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  4. S-layer-streptavidin fusion proteins as template for nanopatterned molecular arrays

Ucisik , Uwe B. Sleytr , Bernhard Schuster Current pharmaceutical biotechnology Egelseer Archives of Microbiology Purification, characterization and preparation immunomatrixes of S-layer proteins of Thermobifida fusca. Sleytr International journal of molecular sciences The structure and binding behavior of the bacterial cell surface layer protein SbsC. Hence, a straightforward approach is the utilization of SUMs with the S-layer acting as a stabilizing and anchoring structure and as a smoothening layer, which reduces the roughness of the porous support [ , , ].

S-layer fragments deposited as a coherent layer on microfiltration membranes are forming the SUM [ , , ]. The mechanical and chemical stability of the SUM is subsequently introduced by chemical inter- and intramolecular cross-linking [ , , , , ]. The S-layer lattice with its uniformity of functional groups on the surface and within the pore area permits the very accurate chemical modifications in the sub-nanometer range.

These modifications enable the tuning of the molecular sieving as well as antifouling characteristics of SUMs [ , , ]. Moreover, SUMs can be prepared with different net charges and hydrophobic or hydrophilic surface properties. These features make SUMs very attractive as stabilizing and supporting structures for the functional lipid membranes [ 15 , 41 , 94 , 97 , ]. SUM-supported phospholipid bilayers were highly isolating structures with a lifetime of up to 17 h [ , , ], whereas BLMs on plain microfiltration membranes revealed a life-time of only approximately 3 h.

The lifetime increased significantly to about 24 h by the attachment of an S-layer lattice on both sides of the lipid membrane Figure 3 F [ , ]. The crosslinking of those lipid head groups, which are direct in contact with domains on the S-layer protein may result in a further increase of the longevity of this composite membrane architecture, and is thus a promising strategy for generating stable and fluid lipid membranes [ 86 ]. Investigations on the incorporation behavior of the membrane-active peptides valinomycin, alamethicin, gramicidin D, and the negatively charged antimicrobial peptide analogue peptidyl-glycine-leucine-carboxyamide PGLa - permit drawing conclusions on the functionality of SsLMs resting on solid supports Table 2 [ , , ].

SsLMs with incorporated valinomycin, which is a potassium-selective ion carrier, exhibited in sodium buffer a remarkable high membrane resistance. However, the change to potassium buffer resulted in a decrease of the membrane resistance by a factor of because now the valinomycin-mediated potassium transport could take place [ ].

The results of this study indicated toroidal pore formation in a concentration-dependent manner [ ]. In addition, electrochemical measurements on gramicidin, which is a membrane-active peptide Table 2 , revealed the formation of functional gramicidin pores in all of the mentioned SsLMs [ ]. Moreover, the SsLMs allowed for tracing of even single gramicidin pores. Thus, SsLMs are promising lipid membrane platforms for studying the interaction and insertion of membrane-active antimicrobial peptides in model lipid membranes [ , ].

Finally, SsLMs provided also a proper matrix for the functional incorporation of alamethicin Table 2. The addition of amiloride, which is an inhibitor for alamethicin, resulted in a specific blocking of the alamethicin channels as increasing amounts of amiloride gave rise to a significant increase in membrane resistance [ ].

In future, the ability to reconstitute membrane-active peptides like antimicrobial ones in defined structures on, e. The reconstitution of integral membrane proteins in SsLMs was also successful Table 3. The reconstitution of the integral ryanodine receptor, RyR1 Table 3 , which was isolated from rabbit muscle cells was also successful in SsLMs [ ].

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Depending on the type of measurement, the SsLM formation occurred either on glass surfaces for fluorescence experiments or on gold sensors for QCM-D measurements. Preliminary measurements clearly indicated that incorporation of RyR1 took place. This was verified by control experiments to exclude misinterpretation due to unspecific adsorption of RyR1 to the bilayer or the S-layer lattice [ ]. Nevertheless, further experiments, like, e.

