Increasing the integration density of electron device components will necessitate the use of new nanofabrication paradigms that complement and extend existing technologies. One potential approach to overcome the current limitations of electron-beam lithography may involve the use of hybrid systems, in which existing lithographic techniques are coupled with “bottom up” approaches such as supramolecular self-assembly. In this respect, biological systems offer some unique possibilities as they combine both self-organization and spatial patterning at the nanometer length scale. In particular, Surface Layer Proteins (S-layers) can facilitate high order organization and specific orientation of inorganic structures as they are two-dimensional porous crystalline membranes with regular structure at the nanometer scale. In this framework, the aim of the present work was the characterization of the S-layer of Sporosarcina ureae ATCC 13881 (SslA) with respect to its self-assembling properties and modification that would allow it to be employed as a patterning element and a new building block for nanobiotechnology. In vitro recrystallization experiments have shown that wild type SslA self-assembles into monolayers, multilayers or tubes. Factors such as initial monomer concentration, Ca2+ ions, pH of the recrystallization buffer and the presence of a silicon substrate have a strong influence on the recrystallization process. SslA monolayers proved to be an excellent biotemplate for ordered assembly of gold nanoparticle arrays. The recombinant SslA after expression and purification formed micrometer sized, crystalline monolayers exhibiting the same lattice structure as the wild type protein (p4 symmetry). This remarkable property of self-assembling has been preserved even when SslA was truncated. The deletion of both, N- and C-terminal SslA domains does not hinder self-assembly; the resulting protein is able to form extended monolayers that exhibit the p4 lattice symmetry. The central SslA-domain is self sufficient for the self-assembly. The possibility to change the natural properties of S-layers by genetic engineering techniques opens a new horizon for the tuning of their structural and functional features. The SslA-streptavidin fusion protein combines the remarkable property of self-assembling with the ligand i.e. biotin binding function. On silicon wafers, this chimeric protein recrystallized into coherent protein layers and exposes streptavidin, fact demonstrated by binding studies using biotinylated quantum dots. In this way, it can serve as a functional surface for controlled immobilization of biologically active molecules but also as a platform for the synthesis of planar arrays of quantum dots. Furthermore, the results open up exciting possibilities for construction of hybrid S-layers, structures that may ultimately promote the fabrication of miniaturized, nanosized electronic devices.

In numerous Gram-negative and Gram-positive bacteria as well as in Archaea SL proteins form the outermost layer of the cell envelope. SL (glyco)monomers self-assemble with oblique (p2), tetragonal (p4), or hexagonal (p3, p6) symmetries [12]. SL subunits interact with each other and with the underlying cell surface by relatively weak non-covalent forces such as hydrogen-bonds, ionic bonds, salt-bridges or hydrophobic interactions. This makes them easy to isolate by applying chaotropic agents like urea and guanidine hydrochloride (GuHCl), chelating chemicals, or by changing the pH of the environment [10]. Upon dialysis in an ambient buffer monomers recrystallize into regular arrays that possess the forms of flat sheets, open ended cylinders, or spheres on solid substrates, at air-water intefaces and on lipid films, making them appealing for nanobiotechnological applications [3, 18]. The aim of this study was to investigate the structure, thermal stability, in vivo self-assembly process, recrystallization and metallization of three different recombinant SL proteins (SslA-eGFP, mSbsC-eGFP and S13240-eGFP) expressed in yeast S. cerevisiae BY4741 which could be further used in nanobiotechnological applications. In order to fulfill this aim, I investigated the in vivo expression of SL proteins (SslA, SbsC, S13240) tagged with eGFP (SL-eGFP) in the yeast S. cerevisiae BY4141. First, I characterized the heterologous expression of SL fusion constructs with growth and fluorescence measurements combined with Western blot analyses. Fluorescence microscopy investigations of overnight grown cultures showed that SslA-eGFP fusion protein was expressed as fluorescent patches, mSbsC-eGFP as tubular networks, and S13240-eGFP as hollow-like fibrillar network structures, while eGFP did not show any distinct structure Thermal stability of in vivo expressed SL-eGFP fusion proteins were investigated by fluorescence microscopy and immunodetection. In vivo self-assembly kinetics during mitosis and meiosis was the second main issue. In parallel, association of in vivo mSbsC-eGFP structures with the cellular components was of interest. A network of tubular structures in the cytosol of the transformed yeast cells that did not colocalize with microtubules or the actin cytoskeleton was observed. Time-resolved analysis of the formation of these structures during vegetative growth and sporulation was investigated by live fluorescence microscopy. While in meiosis ascospores seemed to receive assembled structures from the diploid cells, during mitosis surface layer structures were formed de novo in the buds. Surface layer assembly always started with the appearance of a dot-like structure in the cytoplasm, suggesting a single nucleation point. In order to get these in vivo SL assemblies stably outside the cells (in situ), cell distruption experiments were conducted. The tubular structures formed by the protein in vivo were retained upon bursting the cells by osmotic shock; however their average length was decreased. During dialysis, monomers obtained by treatment with chaotropic agents recrystallized again to form tube-like structures. This process was strictly dependent on calcium ions, with an optimal concentration of 10 mM. Further increase of the Ca2+ concentration resulted in multiple non-productive nucleation points. It was further shown that the lengths of the S-layer assemblies increased with time and could be controlled by pH. After 48 hours the average length at pH 9.0 was 4.13 µm compared to 2.69 µm at pH 5.5. Successful chemical deposition of platinum indicates the potential of recrystallized mSbsC-eGFP structures for nanobiotechnological applications. For example, such metalized protein nanotubes could be used in conductive nanocircuit technologies as nanowires.

