Abstract

Crystalline bacterial cell surface layers (S-layers) have been identified in a great number of different species of bacteria and represent an almost universal feature of archaea. Isolated native S-layer proteins and S-layer fusion proteins incorporating functional sequences self-assemble into monomolecular crystalline arrays in suspension, on a great variety of solid substrates and on various lipid structures including planar membranes and liposomes. S-layers have proven to be particularly suited as building blocks and patterning elements in a biomolecular construction kit involving all major classes of biological molecules (proteins, lipids, glycans, nucleic acids and combinations of them) enabling innovative approaches for the controlled ‘bottom-up’ assembly of functional supramolecular structures and devices. Here, we review the basic principles of S-layer proteins and the application potential of S-layers in nanobiotechnology and biomimetics including life and nonlife sciences.

Introduction

Molecular self-assembly systems that exploit the molecular-scale manufacturing precision of biological systems are prime candidates for controlled ‘bottom-up’ production of defined nanostructures (Whitesides et al., 1991; Goodsell, 2004; Sleytr et al., 2005). Although, self-assembly of molecules is an ubiquitous strategy of morphogenesis in nature, research in the area of molecular nanotechnology, nanobiotechnology and biomimetics are only beginning to exploit its potential for the functionalization of surfaces and interfaces as well as for the production of biomimetic membranes and encapsulation systems. Particularly the immobilization of biomolecules in an ordered fashion on solid substrates and their controlled confinement in defined areas of nanometer dimensions has grown into a scientific and engineering discipline that crosses the boundaries of several established fields in the nanosciences.

In this context, crystalline bacterial cell surface layers (S-layers), a unique self-assembly system optimized during billions of years of biological evaluation fulfil key requirements for controlled assembly of supramolecular materials (Sleytr, 1997; Pum & Sleytr, 1999; Sleytr et al., 1999, 2003a, 2004a, 2005; Sára et al., 2005).

S-layers have been observed in species of nearly every taxonomical group of walled bacteria and represent an almost universal feature of archaeal envelopes (Sleytr, 1978; König, 1988; Beveridge & Konval, 1993; Sleytr et al., 1993, 1996b; Sleytr, 1997; Sleytr & Beveridge, 1999; Claus et al., 2005). As the most abundant of bacterial cellular proteins, they represent important model systems for studies of structure, synthesis, transports, assembly, and function of proteinaceous components and evolutionary relationship within the prokaryotic world. Beyond their relevance for fundamental studies, S-layers were demonstrated to possess a great potential for nanobiotechnological applications (Sleytr, 1997; Pum & Sleytr, 1999; Sleytr et al., 2002, 2003b; Sára et al., 2006a; Sára et al., in press; Sleytr et al., in press). Most importantly, the constituent (glyco)protein subunits of the S-layer lattices have the capability to recrystallize into isoporous monolayers in suspension, at liquid-surface interfaces, lipid structures and on solid supports (e.g. polymers, metals, silicon wafers; Fig. 1). S-layer lattices can serve as a matrix for the controlled immobilization of functional biomolecules and more recently, the construction of S-layer fusion proteins have shown to combine the self-assembly principle with a broad spectrum of specific functions (e.g. ligands, antibodies, antigens, enzymes) providing an unsurpassed precision in spatial control and of alignment of functions encoded in proteins (Sleytr et al., 2005; Sára et al., in press).

1

Schematic drawing of the recrystallization procedure of native or recombinant S-layer (fusion) proteins. After isolation and disintegration, the native or recombinant proteins recrystallize either in suspension, on solid supports (optionally precoated with SCWP), at the air-water interface, on lipid-membranes or liposomes and nanocapsules. Modified after Pum (2006), Copyright (2006), with permission from Springer Verlag.

1

Schematic drawing of the recrystallization procedure of native or recombinant S-layer (fusion) proteins. After isolation and disintegration, the native or recombinant proteins recrystallize either in suspension, on solid supports (optionally precoated with SCWP), at the air-water interface, on lipid-membranes or liposomes and nanocapsules. Modified after Pum (2006), Copyright (2006), with permission from Springer Verlag.

In this review, we present an overview of nanostructure technologies using S-layers as patterning elements and basic building blocks for generating more complex supramolecular structures involving all major classes of biological molecules (e.g. proteins, lipids, glycans, nucleic acids, or combinations of them).

General aspects of S-layer proteins

Despite the fact that considerable variation exists in the chemistry and structure of prokaryotic cell envelopes, S-layers have apparently coevolved with these diverse structures (Sleytr & Beveridge, 1999). In certain archaea, the S-layer lattices as exclusive wall component can be even integrated with its inner part into the plasma membrane involving pillar-like domains (Mayr et al., 1996).

In gram-positive bacteria and in archaea, the lattice assembles on the surface of the wall matrix [e.g. peptidoglycan, secondary cell wall polymers (SCWPs), pseudomurein]. In gram-negative bacteria, the S-layer is attached to the lipopolysaccharide component of the outer membrane and some bacteria have been described to produce two superimposed S-layers, each composed of a different subunit species (König, 1988; Beveridge & Konval, 1993; Sleytr et al., 1996b, 2003a, 2005; Sleytr & Beveridge, 1999; Claus et al., 2005).