The addition of the voltage-dependent anion channel VDAC; Table 3 to a preformed SsLM caused a significant decrease in membrane resistance, whereas the membrane capacitance did not change significantly [ ]. VDAC is a voltage-gated channel, which is at high membrane potentials in the closed state and switches to an open state at low membrane potentials less than10 mV [ , , ]. Furthermore, the membrane resistance decreased again after reducing the voltage from 10 mV back to zero, but the resistance remained higher when compared to the first measurement.

The reason for this may be the re-opening of some channels while others remain closed.

In addition, it is conceivable that keeping the channels in the closed state for a long period during the measurements may reduce the rate of re-opening of VDAC and cause some structural rearrangements in order to achieve a more stable closed conformation [ , ]. Moreover, the presence of the nucleotides nicotinamide adenine dinucleotide hydride NADH or nicotinamide adenine dinucleotide phosphate hydrogen induce channel closure, leading to a significantly reduced conductance of the VDAC channels [ , , , ].

All the before mentioned examples for the functional reconstitution of membrane-active peptides and membrane proteins in SsLMs are currently proof-of-principle studies. There is, however, a strong desire to use SsLMs, particularly for probing the function of membrane proteins, e. A direct electrical readout of membrane functions, e. There is no need for any labelling, as it is mandatory for many current state-of-the-art techniques that are used for membrane protein screening. Vesicular lipid structures, like unilamellar liposomes, comprising of a closed, spherical lipid mono- or bilayer with an aqueous inner space and emulsomes, comprising of a solid fat core that is surrounded by lipid layers are mainly used as drug targeting and drug delivery systems [ , ].

However, these lipid nanoparticles can also be used as biosensors for diagnosis purposes if the drug is replaced or is supplemented by a radiotracer, contrast agent, or a fluorescent dye. The use of molecular imaging to non-invasively measure the in vivo distribution of nanomedicines becomes increasingly important [ ]. Labeling the nanoparticle gives also an indication of delivery to the target tissue.

In this context, it is worth mentioning that liposomes and emulsomes can be covered by an S-layer lattice Figure 4 A,B [ , , , , ]. In addition, the S-layer lattice may be functionalized with, e. Moreover, S-layer-coated and with labelling agents loaded liposomes or emulsomes may be used for molecular imaging to detect, e. A highly challenging scientific area is the research at the intersection of biological and engineering sciences for the development of biosensors.

Significant progress in issues like miniaturization, functional sensitivity, simplified read-out, multiplexing, and the utilization of newly discovered physical phenomena further pushed the development of smart devices. Moreover, semiconducting technology has proceeded in a way that in the field of biosensors a rapid infiltration of new bio nanotechnology-based approaches occurred.


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Due to bottom-up self-assembly processes at the nanometer scale, the traditional separation between transducers and bioreceptors is not valid anymore. Indeed, by an integrative approach, the interface architecture plays an important role in the recognition event and the receptors become active transducing elements of the biosensors Figure 1.

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The present paper describes the successful implementation of cell envelope components, like, e. S-layer proteins can be self-assembled to become part of the interface architecture and thereby connecting the bioreceptor to the transducer interface. The recrystallized S-layer lattice provides significant advantages over other coatings, which can be summarized as follows: This becomes very important if one uses surface-sensitive phenomena as transducer, as it is indicated in Figure 1.

Because these techniques have a limited measuring range, the sensitivity decreases with an increasing intermediate layer thickness.

This becomes very important if one uses electrochemical methods as transducer. Due to the electrolyte-filled pores, there is much less limitation of ions and the S-layer lattice itself shows a negligible resistance and capacitance. This property allows for arranging bioreceptors as densely packed and highly directed on the interface. This characteristic is favourable when the measured signal corresponds to the bound and adsorbed biomolecules on the sensor surface. Finally, 5 the S-layer lattice can completely cover areas in the cm 2 -range by a one-step process.

