist of loops and b-sheets. Information on the Molecular construction kit based on S-layers U. B. Sleytr et al. 326 FEBS Journal 274 (2007) 323–334 ª 2006 The Authors Journal compilation ª 2006 FEBS 3D structure of S-layer proteins would open up the possibility of rationally designing S-layer fusion pro- teins incorporating functional domains, for example within the pore areas of the protein lattice. As cell surface components can generally be consid- ered to be nonconservative structures that determine the interaction between the living cell and its environ- ment, the observation of phenotypic S-layer variation was not surprising. S-Layer variation has been repor- ted to occur in pathogens as well as in nonpathogens and leads to the synthesis of alternate S-layer proteins, either by the expression of complete (silent) S-layer genes or by recombination of partial coding sequences [7]. In pathogens, altered cell surface properties prob- ably protect the cells from the lytic activity of the immune system. In Campylobacter fetus, only the C-terminal part of the S-layer protein is exchanged, and the N-terminal region and the S-layer-specific lipo- polysaccharides remain conserved. In nonpathogens, S-layer variation is often induced in response to envi- ronmental stress factors such as increased oxygen supply [23]. In the strain variants, expression of a com- pletely new S-layer protein is accompanied by synthesis of a different type of SCWP, and S-layer variation can also lead to a change in the lattice type. At the molecular level, S-layer variation in G. stearothermo- philus PV72 was found to be based on DNA rear- rangements between the chromosome and a naturally occurring megaplasmid. Regarding the development of S-layer-deficient phenotypes, the importance of inser- tion sequence elements has been demonstrated for at least three different organisms [7]. S-Layer fusion proteins and applications For the production of nanoscale building blocks for the bottom-up fabrication of bio-inspired materials with designed properties, genetic approaches are cur- rently used for the construction of functional S-layer fusion proteins [6,7]. S-Layer fusion proteins based on the S-layer proteins, SbsB, SbsC and SbpA, incorpor- ate an accessible N-terminal SCWP-binding domain, the self-assembly domain, as well as a biologically act- ive sequence fused to the C-terminus. After heterolo- gous expression of the genes encoding chimeric S-layer proteins in Escherichia coli, it could be shown that the self-assembling properties of the S-layer protein moiety as well as the functionality of the fused sequences were retained in all S-layer proteins (Fig. 3). In order to build up functional monomolecular S-layer protein lattices on artificial solid supports such as gold, silicon, glass, indium tin oxide or polymers (Fig. 3A,B), the surface has to be functionalized with covalently attached chemically modified SCWP, to which the S-layer fusion proteins bind with their N-ter- minal part, leaving the C-terminal part with the fused functional sequence exposed to the environment [6]. Such chimeric S-layers recrystallized on solid supports in defined orientation (Fig. 3C) should find application in diagnostics and biochip technology (laboratory- on-a-chip), as well as for the development of specific cell targeting and delivery systems [2,4,6–8,24]. In a first approach, S-layer fusion proteins compri- sing the C-terminally truncated form, recombinant (r)SbpA 31-1068 , and the hypervariable region of heavy chain camel antibodies recognizing lysozyme or a pros- tate-specific antigen (PSA) were constructed [25]. PSA is a useful marker for screening for potential prostate A B C Self-assenbling part of (truncated) S-layer protein Functionality of S-layer fusion protein Fig. 3. Digital image reconstructions of transmission electron micro- graphs of negatively stained preparations of (A) the native S-layer protein, SbsB, from Geobacillus stearothermophilus PV72 ⁄ p2 and (B) the streptavidin fusion protein. In the lattice of the fusion pro- tein (B), the streptavidin heterotetramers show up as additional mass (arrows). Bars correspond to 10 nm. (C) Schematic illustration of the self-assembling parts of S-layer fusion proteins and their well-oriented functional domains. Such arrays theoretically provide the highest possible order (spatial control, orientation and position) of functional domains at the nanometer level. The knights (grey) reassemble the functional domains (antigens, enzymes, antibodies, ligands, etc.) and the cut squares (yellow) represent the S-layer. Modified from Pum et al. [8], with permission from Springer. U. B. Sleytr et al. Molecular construction kit based on S-layers FEBS Journal 274 (2007) 323–334 ª 2006 The Authors Journal compilation ª 2006 FEBS 327 cancer patients. This fusion protein specific for PSA was recrystallized as a monolayer on SCWP-precoated gold chips and used as sensing layer in biochips for surface plasmon resonance (SPR) spectroscopy (Fig. 4). It turned out that at least three of four poss- ible PSA molecules were bound per morphological unit of the square S-layer lattice [25]. To summarize, S-layer fusion proteins incorporating camel antibody sequences can be considered key elements for the development of sensing layers for label-free detection systems such as SPR, surface acoustic wave or quartz crystal microbalance, in which the binding event can be measured directly by mass increase without the need for any labeled molecule. The genes encoding the chimeric S-layer proteins, rSbsC 31-920 ⁄ Bet v1 and rSbpA 31-1068 ⁄ Bet v1, carrying the major birch pollen allergen Bet v1 at the C-terminus maintained the ability to self-assemble as well as the functionality of the fused allergen to bind the Bet v1-specific monoclonal mouse antibody [26]. In a recent study, rSbsC 31-920 ⁄ Bet v1 was shown to contain all rele- vant B and T cell epitopes of Bet v1. Compared with free Bet v1, in cells of birch pollen-allergic individuals, the histamine-releasing capacity caused by the fusion protein was significantly reduced, and no Th2-like immune response was observed like after stimulation with free Bet v1 [27]. Owing to its immunomodulating capacity, this fusion protein is generally considered to be a novel approach to specific treatment of allergic diseases (e.g. as carrier ⁄ adjuvants in the design of vaccines for immunotherapy of type 1 allergy). In order to generate a universal affinity matrix for binding of any kind of biotinylated molecules, mini- mum-sized core streptavidin (118 amino acids) was fused to either N-terminal or C-terminal positions of rSbsB or the C-terminus of rSbpA 31-1068 [24]. After expression of the chimeric genes in E. coli and isolation of the fusion proteins from the host cells, a refolding protocol was applied to obtain heterotetramers consist- ing of one chain of the respective fusion protein and three chains of core streptavidin (Fig. 3A,B). Fluores- cence quenching of tryptophan residues in the binding pockets of streptavidin confirmed that the biotin-bind- ing capacity of soluble heterotetramers correlated with the molecular mass of the appropriate biotinylated pro- teins. As a first approach, monolayers generated by recrystallization of rSbpA 31)1068 ⁄ streptavidin hetero- tetramers on plain gold chips or on gold chips precoat- ed with thiolated SCWP were exploited for binding of biotinylated oligonucleotides (30-mers). SPR studies revealed that nonspecific adsorption of fluorescently labeled oligonucleotides (15-mers) carrying two mismat- ches was negligible. Moreover, it could be demonstra- ted that the hybridization reaction with complementary fluorescently labeled oligonucleotides carrying one mis- match followed the Langmuir isotherm. The detection limit for hybridized oligonucleotides was found to be in the picomolar range [24]. To conclude, hybridization experiments with biotinylated and fluorescently labeled oligonucleotides using SPR spectroscopy indicated that a functional sensor surface could be generated by recrystallization of heterotetramers on gold chips. Such promising structures that combine self-assembly prop- erties of an S-layer protein with the biotin-binding properties of streptavidin should find numerous applications in (nano)biotechnology (Fig. 3C). The S-layer fusion protein, rSbpA 31-1068 ⁄ ZZ, incor- porates two copies of the Fc-binding domain (ZZ), a synthetic analog of the IgG-binding domain of pro- tein A from Staphylococcus aureus [28]. As demonstra- ted by SPR, the amount of human IgG that could be bound on the native rSbpA 31-1068 ⁄ ZZ monolayer was slightly higher than on the rSbpA 31-1068 ⁄ ZZ monolayer cross-linked with the bifunctional imidoester dimethylpi- melinimidate. On average,  66% of the theoretical sat- uration capacity of a planar surface was covered by IgG with the Fab regions in the condensed state. Novel bio- compatible microparticles for the microsphere-based detoxification