S-layers are composed, with a few exceptions, of a single homogeneous protein or glycoprotein species with molecular weights ranging from 40 to 200 kDa (Sleytr et al., 2003a). Glycosylation is a remarkable characteristic of many archaeal and some bacterial S-layer proteins (Schäffer & Messner, 2004). Bacterial S-layers are generally 5–20 nm thick, whereas those of archaea reveal a thickness up to 70 nm. A common feature of S-layers is, with respect to the orientation on the cell, their smooth outer surface and more corrugated inner surface. Since S-layers are in most cases assemblies of identical subunits, they exhibit pores of identical size and morphology. In many S-layers, two or even more distinct classes of pores are present, with diameters in the range of 2–8 nm. The proteinaceous subunits of S-layers are aligned either in lattices with oblique (p1, p2), square (p4) or hexagonal (p3, p6) symmetry. The morphological units are composed of one, two, three, four, or six identical subunits with a center-to-center spacing of c. 5–30 nm (Fig. 2, for review see also Sleytr et al., 1999).

2

Electron micrograph of a freeze-etched preparation of whole cells of Bacillus sphaericus CCM 2177 revealing a square S-layer lattice (a). Schematic drawings of the different S-layer lattice types. The regular arrays either show oblique (p1, p2), square (p4) or hexagonal (p3, p6) lattice symmetry. The morphological units are composed of one (p1), two (p2), three (p3), four (p4) or six (p6) identical subunits (b). Computer image reconstruction of scanning force microscopic images of the topography of the square (p4) S-layer lattice of B. sphaericus CCM 2177 (c) and the oblique (p1) S-layer lattice of Geobacillus stearothermophilus PV72/p2 (d). Bars: 100 nm in (a), 10 nm in (c) and (d).

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Electron micrograph of a freeze-etched preparation of whole cells of Bacillus sphaericus CCM 2177 revealing a square S-layer lattice (a). Schematic drawings of the different S-layer lattice types. The regular arrays either show oblique (p1, p2), square (p4) or hexagonal (p3, p6) lattice symmetry. The morphological units are composed of one (p1), two (p2), three (p3), four (p4) or six (p6) identical subunits (b). Computer image reconstruction of scanning force microscopic images of the topography of the square (p4) S-layer lattice of B. sphaericus CCM 2177 (c) and the oblique (p1) S-layer lattice of Geobacillus stearothermophilus PV72/p2 (d). Bars: 100 nm in (a), 10 nm in (c) and (d).

Provided that other cell-surface components (e.g. capsules and sheaths) are absent, S-layers as the outermost envelope structure represent an important interface between the cell and its environment. Since prokaryotes carrying S-layers are ubiquitous in the biosphere and inhabit the most diverse ecological niches, they fulfil a broad spectrum of functions. It is now recognized that S-layer lattices can provide the organism with a selection advantage by functioning as: (1) structure involving in cell adhesion and surface recognition; (2) protective coats, molecular sieves, and molecule and ion traps and (3) virulence factor in pathogenic organisms. (Messner & Sleytr, 1992; Beveridge & Konval, 1993; Sleytr et al., 1993, 1996b, 2003a; Beveridge, 1994; Sleytr, 1997; Sleytr & Messner, 2003). Moreover, in archaea that possess S-layers as the exclusive wall component, the protein lattices determine cell shape and the cell fission process (Messner et al., 1986; Pum et al., 1991).

Structural and self-assembly properties of S-layers

Information regarding the secondary structure of S-layer proteins is either derived from the amino acid sequence or from circular dichroism (CD) measurements indicating that c. 20% of the amino acids are organized as α-helices and about 40% occur as β-sheets. Aperiodic foldings and β-turn content may vary between 5% and 45%. More detailed studies were performed with the S-layer proteins rSbsB from Geobacillus stearothermophilus PV72/p2 and rSbpA from Bacillus sphaericus CCM 2177 (Table 1). Truncated forms of these S-layer proteins were constructed comprising either the N-terminal S-layer-homologous (SLH)-domain, which is responsible for binding of the S-layer subunits to the rigid cell wall layer or the middle and C-terminal self-assembly domain required for assembling into two dimensional protein arrays. For both S-layer proteins, CD-spectroscopy studies confirmed that most α-helical segments are arranged in the N-terminal SLH-domain, whereas the middle and C-terminal part could be characterized as a β-sheet protein (Rünzler et al., 2004; Huber et al., 2005).