In numerous Gram-negative and Gram-positive bacteria as well as in Archaea SL proteins form the outermost layer of the cell envelope. SL (glyco)monomers self-assemble with oblique (p2), tetragonal (p4), or hexagonal (p3, p6) symmetries [12]. SL subunits interact with each other and with the underlying cell surface by relatively weak non-covalent forces such as hydrogen-bonds, ionic bonds, salt-bridges or hydrophobic interactions. This makes them easy to isolate by applying chaotropic agents like urea and guanidine hydrochloride (GuHCl), chelating chemicals, or by changing the pH of the environment [10]. Upon dialysis in an ambient buffer monomers recrystallize into regular arrays that possess the forms of flat sheets, open ended cylinders, or spheres on solid substrates, at air-water intefaces and on lipid films, making them appealing for nanobiotechnological applications [3, 18]. The aim of this study was to investigate the structure, thermal stability, in vivo self-assembly process, recrystallization and metallization of three different recombinant SL proteins (SslA-eGFP, mSbsC-eGFP and S13240-eGFP) expressed in yeast S. cerevisiae BY4741 which could be further used in nanobiotechnological applications. In order to fulfill this aim, I investigated the in vivo expression of SL proteins (SslA, SbsC, S13240) tagged with eGFP (SL-eGFP) in the yeast S. cerevisiae BY4141. First, I characterized the heterologous expression of SL fusion constructs with growth and fluorescence measurements combined with Western blot analyses. Fluorescence microscopy investigations of overnight grown cultures showed that SslA-eGFP fusion protein was expressed as fluorescent patches, mSbsC-eGFP as tubular networks, and S13240-eGFP as hollow-like fibrillar network structures, while eGFP did not show any distinct structure Thermal stability of in vivo expressed SL-eGFP fusion proteins were investigated by fluorescence microscopy and immunodetection. In vivo self-assembly kinetics during mitosis and meiosis was the second main issue. In parallel, association of in vivo mSbsC-eGFP structures with the cellular components was of interest. A network of tubular structures in the cytosol of the transformed yeast cells that did not colocalize with microtubules or the actin cytoskeleton was observed. Time-resolved analysis of the formation of these structures during vegetative growth and sporulation was investigated by live fluorescence microscopy. While in meiosis ascospores seemed to receive assembled structures from the diploid cells, during mitosis surface layer structures were formed de novo in the buds. Surface layer assembly always started with the appearance of a dot-like structure in the cytoplasm, suggesting a single nucleation point. In order to get these in vivo SL assemblies stably outside the cells (in situ), cell distruption experiments were conducted. The tubular structures formed by the protein in vivo were retained upon bursting the cells by osmotic shock; however their average length was decreased. During dialysis, monomers obtained by treatment with chaotropic agents recrystallized again to form tube-like structures. This process was strictly dependent on calcium ions, with an optimal concentration of 10 mM. Further increase of the Ca2+ concentration resulted in multiple non-productive nucleation points. It was further shown that the lengths of the S-layer assemblies increased with time and could be controlled by pH. After 48 hours the average length at pH 9.0 was 4.13 µm compared to 2.69 µm at pH 5.5. Successful chemical deposition of platinum indicates the potential of recrystallized mSbsC-eGFP structures for nanobiotechnological applications. For example, such metalized protein nanotubes could be used in conductive nanocircuit technologies as nanowires.

Increasing the integration density of electron device components will necessitate the use of new nanofabrication paradigms that complement and extend existing technologies. One potential approach to overcome the current limitations of electron-beam lithography may involve the use of hybrid systems, in which existing lithographic techniques are coupled with “bottom up” approaches such as supramolecular self-assembly. In this respect, biological systems offer some unique possibilities as they combine both self-organization and spatial patterning at the nanometer length scale. In particular, Surface Layer Proteins (S-layers) can facilitate high order organization and specific orientation of inorganic structures as they are two-dimensional porous crystalline membranes with regular structure at the nanometer scale. In this framework, the aim of the present work was the characterization of the S-layer of Sporosarcina ureae ATCC 13881 (SslA) with respect to its self-assembling properties and modification that would allow it to be employed as a patterning element and a new building block for nanobiotechnology. In vitro recrystallization experiments have shown that wild type SslA self-assembles into monolayers, multilayers or tubes. Factors such as initial monomer concentration, Ca2+ ions, pH of the recrystallization buffer and the presence of a silicon substrate have a strong influence on the recrystallization process. SslA monolayers proved to be an excellent biotemplate for ordered assembly of gold nanoparticle arrays. The recombinant SslA after expression and purification formed micrometer sized, crystalline monolayers exhibiting the same lattice structure as the wild type protein (p4 symmetry). This remarkable property of self-assembling has been preserved even when SslA was truncated. The deletion of both, N- and C-terminal SslA domains does not hinder self-assembly; the resulting protein is able to form extended monolayers that exhibit the p4 lattice symmetry. The central SslA-domain is self sufficient for the self-assembly. The possibility to change the natural properties of S-layers by genetic engineering techniques opens a new horizon for the tuning of their structural and functional features. The SslA-streptavidin fusion protein combines the remarkable property of self-assembling with the ligand i.e. biotin binding function. On silicon wafers, this chimeric protein recrystallized into coherent protein layers and exposes streptavidin, fact demonstrated by binding studies using biotinylated quantum dots. In this way, it can serve as a functional surface for controlled immobilization of biologically active molecules but also as a platform for the synthesis of planar arrays of quantum dots. Furthermore, the results open up exciting possibilities for construction of hybrid S-layers, structures that may ultimately promote the fabrication of miniaturized, nanosized electronic devices.