SymProFold: Structural prediction of symmetrical biological assemblies.

Symmetry in nature often emerges from self-assembly processes and serves a wide range of functions. Cell surface layers (S-layers) form symmetrical lattices on many bacterial and archaeal cells, playing essential roles such as facilitating cell adhesion, evading the immune system, and protecting against environmental stress. However, the experimental structural characterization of these S-layers is challenging due to their self-assembly properties and high sequence variability.

Scalable biomimetic sensing system with membrane receptor dual-monolayer probe and graphene transistor arrays.

Affinity-based biosensing can enable point-of-care diagnostics and continuous health monitoring, which commonly follows bottom-up approaches and is inherently constrained by bioprobes' intrinsic properties, batch-to-batch consistency, and stability in biofluids. We present a biomimetic top-down platform to circumvent such difficulties by combining a "dual-monolayer" biorecognition construct with graphene-based field-effect-transistor arrays. The construct adopts redesigned water-soluble membrane receptors as specific sensing units, positioned by two-dimensional crystalline S-layer proteins as dense antifouling linkers guiding their orientations.

Isolation and Characterization of Cell Envelope Fragments Comprising Archaeal S-Layer Proteins.

The outermost component of cell envelopes of most bacteria and almost all archaea comprise a protein lattice, which is termed Surface (S-)layer. The S-layer lattice constitutes a highly porous structure with regularly arranged pores in the nm-range. Some archaea thrive in extreme milieus, thus producing highly stable S-layer protein lattices that aid in protecting the organisms.

A New Method for Dispersing Pristine Carbon Nanotubes Using Regularly Arranged S-Layer Proteins.

Homogeneous and stable dispersions of functionalized carbon nanotubes (CNTs) in aqueous solutions are imperative for a wide range of applications, especially in life and medical sciences. Various covalent and non-covalent approaches were published to separate the bundles into individual tubes. In this context, this work demonstrates the non-covalent modification and dispersion of pristine multi-walled carbon nanotubes (MWNTs) using two S-layer proteins, namely, SbpA from CCM2177 and SbsB from PV72/p2.

S-Layer Ultrafiltration Membranes.

Monomolecular arrays of protein subunits forming surface layers (S-layers) are the most common outermost cell envelope components of prokaryotic organisms (bacteria and archaea). Since S-layers are periodic structures, they exhibit identical physicochemical properties for each constituent molecular unit down to the sub-nanometer level. Pores passing through S-layers show identical size and morphology and are in the range of ultrafiltration membranes.

CRISPR-mediated gene silencing reveals involvement of the archaeal S-layer in cell division and virus infection.

The S-layer is a proteinaceous surface lattice found in the cell envelope of bacteria and archaea. In most archaea, a glycosylated S-layer constitutes the sole cell wall and there is evidence that it contributes to cell shape maintenance and stress resilience. Here we use a gene-knockdown technology based on an endogenous CRISPR type III complex to gradually silence slaB, which encodes the S-layer membrane anchor in the hyperthermophilic archaeon Sulfolobus solfataricus.

Nanotechnology with S-layer Proteins.

Nanosciences are distinguished by the cross-fertilization of biology, chemistry, material sciences, and solid-state physics and, hence, open up a great variety of new opportunities for innovation. The technological utilization of self-assembly systems, wherein molecules spontaneously associate under equilibrium conditions into reproducible supramolecular structures, is one key challenge in nanosciences for life and non-life science applications. The attractiveness of such processes is due to their ability to build uniform, ultra-small functional units with predictable properties down to the nanometer scale.

Acoustic and hybrid 3D-printed electrochemical biosensors for the real-time immunodetection of liver cancer cells (HepG2).

This study presents an efficient acoustic and hybrid three-dimensional (3D)-printed electrochemical biosensors for the detection of liver cancer cells. The biosensors function by recognizing the highly expressed tumor marker CD133, which is located on the surface of liver cancer cells. Detection was achieved by recrystallizing a recombinant S-layer fusion protein (rSbpA/ZZ) on the surface of the sensors.