Hence, a coherent proteinaceous coating can functionalize the whole surface of commonly used transducers. Although only little material is necessary, a possible drawback of using S-layer proteins is the need for the scale-up of the cultivation of bacteria and the isolation, subsequent purification, and the storage of S-layer proteins. However, recombinant production of S-layer proteins may help to overcome this issue. Moreover, physicists have to consider adopting certain protocols to meet the demands of biological materials. As previously mentioned, because of their structural features, S-layer lattices are highly suitable to immobilize biosensor-relevant molecules, like enzymes, dyes, fluorophores, and receptors.

In another approach, the bioreceptor may be fused to the S-layer protein. The S-layer protein recrystallizing part ensures a layer presenting the bioreceptor molecules, like enzymes, antibodies, IgG-binding domains, and peptide mimotopes in a tight packing and rectified orientation.

In contrast to tethered lipid membranes, where a precisely balanced mixture of tether and spacer molecules have to be assembled on the sensor surface, only one type of biomolecules, the S-layer protein, is sufficient to provide few repetitive anchoring points for the lipid membrane. Moreover, biologically inspired lipid membrane-based platforms enabled the unprecedented signal amplification down to single-molecule sensitivity.

This was achieved by the creation of mechanically and chemically stable membrane platforms with a high longevity. A further crucial property of membrane platforms is their ability to host membrane-associated and -integrated biomolecules like membrane-active peptides, ionophores, pore-forming proteins, ion channels or G-protein coupled receptors in a functional form.

All these, in many cases highly sensitive biomolecules distinguish themselves by operating at very low concentrations of, e. For instance, biological nanomachines, like G-protein coupled receptors and ion channels, are very successful in solving the problem of selective and efficient amplification of binding events.

Miniaturization of membrane platforms in a chip format reduces the volume of needed biological material and allows for very sensitive recording of single membrane protein activities. Miniaturized membrane platforms are very promising in the field of drug discovery due to the possibility to directly record membrane protein functionality when they are exposed, e.

At present, the research on biosensor is not only driving the ever-accelerating race to construct devices that are more efficient, smaller, and cheaper, but may also ultimately result in the successful integration of electronic and biological systems, and hence, in novel electronic sensing technologies. Elements and selected components of an S-layer protein-based quartz crystal microbalance with dissipation monitoring QCM-D biosensor.

A A biosensing element or bioreceptor comprising of accessible functions like, e. B An interface architecture comprising of a QCM-D sensor surface covered by a recrystallized S-layer lattice, which provides an environment for the proper functioning of the biosensing element. Here, the specific biological event takes place, which gives rise to a certain physical phenomenon. D Associated electronics comprising of signal amplifier, signal processor and a display allowing for a user-friendly visualization and evaluation of the data.

Transmission electron microscopy TEM image of a freeze-etched and metal shadowed preparation of a an archaeal cell from Methanocorpusuculum sinense , and b a bacterial cell from Desulfotomaculum nigrificans. Scheme of natural and surface layer S-layer supported lipid membranes. Supramolecular structure of an archaeal cell envelope comprising of a cytoplasma membrane, archaeal S-layer proteins incorporated in the lipidic matrix and integral membrane proteins A.

Schematic illustrations of various S-layer-supported lipid membranes. C A tetraether lipid monolayer membrane is generated across an orifice of a patch clamp pipette by the tip—dip method. Subsequently a closely attached S-layer lattice is formed by bacterial S-layer proteins on one side of the lipid membrane.

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In D , a folded or painted bilayer phospholipid membrane spanning a Teflon aperture is shown. A closed bacterial S-layer lattice can be self-assembled on either one or both not shown sides of the lipid membrane. E Schematic drawing of a solid support where a closed bacterial S-layer lattice has been assembled.


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  8. On this biomimetic structure, a tetraether lipid membrane was generated by the modified Langmuir-Blodgett method. Optionally as shown on the left side, a bacterial S-layer lattice can be attached on the external side of the solid supported lipid membrane. F Scheme of a bilayer lipid membrane generated on an S-layer ultrafiltration membrane.