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Nanobiotechnologically relevant S-layer proteins (for review see Sára et al., 2005)

Organism S-layer protein Amino acids Molecular weight (Da) Lattice type 
Geobacillus stearothermophilus PV72/p2 SbsB 32-920 94 000 Oblique 
Bacillus sphaericus CCM 2177 SbpA 31-1068 130 000 Square 
Geobacillus stearothermophilus ATCC 12980 SbsC 31-1099 121 000 Oblique 
Organism S-layer protein Amino acids Molecular weight (Da) Lattice type 
Geobacillus stearothermophilus PV72/p2 SbsB 32-920 94 000 Oblique 
Bacillus sphaericus CCM 2177 SbpA 31-1068 130 000 Square 
Geobacillus stearothermophilus ATCC 12980 SbsC 31-1099 121 000 Oblique 

The fact that no structural model at atomic resolution of an S-layer protein has been available until now, may be explained by the molecular mass of the subunits being too large for nuclear magnetic resonance analysis, as well as by the intrinsic property of S-layer proteins to self-assemble into two dimensional lattices, thereby hindering the formation of isotropic three dimensional crystals as required for X-ray crystallography. In addition, the low solubility of S-layer proteins is a general hindrance for both methods. However, in the case of the S-layer protein SbsC from G. stearothermophilus ATCC 12980 (Table 1), water soluble N- or C-terminally truncated forms were used for the first three-dimensional crystallization experiments. Native and heavy atom derivative data confirmed results of the secondary structure prediction, which indicated that the N-terminal region comprising the first 257 amino acids of the mature S-layer protein is mainly organized as α-helices, whereas the middle and C-terminal part of SbsC consist of loops and β-sheets (Pavkov et al., 2003).

S-layer proteins of bacteria are not covalently linked to each other and to the supporting cell wall component. Thus, complete solubilization of S-layers into their constituent subunits and release from the bacterial cell envelope can be achieved by treatment with high concentrations of hydrogen-bond breaking agents (e.g. guanidine hydrochloride), by dramatic changes in the pH-value or in the salt concentration (Sleytr et al., 1996a, 2005). S-layer proteins reassemble into two-dimensional arrays upon removal of the disrupting agent used in the dissolution procedure. Self-assembly products may have the form of flat sheets, open ended cylinders or closed vesicles (Sleytr, 1978; Sleytr et al., 1996a, 2005) (Fig. 1). Crystal growth is initiated simultaneously at many randomly distributed nucleation points and proceeds in-plane until the crystalline domains meet, thus leading to a closed, coherent mosaic of individual several micrometer large S-layer domains (Pum et al., 1993; Pum & Sleytr, 1995; Sleytr et al., 1996a).

Reassembly of isolated S-layer proteins into larger crystalline arrays can also be induced on technologically relevant solid supports such as silicon wafers, carbon-, platinum- or gold electrodes and on synthetic polymers (Pum & Sleytr, 1994, 1995; Sleytr et al., 1996a, 1999, 2003a, 2005; Pum & Sleytr, 1999; Györvary et al., 2003) (Fig. 1). The formation of coherent crystalline arrays strongly depends on the S-layer protein species, the environmental conditions of the bulk phase and, in particular, on the surface properties of the substrate.

Reassembly of isolated S-layer subunits at the air/water interface and on Langmuir–Blodgett (LB) films has proven to be an easy and reproducible way for generating coherent S-layer lattices on large scales (Fig. 1). In accordance with S-layer proteins recrystallized on solid surfaces, the orientation of the protein arrays at liquid interfaces was determined by the anisotropy in the physicochemical surface properties of the protein lattice. Electron microscopic examination revealed that recrystallized S-layer proteins from Bacillaceae (Table 1) were oriented with their outer charge neutral, less hydrophilic surface against the air/water interface and with their negatively charged, more hydrophilic inner surface against the positively charged or zwitterionic head groups of phospholipid or tetraether lipid films (Pum et al., 1993; Pum & Sleytr, 1994; Györvary et al., 2003).