system used for extracorporeal blood purification of patients suffering from autoimmune A B Fig. 4. (A) Schematic drawing illustrating a biosensor based on rSbpA 31-1068 ⁄ cAb-PSA-N7 recrystallized on gold chips precoated with thiolated SCWP. The monomolecular protein lattice was able to specifically bind PSA on the outermost surface. (B) Sensorgram showing association (—) and dissociation (– ) – –) between PSA and rSbpA 31-1068 ⁄ cAb-PSA-N7 recrystallized on a SCWP-coated gold chip. The sensorgram indicates specific binding of PSA to the S-layer fusion protein. Molecular construction kit based on S-layers U. B. Sleytr et al. 328 FEBS Journal 274 (2007) 323–334 ª 2006 The Authors Journal compilation ª 2006 FEBS disease could be generated by recrystallization of the S-layer fusion protein on SCWP-coated microbeads [28]. As only a marginal loss of IgG-binding capacity has been observed, the protein lattice was cross-linked with dimethylpimelinimidate. Thus, regeneration of the binding matrix under acidic pH conditions could be performed and the affinity microparticles could be repeatedly used for extracorporeal blood purification. S-Layer-stabilized lipid membranes and liposomes The advances in genome mapping have revealed that approximately one-third of all genes in an organism encode membrane proteins such as pores, ion channels, receptors, and membrane-bound enzymes. These pro- teins are key factors in the cell’s metabolism and thus are the preferred target for pharmaceuticals. Currently more than 60% of drugs consumed act on membrane proteins [10,29]. Therefore not only biological mem- branes, but also the biomimetic approach to generate stabilized lipid membranes with functional membrane proteins has attracted much interest in recent years. The latter poses a challenge to apply membrane pro- teins as key elements in drug discovery, protein–ligand screening, and biosensors. A promising approach for the generation of biomi- metic membrane systems includes stabilization of lipid membranes with S-layer lattices (Fig. 5). These com- posite structures mimic the supramolecular assembly B Teflon aperture A water Patch clamp pipette air E (a) (b) (c) (d) (e) (f) D C porous support solid support Fig. 5. Schematic illustrations of various S-layer-supported lipid membranes. (A) Bilayer lipid membranes have been generated across an aperture of a patch clamp pipette by the tip-dip method, and a closed S-layer has been recrystallized from the aqueous subphase. In (B), a folded or painted membrane has been generated to span a Teflon aperture. Subsequently S-layer protein can be injected into one or both compartments (not shown) whereby the protein self-assembles to form closely attached S-layer lattices on the lipid membranes. (C) On an S-layer ultrafiltration membrane (SUM), a lipid membrane can be generated by a modified Langmuir–Blodgett (LB) technique. As a further option, a closed S-layer lattice can be attached on the external side of the SUM-supported lipid membrane (left part). (D) Solid supports can be covered by a closed S-layer lattice, and subsequently lipid membranes can be generated using combinations of the LB and Langmuir– Schaefer technique, and vesicle fusion. As shown in (C), a closed S-layer lattice can be recrystallized on the external side of the solid-suppor- ted lipid membrane (left part). (E) Schematic drawing of (a) an S-liposome with entrapped water-soluble (blue) or lipid-soluble (brown) functional molecules and (b) functionalized by reconstituted integral proteins. S-Liposomes can be used as immobilization matrix for functional molecules (e.g. IgG) by direct binding (c) or immobilization via the Fc-specific ligand protein A (d), or biotinylated ligands can be bound to the S-liposome via the biotin–streptavidin system (e). (f) Alternatively, liposomes can be coated with genetically modified S-layer proteins incorporating functional domains. Modified from Sleytr et al. [4] and Sleytr et al [9], with permission from Wiley-VCH. U. B. Sleytr et al. Molecular construction kit based on S-layers FEBS Journal 274 (2007) 323–334 ª 2006 The Authors Journal compilation ª 2006 FEBS 329 of archaeal cell envelopes, as the latter are composed of a cytoplasmic membrane and a closely associated S-layer as exclusive cell wall component [3,4,10,11]. In this biomimetic architecture, artificial lipids replace the cytoplasmic membrane, and isolated or recombinant S-layer proteins derived from Bacillaceae are attached on either one or