    Optionally as shown on the left side, a bacterial S-layer lattice can be attached on the external side of the S-layer ultrafiltration membrane SUM -supported lipid membrane. In B to F, the head groups of the lipid molecules interacting with the S-layer protein are marked in dark. As indicated in C to F, all S-layer-supported model lipid membranes can be functionalized by biomolecules like membrane-active peptides and integral membrane proteins.

    Modified after [ 63 ], copyright with permission from Wiley-VCH. S-layer-coated liposomes and emulsomes. The bars correspond to nm. Adopted from [ ], copyright with permission from Wiley-VCH. C Schematic drawing of 1 an S-layer coated emulsomes left and Iiposome right with entrapped functional molecules and 2 functionalized by reconstituted integral proteins.

    Note, S-layer coated emulsomes can only transport hydrophobic molecules but with a much higher transport capacity. S-layer coated emulsomes and liposomes can be used as immobilization matrix for functional molecules e. Alternatively, emulsomes or liposomes can be coated with genetically modified S-layer subunits incorporating functional domains 6.

    Modified after [ 61 ], copyright with permission from Wiley-VCH. Efforts to use proteins as patterning elements and building blocks in nanotechnology included making fusion proteins that self-assemble into large symmetrical nanomaterials 4. In this study, we designed chimeric proteins by fusing streptavidin to a crystalline bacterial cell surface layer S-layer protein. S-layer proteins have evolved to form two-dimensional protein crystals as the outermost component of bacterial cell envelopes 5—7.

    Their intrinsic ability to self-assemble allows the in vitro formation of monomolecular protein lattices in suspension, on lipid films, on liposomes, and on solid supports including silicon wafers, metals, and polymers 8 , 9. Self-assembly of the S-layer protein moiety was exploited to arrange streptavidin in defined order and orientation in two-dimensional protein crystals. Owing to the versatile applications of the streptavidin—biotin interaction as a biomolecular coupling system, the chimeric S-layer was designed to serve as a compatible patterning element for any biotinylated targets.

    SbsB is generated from a aa preprotein by cleavage of a aa signal peptide It contains an S-layer homology SLH domain 12 at its N terminus, which is responsible for anchoring the protein to the cell surface by binding to an accessory cell wall polymer SbsB forms a 4. Because in a p1 lattice one morphological unit consists of a single protein and there are no internal symmetries, a protein moiety that is fused to any position of SbsB, provided that it does not interfere with lattice formation, will be presented with this same spacing.

    When SbsB crystallizes in vitro on solid supports or lipid films, its orientation is reversed, and the inner face with the SLH domain is exposed to the solvent. However, on surfaces presenting the specific accessory cell wall polymer, SbsB crystallizes in its natural orientation. Therefore, two types of building blocks with streptavidin at the outer and at the inner face of SbsB were desired.

    Until now, no structural model at atomic resolution of SbsB or any other S-layer protein has been available. Therefore, an understanding of the protein's structure—function relationship was gained in a preliminary study from the production and analysis of truncated forms, sequence analysis, and electron microscopy. The knowledge was used to select sequence positions for functional fusion. The proteins were refolded to heterotetramers consisting of one chain of fusion protein and three chains of streptavidin.

    The self-assembly capability of chimeric S-layer proteins in suspension, on liposomes, on silicon wafers, and on cell wall fragments was demonstrated, and their ability to bind d -biotin and two biotinylated marker proteins, peroxidase and ferritin, was studied. This section can be found in Supporting Materials and Methods , which is published as supporting information on the PNAS web site, www.

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    Biomass was fixed, embedded, and ultrathin-sectioned as described Core streptavidin and N-terminal fusion proteins were isolated according to a published protein-isolation protocol 16 from half a liter of expression culture each, mixed and dissolved in 10 ml of 7 M guanidine hydrochloride GHCl; pH 1. Tetramers of the desired stoichiometry were separated by gel permeation chromatography Superdex resin, Amersham Pharmacia and purified by affinity chromatography 2-iminobiotin resin, Sigma according to the manufacturers' instructions, except that all buffers for N-terminal fusion proteins contained 2 M GHCl.