Molecular biology and genetics of S-layers

Since S-layers are ubiquitous and represent one of the most abundant cellular proteins, numerous S-layer genes from quite different taxonomical positions have been sequenced and cloned (Sleytr et al., 1999, 2003a; Sára & Sleytr, 2000; Akca et al., 2002; Avall-Jaaskelainen & Palva, 2005). Thereby, for S-layer proteins of Bacillaceae, common structural organization principles could be identified. The N-terminal region was found to be responsible for anchoring the S-layer subunits to the underlying rigid cell envelope layer in a defined orientation by binding to a heteropolysaccharide, termed SCWP (Ries et al., 1997; Sára & Sleytr, 2000; Sára, 2001; Schäffer & Messner, 2005). In gram-positives, at least two types of binding mechanisms between S-layer proteins and SCWPs have been identified. The first one involves so-called SLH domains and pyruvylated SCWPs (Ries et al., 1997; Lemaire et al., 1998; Sára et al., 1998; Chauvaux et al., 1999; Ilk et al., 1999; Mesnage et al., 1999, 2000; Sára, 2001; Cava et al., 2004; Mader et al., 2004; Rünzler et al., 2004; Huber et al., 2005). For this binding mechanism, the necessity of SCWP pyruvylation for binding of SLH-domain carrying proteins was demonstrated by the construction of knock-out mutants in Bacillus anthracis and Thermus thermophilus in which the gene encoding a putative pyruvyl transferase was deleted as well as by surface plasmon resonance (SPR) spectroscopy measurements using native and chemically modified SCWPs devoid of pyruvic acid residues. In the case of the S-layer protein SbsB of G. stearothermophilus PV72/p2 (Table 1), the SLH-domain corresponds to the SCWP-binding domain, whereas the larger C-terminal part represents the self-assembly domain (Mader et al., 2004; Rünzler et al., 2004). The C-terminal part of SbsB was highly sensitive against deletions, and the removal of even <15 amino acids led to water-soluble S-layer protein forms (Howorka et al., 2000; Moll et al., 2002). In contrast to SbsB, in SbpA, the S-layer protein of B. sphaericus CCM 2177 (Table 1), the three SLH-motifs and an additional 58 amino acids long SLH-like motif were required for reconstituting the functional SCWP-domain (Huber et al., 2005). In the C-terminal part of this S-layer protein, up to 237 amino acids could be deleted without interfering with the formation of the square lattice structure. Interestingly, the deletion of further 113 C-terminal amino acids led to a change of the lattice structure from square (p4) to oblique (p1) (Huber et al., 2005). As derived from SPR measurements, the binding mechanism between SLH-domains and pyruvylated SCWPs is highly specific (Mader et al., 2004; Huber et al., 2005) and therefore is exploited for a biomimetic approach for an oriented recrystallization of S-layer (fusion) proteins on various SCWP-coated solid supports as required for many nanobiotechnological applications (Sleytr et al., 1999, 2000, 2001, 2004a, b; Sára et al., 2005).

The second type of binding mechanism between S-layer proteins and SCWPs has been described for G. stearothermophilus wild-type strains and involves an SCWP that contains 2,3-dideoxy-diacetamido mannosamine uronic acid as the negatively charged component and a highly conserved N-terminal region, which is devoid of an SLH-domain (Egelseer et al., 1998; Jarosch et al., 2000; Schäffer et al., 2002). In these S-layer proteins, arginine and tyrosine, which typically occur in carbohydrate binding proteins, such as lectins, are accumulated in the N-terminal part (Weis, 1997). The production of different truncated forms of the S-layer protein SbsC of G. stearothermophilus ATCC 12980 (Table 1) confirmed that the N-terminal part comprising amino acids 31–257 is exclusively responsible for cell wall binding. This positively charged segment is not involved in the self-assembly process (Jarosch et al., 2001) and seems to fold independently of the remainder of the protein sequence.

S-layer variation developed as a pivotal mechanism to respond to changing environmental conditions in the course of evolution and has been described to occur in pathogens as well as in nonpathogens (Sára et al., 1994, 1996; Boot et al., 1995, 1996; Dworkin & Blaser, 1997; Cerquetti et al., 2000; Thompson & Blaser, 2000; Calabi et al., 2001; Karjalainen et al., 2001; Mignot et al., 2002). In the latter, S-layer variation is frequently induced in response to environmental stress factors, such as increased oxygen supply (Sára & Sleytr, 1994; Sára et al., 1996; Jakava-Viljanen et al., 2002; Avall-Jaaskelainen & Palva, 2005). In G. stearothermophilus strain variants, expression of a completely new type of S-layer protein is accompanied by synthesis of a different type of SCWP and S-layer variation may also lead to a change in the lattice type (Sára et al., 1994, 1996). Regarding the development of S-layer-deficient strain variants, the importance of insertion sequence (IS) elements has been demonstrated for various organisms (Egelseer et al., 2000; Scholz et al., 2000).

S-layer fusion proteins and applications

Since one of the most relevant areas of research in nanobiotechnology concerns technological utilization of self-assembly systems, S-layer technology was advanced by the construction of functional S-layer fusion proteins (Sára et al., 2006a, b) (Table 2). All chimeric proteins comprised (1) an accessible N-terminal SCWP-binding domain, which could be exploited for oriented binding and recrystallization on artificial solid supports precoated with SCWP, (2) the self-assembly domain and (3) a functional sequence fused to the C-terminal end of the S-layer protein (Sára et al., 2005). A broad spectrum of functional S-layer fusion proteins based on the S-layer proteins SbpA, SbsB, and SbsC were cloned and heterologously expressed in Escherichia coli. Using electron and scanning probe microscopy as well as functional tests, it could be demonstrated that the recrystallization properties conferred by the S-layer protein moiety as well as the functionality of the fused peptide sequence were retained in all S-layer fusion proteins (Sára et al., 2006a).