both sides of the lipid membrane. Closed S-layer lattices can be generated for instance at Langmuir lipid monolayers, planar lipid membranes (Fig. 5A–D), liposomes (Fig. 5E), emulsomes, or lipid- coated nanocapsules [8,10,11,30]. The interaction of S-layer proteins with lipid mole- cules has been demonstrated to be noncovalent. Elec- trostatic interaction between exposed carboxy groups on the inner face of the S-layer lattice and the zwitter- ionic lipid head groups is primarily responsible for the binding and defined orientation of the S-layer subunits to form a closed lattice structure. For such an align- ment, it has been suggested that there are at least two to three contact points between the lipid film and the attached S-layer protein. Therefore, only a few lipid molecules are anchored via their head groups to pro- tein domains on the S-layer lattice, whereas the remaining scores of lipid molecules diffuse freely in the membrane between the pillars consisting of anchored lipid molecules. Because of its widely retained fluid characteristic, this nano-patterned type of lipid mem- brane is also referred to as ‘semifluid membrane’ [31]. However, most importantly, the attached S-layer lattices reveal no effect on the hydrophobic lipid acyl chains. Thus, S-layer lattices constitute unique scaffolding for lipid membranes [11,29,30]. This observation has been confirmed by the functional reconstitution of transmembrane proteins. Supported lipid membranes can also be generated on S-layer ultrafiltration membranes (SUMs; Fig. 5C), with S-layer fragments deposited in microfiltration membranes as an active filtration layer [32], and on S-layer-coated electrodes or structured silicon chips (Fig. 5D), with the S-layer as a stabilizing biomimetic layer [10,11]. S-Layer-coated silicon chips with attached lipid membranes are also referred to as lipid chips, and, in combination with microfluidics, these platforms constitute a prerequisite for the Laboratory- on-a-Chip technology [33]. The electrochemical proper- ties of S-layer-supported lipid membranes on porous and solid supports (Fig. 5C,D, respectively) are com- parable with those of free-standing lipid membranes (Fig. 5A,B). In addition, membranes on S-layer- covered gold electrodes exhibit remarkable long-term robustness of up to 1 week, which is far from feasible with any other stabilization technique. The functionality of lipid membranes resting on SUMs and S-layer-covered gold electrodes has been demonstrated by reconstituting the pore-forming pro- tein, a-hemolysin, and the membrane-active peptides, alamethicin, gramicidin A, and valinomycin. Recently, even single-pore recordings have been performed with a-hemolysin and gramicidin A reconstituted in S-layer- supported lipid membranes (Fig. 6) [10,11,30]. These results demonstrate that the biomimetic approach of copying the supramolecular architecture of archaeal cell envelopes opens up new possibilities for exploiting functional lipid membranes at the mesoscopic and macroscopic level. Moreover, this technology has the potential to initiate a broad spectrum of developments in many areas such as diagnostics, high-throughput screening for drug discovery, sensor technology, and Fig. 6. Opening and closing of single gramicidin pores. Gramicidin has been incorporated into an S-layer ultrafiltration membrane-supported lipid membrane composed of the main tetraether phospholipid isolated from Thermoplasma acidophilum. The schematic drawing on the left indicates the formation of dimeric gramicidin pores. Because of pore formation, a cascaded increase in electric current is observed. The schematic drawing on the right indicates the dissociation of gramicidin dimers. Gramicidin monomers do not form pores, and thus the con- ductance decreases cascaded for each dissociated gramicidin dimer. Conditions: 1 M KCl; pH ¼ 5.8; V m ¼ +150 mV; T ¼ 22 °C. Molecular construction kit based on S-layers U. B. Sleytr et al. 330 FEBS Journal 274 (2007) 323–334 ª 2006 The Authors Journal compilation ª 2006 FEBS electronic or optical devices, and might even find appli- cation in DNA sequencing [10,11,29,30]. Artificial lipid vesicles termed liposomes are widely used as delivery systems for enhancing the efficiency of various biological active molecules. S-Layer-coated liposomes (S-liposomes) represent simple model sys- tems resembling features of archaeal cell or virus enve- lopes (Fig. 5E). S-Layer proteins, once crystallized on liposomes, can be cross-linked