In Vitro Characterization of the Two-Stage Non-Classical Reassembly Pathway of S-Layers.

The recombinant bacterial surface layer (S-layer) protein rSbpA of CCM 2177 is an ideal model system to study non-classical nucleation and growth of protein crystals at surfaces since the recrystallization process may be separated into two distinct steps: (i) adsorption of S-layer protein monomers on silicon surfaces is completed within 5 min and the amount of bound S-layer protein sufficient for the subsequent formation of a closed crystalline monolayer; (ii) the recrystallization process is triggered-after washing away the unbound S-layer protein-by the addition of a CaCl₂ containing buffer solution, and completed after approximately 2 h. The entire self-assembly process including the formation of amorphous clusters, the subsequent transformation into crystalline monomolecular arrays, and finally crystal growth into extended lattices was investigated by quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM). Moreover, contact angle measurements showed that the surface properties of S-layers change from hydrophilic to hydrophobic as the crystallization proceeds.

Impact of surface wettability on S-layer recrystallization: a real-time characterization by QCM-D.

Quartz crystal microbalance with dissipation monitoring (QCM-D) has been employed to study the assembly and recrystallization kinetics of isolated SbpA bacterial surface proteins onto silicon dioxide substrates of different surface wettability. Surface modification by UV/ozone oxidation or by vapor deposition of 1,1,2,2-perfluorododecyltrichlorosilane yielded hydrophilic or hydrophobic samples, respectively. Time evolution of frequency and dissipation factors, either individually or combined as the so-called plots, showed a much faster formation of crystalline coatings for hydrophobic samples, characterized by a phase-transition peak at around the 70% of the total mass adsorbed.

Inspired and stabilized by nature: ribosomal synthesis of the human voltage gated ion channel (VDAC) into 2D-protein-tethered lipid interfaces.

We present an elegant synthesis and reconstitution approach for functional studies of voltage responsive membrane proteins. For such studies, we propose a planar architecture of an S-layer-supported lipid membrane as a suitable matrix for presenting unmodified membrane protein species, and here we focus on the voltage-dependent anion channel (VDAC) from human mitochondria. The presented cell-free strategy, in which VDAC proteins are synthesized in bacterial cell lysate, into a membrane structure, offers a great advantage in the study of such subtle membrane proteins over the conventional, cell-based synthesis approach in terms of reproducibility.

Relevance of glycosylation of S-layer proteins for cell surface properties.

Elucidating the building principles and intrinsic features modulating certain water-associated processes (e.g., surface roughness in the nanometer scale, surface hydration and accompanied antifouling property, etc.

S-layer fusion protein as a tool functionalizing emulsomes and CurcuEmulsomes for antibody binding and targeting.

Selective targeting of tumor cells by nanoparticle-based drug delivery systems is highly desirable because it maximizes the drug concentration at the desired target while simultaneously protecting the surrounding healthy tissues. Here, we show a design for smart nanocarriers based on a biomimetic approach that utilizes the building principle of virus envelope structures. Emulsomes and CurcuEmulsomes comprising a tripalmitin solid core surrounded by phospholipid layers are modified by S-layer proteins that self-assemble into a two-dimensional array to form a surface layer.

Emulsomes meet S-layer proteins: an emerging targeted drug delivery system.

Here, the use of emulsomes as a drug delivery system is reviewed and compared with other similar lipidic nanoformulations. In particular, we look at surface modification of emulsomes using S-layer proteins, which are self-assembling proteins that cover the surface of many prokaryotic organisms. It has been shown that covering emulsomes with a crystalline S-layer lattice can protect cells from oxidative stress and membrane damage.

Probing peptide and protein insertion in a biomimetic S-layer supported lipid membrane platform.