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Areas of application for S-layer fusion proteins

  Diagnostic systems and label-free detections systems (Surface Plasmon Resonance Spectroscopy, Surface Acoustic Wave, Quartz Crystal Microbalance with Dissipation Monitoring) 
  Biosensors 
  High density affinity coatings (e.g. biocatalysis and immobilized enzymes, downstream-processing, blood purification) 
  Immunogenic and immunomodulating structures (e.g. antiallergic vaccines) 
  Stabilization of functional lipid membranes 
  Functionalization of liposomes and emulsomes as targeting and delivery systems 
  Binding of nanoparticles (e.g. molecular electronics, nonlinear optics, catalysts) 
  Biomineralization 
  Isoporous ultrafiltration membranes 
  Diagnostic systems and label-free detections systems (Surface Plasmon Resonance Spectroscopy, Surface Acoustic Wave, Quartz Crystal Microbalance with Dissipation Monitoring) 
  Biosensors 
  High density affinity coatings (e.g. biocatalysis and immobilized enzymes, downstream-processing, blood purification) 
  Immunogenic and immunomodulating structures (e.g. antiallergic vaccines) 
  Stabilization of functional lipid membranes 
  Functionalization of liposomes and emulsomes as targeting and delivery systems 
  Binding of nanoparticles (e.g. molecular electronics, nonlinear optics, catalysts) 
  Biomineralization 
  Isoporous ultrafiltration membranes 

The S-layer fusion protein rSbpA31−1068/cAb-prostate-specific antigen (PSA)-N7 carrying the camel antibody sequence recognizing the PSA, was recrystallized on gold chips precoated with thiolated SCWP, and exploited as nanopatterned sensing layer in SPR to detect PSA (Pleschberger et al., 2003, 2004). Furthermore, the S-layer fusion protein rSbpA31−1068/ZZ incorporating two copies of the Z-domain, (a synthetic analogue of the B-domain of Protein A, capable of binding the Fc-part of IgGs), could be recrystallized on biocompatible, SCWP-coated microbeads composed of different polymers, which shall find application in the microsphere-based detoxification system for extracorporeal blood purification (Völlenkle et al., 2004). Chimeric S-layer proteins comprising the sequence of the allergen Bet v1 are generally considered as a novel approach to specific treatment of allergic diseases due to their immunomodulating capacity (e.g. development of vaccines for immunotherapy of type 1 allergy) (Breitwieser et al., 2002; Ilk et al., 2002; Bohle et al., 2004). In order to visualize the uptake of S-layer coated liposomes (S-liposomes) into eukaryotic cells, an S-layer fusion protein incorporating the sequence of enhanced green fluorescent protein (EGFP) (rSbpA31−1068/EGFP) was recrystallized on positively charged liposomes (Ilk et al., 2004). Furthermore, for generating a universal affinity matrix for binding of any kind of biotinylated molecule, minimum-sized core-streptavidin (118 amino acids) was fused either to N- or C-terminal positions of rSbsB or to a C-terminally truncated form of rSbpA (Moll et al., 2002; Huber et al., 2006a, b) (Fig. 3). As biologically active streptavidin occurs as a tetramer, heterotetramers consisting of one chain fusion protein and three chains core streptavidin were prepared by applying a special refolding procedure. A biotin binding capacity of about 75% could be determined for soluble heterotetramers indicating that three of four biotin binding sites were active. Hybridization experiments with biotinylated and fluorescent labelled oligonucleotides using surface-plasmon-field-enhanced fluorescence spectroscopy indicated that a functional sensor surface could be successfully generated by recrystallization of heterotetramers on gold chips. Such promising structures could be exploited for the development of DNA or protein chips as required for many (nano)biotechnological applications (Huber et al., 2006a, b).

3

Digital image reconstruction from transmission electron micrographs of negatively stained preparations from self-assembly products of SbsB (a) and N-terminal SbsB-streptavidin heterotetramers (b) (bars: 10 nm). The thin white arrows indicate the base vectors of the oblique p1 lattice. In the lattice of the fusion protein, streptavidin showed up as an additional protein mass (thick white arrow) attached to the N-terminal SLH-domain. Lattices generated from C-terminal SbsB-streptavidin heterotetramers were capable of binding biotinylated ferritin as a superlattice (c). The black arrows indicate the base vectors of the oblique p1 lattice (bar: 100 nm). Cartoon illustrating self-assembled S-layer-fusion proteins with target molecules or nanoparticles bound in defined spacing and orientation (d). Modified after Schuster (2006), Copyright (2006), with permission from Bentham Science Publishers Ltd.

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Digital image reconstruction from transmission electron micrographs of negatively stained preparations from self-assembly products of SbsB (a) and N-terminal SbsB-streptavidin heterotetramers (b) (bars: 10 nm). The thin white arrows indicate the base vectors of the oblique p1 lattice. In the lattice of the fusion protein, streptavidin showed up as an additional protein mass (thick white arrow) attached to the N-terminal SLH-domain. Lattices generated from C-terminal SbsB-streptavidin heterotetramers were capable of binding biotinylated ferritin as a superlattice (c). The black arrows indicate the base vectors of the oblique p1 lattice (bar: 100 nm). Cartoon illustrating self-assembled S-layer-fusion proteins with target molecules or nanoparticles bound in defined spacing and orientation (d). Modified after Schuster (2006), Copyright (2006), with permission from Bentham Science Publishers Ltd.