and exploited as a mat- rix for the covalent attachment of functional molecules as required for drug-targeting or immunodiagnostic assays ([5,34] and references therein). In a recent study, the fusion protein, rSbpA 31)1068 ⁄ EGFP, carrying the sequence of enhanced green fluorescent protein (EGFP) at the C-terminus was recrystallized on positively charged liposomes. Because of the ability of EGFP to fluoresce, positively charged liposomes coated with rSbpA 31-1068 ⁄ EGFP represent a useful tool for visualizing the uptake of S-liposomes into eukaryotic cells, which can then be investigated by confocal scanning microscopy [34]. The high mechanical and thermal stability of S-layer-coated liposomes and the possibility for immo- bilizing or entrapping biologically active molecules [34] reveal a broad application potential, particularly as carrier and ⁄ or drug delivery, as artificial virus, and, for medicinal applications, as drug targeting system or in gene therapy (Fig. 5E) [4,8,10,11,30]. Controlled binding of nanoparticles The S-layer approach provides, for the first time, a biologically based fabrication technology for the self-assembly of molecular catalysts, templates and scaffolds for the generation of ordered large-scale nanoparticle arrays for applications in electronic or optic devices. Wet chemical approaches From the investigation of mineral formation by bac- teria in natural environments, it is apparent that S-layer lattices can also be used in wet chemical pro- cesses for the precipitation of metal ions from solution [35,36]. The first example of exploitation of this tech- nique was the precipitation of CdS on S-layer lattices composed of the S-layer protein from G. stearothermo- philus NRS 2004 ⁄ 3a variant 1 (SgsE), and on SbpA [35]. CdS and gold nanoparticles were 4–5 nm in size, and their superlattice resembled the oblique lattice symmetry of SgsE. SbpA was also used to generate superlattices of 4–5 nm-sized gold particles [36]. Gold nanoparticles were formed either by reduction of the metal salt with H 2 S or under the electron beam in a transmission electron microscope. The latter approach is technologically important, as it allows the definition of areas where nanoparticles are eventually formed. As determined by electron diffraction, the gold nano- particles were crystalline but their ensemble was not crystallographically aligned. Later, the wet chemical approach was used in the formation of Pd–, Ni–, Pt–, Pb–, and Fe–nanoparticle arrays. Recently, small spot X-ray photoelectron emission spectroscopy was used to characterize the elemental composition of the nano- clusters. This technique demonstrated that they consis- ted primarily of elemental gold [37]. Binding of preformed nanoparticles Although wet chemical methods lead to crystalline arrays of nanoparticles with spacing in register with the underlying S-layer lattice, they do not allow parti- cle size to be precisely controlled and hence the con- tact distances of neighboring particle surfaces, both of which are important for studying and exploiting quan- tum phenomena. Thus, the binding of preformed nanoparticles into regular arrays on S-layers has significant advantages over wet chemical approaches for the development of nanoscale electronic devices. Studies on binding of biomolecules, such as enzymes and antibodies [4,5], to S-layers have shown that metallic and semiconducting nanoparticles can be bound in regular arrangements on S-layers [38]. This is because, with S-layers, the properties of a single constituent unit are replicated with the periodicity of the lattice and thus define the characteristics of the whole 2D array. The pattern of bound molecules often reflects the lattice symmetry, the size of the morphological units, and the physicochemical proper- ties of the array. Specific binding of molecules to S-layer lattices can be induced by noncovalent and covalent forces. For example, the distribution of net negatively charged domains on S-layers could be visu- alized by electron-microscopic methods after labeling with positively charged topographical markers, such as polycationic ferritin (diameter 12 nm) [2]. Recently, citrate-stabilized negatively charged gold nanoparticles 5 nm in diameter were bound by electrostatic interac- tions at the inner S-layer face of SbpA, forming extended superlattices [38]. Furthermore, amino func- tionalized CdSe has been bound to the outer face of SbpA monolayers recrystallized on hydrophobic silicon