    Silicon wafers were cut to 0. Recordings were made 30 s after each addition over a s integration time.

    S-layer-streptavidin fusion proteins as template for nanopatterned molecular arrays

    In the titration profiles, the breakpoint between progressive quenching and the subsequent plateau was used to calculate the amount of d -biotin needed for saturation of all biotin-binding sites. Liposomes coated with an S-layer were suspended in PBS at a concentration corresponding to Peroxidase-biotinamidocaproyl conjugate Sigma was added to a concentration of 2. Biotinylated ferritin was incubated with S-layer-carrying liposomes Liposomes and cell wall fragments carrying an S-layer of recombinant SbsB served as negative controls.

    A preliminary study revealed that truncated forms of SbsB lacking the N-terminal SLH domain self-assembled to form an S-layer lattice with unchanged lattice parameters, whereas deletion of 15 amino acids or more from the C terminus prevented lattice formation. Based on these results, the following six fusion proteins were designed a schematic illustration can be found in Fig.

    The amino acid numbers refer to the mature protein sequences of SbsB 10 and streptavidin 21 with reverse and negative numbering for the signal peptide sequences. The additional eight amino acids in BS2 were intended to serve as a flexible linker The expression levels of all six fusion protein constructs in E. The four N-terminal fusion proteins formed self-assembly products in the cytoplasm of the host cells, which could be visualized by TEM of ultrathin-sectioned preparations Fig. The crystalline sheets readily endured an enzymatic lysis procedure and could be isolated by centrifugation and washing.

    However, the C-terminal fusion proteins appeared in the soluble protein fraction, and no assembly-like structures were observed. Without a fusion partner, core streptavidin accumulated as inclusion bodies. Cytoplasmic self-assembly products and inclusion bodies were isolated, mixed, and subjected to a refolding procedure. The two C-terminal fusion proteins were applied to the same refolding procedure as crude cell lysates. Refolding was performed by denaturing and renaturing a mixture of fusion protein with excess core streptavidin, allowing the formation of two dominant tetramer species; one consisted of the fusion protein and core streptavidin at a 1: Heterotetramers of the desired stoichiometry were purified by gel permeation chromatography and affinity chromatography on 2-iminobiotin resin.

    Similar to SbsB Fig. After Fourier transformation of electron micrographs taken from negatively stained uranyl acetate preparations of self-assembly products, analysis showed that the lattice parameters of SbsB were unchanged in all four chimeric S-layers. Digital image reconstructions of the S1 3 S1B1 lattice in comparison with the SbsB lattice showed the additional protein mass of streptavidin linked to the SLH-domain, to which it was fused Fig. S1 3 S1B1 could also crystallize on silicon wafers Fig. Monocrystalline areas on silicon were typically in the range of nm.

    The two C-terminal fusion proteins could not self-assemble in suspension. However, both of them formed a monomolecular lattice on accessory cell wall polymer-containing cell wall fragments of G. The lattice parameters were identical to those of SbsB, as was confirmed by Fourier processing of electron micrographs taken from negatively stained preparations.

    A and B The crystalline sheets were formed in suspension and negatively stained with uranyl acetate for TEM. The arrows indicate the base vectors of the oblique p1 lattice. C and D The digital image reconstructions were made by Fourier processing of electron micrographs not identical to the ones shown in A and B.

    In the lattice of the fusion protein, streptavidin showed up as additional protein mass D , thick arrow attached to the SLH domain. Fusion protein S1 3 S1B1 crystallized on silicon wafers. Liposomes carrying a chimeric S-layer formed by the fusion protein S1 3 S1B1 A were capable of binding biotinylated ferritin B. Preparations were negatively stained with uranyl acetate for TEM. C The cartoon shows the orientation of S1 3 S1B1 on liposomes with the streptavidin-carrying inner face of the S-layer exposed.