The repetitive features of S-layers have led to their applications in the production of functional S-layer ultrafiltration membranes (SUMs) as supports for a defined covalent attachment of functional molecules (e.g. enzymes, antibodies, antigens, protein A, biotin, and avidin) as required for affinity and enzyme membranes, in the development of solid-phase immunoassays or in biosensors (Sára & Sleytr, 1989; Sleytr et al., 1999, 2000, 2003a; Sára et al., 2005, 2006a, b).

S-layer stabilized lipid membranes and liposomes

Biological membranes have attracted lively interest in recent years as the advances in genome mapping revealed that approximately one-third of all genes in an organism encode for membrane proteins like pores, ion channels, receptors, and membrane-bound enzymes (Gerstein & Hegyi, 1998; Sigworth & Klemic, 2005). These proteins are key factors in the cell's metabolism and thus, are a preferred target for pharmaceuticals. Currently, more than 60% of consumed drugs act on membrane proteins (Ellis & Smith, 2004). Therefore, the generation of stabilized lipid membranes with functional membrane proteins poses a challenge to apply membrane proteins as key elements in drug discovery, protein-ligand screening, and biosensors.

One promising approach includes the stabilization of lipid membranes with S-layer lattices. These composite structures mimic the supramolecular assembly of archaeal cell envelopes as the latter are composed of a cytoplasmic membrane and a closely associated S-layer as exclusive wall component (Fig. 4a) (Kandler, 1982; König, 1988; Sleytr & Beveridge, 1999; Schuster & Sleytr, 2005, 2006). In this biomimetic architecture, artificial lipids replace the cytoplasmic membrane and isolated or recombinant S-layer proteins derived from Bacillaceae are attached either on one or both sides of the lipid membrane (Fig. 4b and c). Closed S-layer lattices can be generated for instance at Langmuir lipid monolayers (Pum et al., 1993; Diederich et al., 1996; Wetzer et al., 1998; Schuster et al., 2003a), planar lipid membranes (Schuster et al., 1998a, b, 1999, 2001, 2004a; Schuster & Sleytr, 2006), liposomes (Küpcü, 1995; Mader et al., 2000), or emulsomes.

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(a) Schematic illustration of an archaeal cell envelope structure composed of the cytoplasmic membrane with integral membrane proteins and an S-layer lattice, integrated into the cytoplasmic membrane. Modified after ref. Sleytr (2003a), Copyright (2002), with permission from Wiley-VCH. (b, c) Schematic illustrations of various S-layer-supported lipid membranes. (b) On an SUM a lipid membrane can be generated by a modified 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). (c) 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 supported lipid membrane (left part). Modified after Sleytr (2004a), Copyright (2004), with permission from Wiley-VCH.

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(a) Schematic illustration of an archaeal cell envelope structure composed of the cytoplasmic membrane with integral membrane proteins and an S-layer lattice, integrated into the cytoplasmic membrane. Modified after ref. Sleytr (2003a), Copyright (2002), with permission from Wiley-VCH. (b, c) Schematic illustrations of various S-layer-supported lipid membranes. (b) On an SUM a lipid membrane can be generated by a modified 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). (c) 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 supported lipid membrane (left part). Modified after Sleytr (2004a), Copyright (2004), with permission from Wiley-VCH.

S-layer proteins interact noncovalently with lipid molecules. Electrostatic interaction between exposed carboxyl groups on the inner face of the S-layer lattice (Pum et al., 1993) and the zwitterionic lipid head groups are primarily responsible for the binding and the defined orientation of the subunits (Küpcü, 1995; Hirn et al., 1999; Schuster et al., 1999). For such an alignment, it has been suggested that there are at least two to three contact points between the lipid film and the attached S-layer protein (Wetzer et al., 1998). Thus, only a few lipid molecules are anchored to protein 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 membrane is also referred to as ‘semifluid membrane’ (Pum & Sleytr, 1994). But most important, attached S-layer lattices reveal no impact on the hydrophobic lipid acyl chains (Schuster et al., 1998b; Weygand et al., 1999; Weygand et al., 2002). Thus, S-layer lattices constitute unique supporting scaffoldings for lipid membranes (Figs 4 and 5) (Schuster & Sleytr, 2000, 2002a, 2006; Schuster et al., 2004b; Schuster, 2005). This observation has also been proven by the functional reconstitution of transmembrane proteins.

5

(a) Schematic drawing of (1) an S-liposome with entrapped functional molecules and (2) functionalized by reconstituted integral proteins. S-liposomes can be used as immobilization matrix for functional molecules (e.g. IgG) either by direct binding (3), by immobilization via the Fc-specific ligand protein A (4), or biotinylated ligands can be bound to the S-liposome via the biotin–streptavidin system (5). (6) Alternatively, liposomes can be coated with genetically modified S-layer proteins incorporating functional domains. (b) Electron micrograph of a freeze-etched preparation of an S-liposome. Bar: 100 nm. Reprinted with permission from Sleytr (2003a), Copyright (2002) Wiley-VCH.