surfaces after carbodi-imide activation of the carboxy residues. A major breakthrough in the regular binding of metallic and semiconducting nanoparticles was U. B. Sleytr et al. Molecular construction kit based on S-layers FEBS Journal 274 (2007) 323–334 ª 2006 The Authors Journal compilation ª 2006 FEBS 331 achieved by the successful design and expression of S-layer–streptavidin fusion proteins [7,15]. The 2D pro- tein crystals displayed streptavidin in defined repetitive spacing, and were capable of binding d-biotin and biotinylated peptides and proteins, in particular fer- ritin. Furthermore, metal-binding peptides can also be used as fusion partners in the design and expression of S-layer fusion proteins. Conclusions and perspectives The cross-fertilization of biology, genetics, chemistry, and material sciences is opening up a great variety of opportunities in nanobiotechnology and biomimetics. S-Layer research has clearly demonstrated that nature provides most elegant examples of nanometer-sized, molecular self-assembly systems (Figs 1 and 2) as required for generating bottom-up nanostructured materials, which may be exploited at mesoscopic and macroscopic levels. Of particular importance is the possibility to change the natural properties of S-layer proteins by genetic manipulation and to incorporate single-functional or multifunctional domains in S-layer lattices (Fig. 3). The spontaneous association of identi- cal S-layer (glyco)protein subunits in suspension or on surfaces or interfaces (Fig. 2) results in stable well- defined isoporous matrices, which can be considerably strengthened by introducing intermolecular and ⁄ or intramolecular bonds. Moreover, even native S-layers of some archaea can assemble and function in the most extreme environmental conditions in which the particular organisms are able to dwell (e.g. tempera- tures up to 120 °C, pH ¼ 0, concentrated salt solution, high hydrostatic pressure). An important line of development is the combining of S-layer and lipid membrane technologies (Fig. 5). This biomimetic approach – copying the supramolecu- lar principle of cell envelopes of archaea or envelopes of a great variety of viruses – is expected to enable the exploitation of functional principles of lipid mem- branes at mesoscopic and even macroscopic levels and the development of new targeting and delivery systems [10,11]. Although, the development of S-layer technol- ogies has focused primarily on life sciences, an import- ant field of future applications relates to nonlife sciences, including S-layer lattice-induced biominerali- zation, nonlinear optics, molecular electronics, and cat- alysts [39]. The dramatic reduction in size is one of the benefits promised by molecular electronics. Individual molecules are hundreds of times smaller than the smallest features conceivably attainable by semicon- ductor technology. For this reason, electronic devices constructed from molecules promise to be thousands of times smaller than their semiconductor-based coun- terparts. S-Layer lattices have proven to be perfect matrices for the generation of large-scale 2D arrays of metallic and semiconducting nanoparticles. Further- more, one of the big promises of molecular electronics is based on the possibility of fabricating 3D structures because of the extremely low power consumption com- pared with silicon-based integrated circuitry. Finally, some of the molecular electronic approaches are inher- ently digital and immune to soft errors, and some are inherently nonvolatile. As with many other technol- ogies based on unique biomaterials, many further areas in which S-layer lattices are of relevance may yet emerge (e.g. neoglycobiology) [14]. Acknowledgements This work was supported by the Austrian Science Fund (FWF, projects P16295-B10, P17170-B10, and P18510-B12), by the Erwin Schro ¨ dinger Society for Nanosciences, by the Austrian Federal Ministry of Transport, Innovation and Technology (MNA-Net- work), by the EU project NAS-SAP, and by the US Air Force Office of Scientific Research (projects F49620-03-1-0222 and FA9550-06-1-0208). 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MINIREVIEW S-Layers as a basic building block in a molecular construction kit Uwe B. Sleytr, Eva M. Egelseer, Nicola Ilk, Dietmar Pum and Bernhard Schuster Center. [2,3,5,6,9–11]. General aspects of S-layers S-Layer proteins are widely distributed in the major lineages of archaea and in Gram-positive and Gram- negative bacteria [2–4,7].