5

(a) Schematic drawing of (1) an S-liposome with entrapped functional molecules and (2) functionalized by reconstituted integral proteins. S-liposomes can be used as immobilization matrix for functional molecules (e.g. IgG) either by direct binding (3), by immobilization via the Fc-specific ligand protein A (4), or biotinylated ligands can be bound to the S-liposome via the biotin–streptavidin system (5). (6) Alternatively, liposomes can be coated with genetically modified S-layer proteins incorporating functional domains. (b) Electron micrograph of a freeze-etched preparation of an S-liposome. Bar: 100 nm. Reprinted with permission from Sleytr (2003a), Copyright (2002) Wiley-VCH.

Supported lipid membranes can also be generated on SUMs (Fig. 4b), with S-layer fragments deposited in microfiltration membranes as the active filtration layer (Sára & Sleytr, 1987; Weigert & Sára, 1995, 1996) or S-layer coated electrodes or structured silicon chips (Fig. 4c) with the S-layer as stabilizing and biomimetic layer (Schuster et al., 2001; Gufler et al., 2004). The latter are also referred to as lipid chips and combined with microfluidics these platforms constitute the prerequisite for lab-on-a-chip technology (Bayley & Cremer, 2001; Sigworth & Klemic, 2005). Lipid membranes on S-layer covered gold electrodes exhibited a remarkable long-term robustness of up to 1 week, which is not feasible with any other stabilization technique. The functionality of lipid membranes resting on SUMs and S-layer covered gold electrodes has been demonstrated by the reconstitution of the pore-forming protein α-hemolysin, alamethicin, gramicidin A, and valinomycin (Schuster et al., 1998a, 2001, 2003b; Gufler et al., 2004). Recently, even single pore recordings have been performed with α-hemolysin and gramicidin A reconstituted in S-layer supported lipid membranes (Schuster et al., 2001, 2003b; Schuster & Sleytr, 2002b).

These results have demonstrated that the biomimetic approach of copying the supramolecular architecture of archaeal cell envelopes opens new possibilities for exploiting functional lipid membranes at meso- and macroscopic scale. Particularly with an S-layer cover (Fig. 4), these membranes revealed a remarkable long-term stability. Hence, this technology has the potential to initiate a broad spectrum of developments in many areas like diagnostics, high-throughput screening for drug discovery, sensor technology, electronic or optical devices, and might even find application in DNA-sequencing (Kasianowicz et al., 1996; Sleytr et al., 1999, 2004a; Meller et al., 2000; Vercoutere et al., 2001; Schuster & Sleytr, 2005, 2006).

S-liposomes are biomimetic structures that resemble the supramolecular principle of archaeal cell or virus envelopes (Fig. 5). S-layer proteins, once crystallized on liposomes, can be cross-linked and exploited as a matrix for the covalent attachment of functional molecules as required for drug-targeting or immunodiagnostic assays (Küpcü, 1995, 1996; Mader et al., 2000). Furthermore, a general stabilization of the whole composite structures could be achieved by coating liposomes with S-layer proteins (Küpcü, 1998; Mader et al., 1999).

The high mechanical and thermal stability of S-liposomes and the possibility for immobilizing or entrapping biologically active molecules (Mader et al., 2000; Moll et al., 2002) reveal a broad application potential, particularly as carrier and/or drug delivery (as artificial viruses), and for medicinal applications as drug targeting system or in gene therapy (Fig. 5) (Sleytr et al., 1999; Schuster & Sleytr, 2000, 2006).

Controlled immobilization of nanoparticles

Based on the investigation of mineral formation by bacteria in natural environments (Douglas & Beveridge, 1998), S-layer lattices can be used in wet chemical processes for the precipitation of metal ions from solution, too. In this approach, self-assembled S-layer structures were exposed to metal-salt solutions followed by slow reaction with a reducing agent, such as hydrogen sulfide (H2S), or under the electron beam in a transmission electron microscope (TEM) (Shenton et al., 1997; Dieluweit et al., 1998; Mertig et al., 1999, 2001; Wahl et al., 2001). Nanoparticle superlattices were formed according to the lattice spacing and symmetry of the underlying S-layer. Furthermore, since the precipitation of the metals was confined to the pores of the S-layer, the nanoparticles also resembled the morphology of the pores. The first example exploiting this technique was the precipitation of cadmium sulfide (CdS) (Shenton et al., 1997). The generated CdS nanoparticles were 4–5 nm in size and their superlattice resembled the oblique lattice symmetry of the S-layer used. In a similar approach, a superlattice of 4–5 nm sized gold particles was formed using an S-layer with square lattice symmetry (with previously induced thiol groups) as a template for the precipitation of a tetrachloroauric (III) acid (HAuCl4) solution (Dieluweit et al., 1998). Gold nanoparticles were formed under the electron beam in a TEM. The latter approach is technologically important since it allows the definition of areas where nanoparticles are eventually formed (Dieluweit et al., 1998; Wahl et al., 2001). As determined by electron diffraction, the gold nanoparticles were crystalline but their ensemble was not crystallographically aligned. In addition, the wet chemical approach was used in the formation of Pd- (salt: PdCl2), Pt- (KPtCl6), nanoparticle arrays, too (Mertig et al., 1999, 2001; Wahl et al., 2001).

A much more controlled and specific way of making highly ordered nanoparticle arrays uses genetic approaches for the construction of chimeric S-layer fusion proteins incorporating unique polypeptides, which have been demonstrated to be responsible for biomineralization processes. The precipitation of metal ions or the binding of metal nanoparticles is then confined to specific and precisely localized positions in the S-layer lattice. Currently, several silver and cobalt precipitating peptides are under investigation.

Although wet chemical methods lead to crystalline arrays of nanoparticles with spacing in register with the underlying S-layer lattice, they do not allow one to precisely control particle size and hence the contact distances of neighbouring particle surfaces, both of which are important for studying and exploiting quantum phenomena. Thus, the binding of preformed nanoparticles into regular arrays on S-layers has significant advantages for the development of nanoscale electronic devices. Based on the work on binding biomolecules, such as enzymes or antibodies, onto S-layers, it has already been demonstrated that metallic and semiconducting nanoparticles can be bound in regular arrangements on S-layers (Hall et al., 2001; Bergkvist et al., 2004; Györvary et al., 2004). Specific binding of molecules on S-layer lattices may be induced by noncovalent and covalent forces. Recently, gold and amino functionalized cadmium selenide (CdSe) had been bound onto S-layer protein monolayers and self-assembly products (Hall et al., 2001; Bergkvist et al., 2004; Györvary et al., 2004). Citrate stabilized negatively charged gold nanoparticles of 5 nm in diameter were bound by electrostatic interactions while amino-functionalized 4 nm sized CdSe particle were bound after carbodiimide (EDC) activation of the carboxyl groups. A major breakthrough in the regular binding of metallic and semiconducting nanoparticles was achieved by the successful design and expression of S-layer-streptavidin fusion proteins, which allowed a specific binding of biotinylated ferritin molecules into regular arrays (Moll et al., 2002). Furthermore, it could be demonstrated that all fused streptavidin functionalities had the same position and orientation within the unit cell and were exposed. Such chimeric S-layer protein lattices can be used as self-assembling nanopatterned molecular affinity matrices capable of arranging biotinylated compounds in ordered arrays on surfaces.

Conclusion

The cross-fertilization of biology, chemistry, and material sciences is opening up a great variety of opportunities for innovation in nanobiotechnology and biomimetics. Most important, the study of biological self-assembly systems has developed into a rapidly growing scientific and engineering field that crosses the boundaries of existing disciplines. In this context, S-layers represent particularly versatile structures. Since their construction principle is based on a single constituent protein or glycoprotein subunit with the intrinsic ability to assemble into closed isoporous lattices on cell surfaces, they represent the simplest type of protein membranes developed during biological evolution (Sleytr, 1975). Considerable knowledge has now accumulated concerning the isolation, purification, and genetics of S-layer proteins as well as assembly of coherent S-layer lattices in defined orientation on flat or porous solid supports, lipid membranes, and liposomes.

Although currently the most extensively studied S-layer proteins are those of G. stearothermophilus PV72/p2 and B. sphaericus CCM 2177, in future many relevant nanobiotechnological applications can be expected from the utilization of S-layer proteins from other organisms, in particular Lactobacillus. (For detailed reviews see Avall-Jaaskelainen & Palva, 2005).

The attractiveness of ‘bottom-up’ processes involving S-layer self-assembly systems lies in their capability to build uniform, ultra small functional units, and the possibility to exploit such structures at the meso- and macroscopic scale. Moreover, S-layers represent very versatile assembly systems with unique features as structural basis for a complete supramolecular construction kit involving all major species of biological (macro)molecules. Particularly the biomimetic approach copying the supramolecular principle of S-layer associated and stabilized plasma membranes developed by archaea will lead to novel technologies for stabilizing functional lipid membranes as required for applications in biosensing devices (e.g. lipid chips) or high throughput screening methods for components acting on membrane proteins (e.g. ion channels).

The possibility to change the natural proportion of S-layer proteins by genetic manipulation and to incorporate single or multifunctional domains in S-layer lattices will significantly influence the development of applied S-layer research. Functionalized S-layer proteins maintaining the self-assembly capability could lead to new affinity matrices, diagnostics and biosensors, targeting and delivery systems, vaccines, biological templating, or specific biomineralization strategies. Besides application in life sciences S-layers will enable a well-defined mean spacing of deposited metal particles and semiconductors as required for nanoelectronic or optical applications (Pum & Sleytr, in press).

Acknowledgements

This work was supported by the Austrian Science Fund (FWF), projects P 16295-B10, P 17170-B10, and P 18510-B12, by the EU project NAS-SAP, by the Austrian Federal Ministry of Transport, Innovation and Technology (MNA-Network), and the US Air Force Office of Scientific Research (Project F49620-03-1-0222).

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Author notes

Editor: Ian Henderson