The most important aspect of synthetic lipid membrane architectures is their ability to study functional membrane-active peptides and membrane proteins in an environment close to nature. Here, we report on the generation and performance of a biomimetic platform, the S-layer supported lipid membrane (SsLM), to investigate the structural and electrical characteristics of the membrane-active peptide gramicidin and the transmembrane protein α-hemolysin in real-time using a quartz crystal microbalance with dissipation monitoring in combination with electrochemical impedance spectroscopy. A shift in membrane resistance is caused by the interaction of α-hemolysin and gramicidin with SsLMs, even if only an attachment onto, or functional channels through the lipid membrane, respectively, are formed.

Reassembly of S-layer proteins.

Crystalline bacterial cell surface layers (S-layers) represent the outermost cell envelope component in a broad range of bacteria and archaea. They are monomolecular arrays composed of a single protein or glycoprotein species and represent the simplest biological membranes developed during evolution. They are highly porous protein mesh works with unit cell sizes in the range of 3 to 30 nm, and pore sizes of 2 to 8 nm.

Biomimetic interfaces based on S-layer proteins, lipid membranes and functional biomolecules.

Designing and utilization of biomimetic membrane systems generated by bottom-up processes is a rapidly growing scientific and engineering field. Elucidation of the supramolecular construction principle of archaeal cell envelopes composed of S-layer stabilized lipid membranes led to new strategies for generating highly stable functional lipid membranes at meso- and macroscopic scale. In this review, we provide a state-of-the-art survey of how S-layer proteins, lipids and polymers may be used as basic building blocks for the assembly of S-layer-supported lipid membranes.

Fluorescent sensors based on bacterial fusion proteins.

Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker.

The grab-and-drop protocol: a novel strategy for membrane protein isolation and reconstitution from single cells.

We present a rapid and robust technique for the sampling of membrane-associated proteins from the surface of a single, live cell and their subsequent deposition onto a solid-supported lipid bilayer. As a proof of principle, this method has been used to extract green fluorescent protein (EGFP) labelled K-ras proteins located at the inner leaflet of the plasma membrane of colon carcinoma cells and to transfer them to an S-layer supported lipid bilayer system. The technique is non-destructive, meaning that both the cell and proteins are intact after the sampling operation, offering the potential for repeated measurements of the same cell of interest.

S-layers: principles and applications.

Monomolecular arrays of protein or glycoprotein subunits forming surface layers (S-layers) are one of the most commonly observed prokaryotic cell envelope components. S-layers are generally the most abundantly expressed proteins, have been observed in species of nearly every taxonomical group of walled bacteria, and represent an almost universal feature of archaeal envelopes. The isoporous lattices completely covering the cell surface provide organisms with various selection advantages including functioning as protective coats, molecular sieves and ion traps, as structures involved in surface recognition and cell adhesion, and as antifouling layers.

Characterization of CurcuEmulsomes: nanoformulation for enhanced solubility and delivery of curcumin.

Background: Curcumin is a polyphenolic compound isolated from the rhizomes of the plant Curcuma longa and shows intrinsic anti-cancer properties. Its medical use remains limited due to its extremely low water solubility and bioavailability. Addressing this problem, drug delivery systems accompanied by nanoparticle technology have emerged.

Exploitation of S-layer anisotropy: pH-dependent nanolayer orientation for cellular micropatterning.

We have developed a tunable, facile, and reliable cell patterning method using a self-assembled crystalline protein monolayer that, depending on its orientation, can exhibit either cell adhesive (cytophilic) or cell repulsive (cytophobic) surface properties. Our technique exploits, for the first time, the inherent biological anisotropy of the bacterial cell wall protein SbpA capable of interacting with its cytophilic inner side with components of the cell wall, while its outer cytophobic side interacts with the environment. By simply altering the recrystallization protocol from a basic to an acidic condition, the SbpA-protein layer orientation and function can be switched from preventing unspecific protein adsorption and cell adhesion to effectively promote cell attachment, spreading, and proliferation.

Surface-layer lattices as patterning element for multimeric extremozymes.

A promising new approach for the production of biocatalysts comprises the use of surface-layer (S-layer) lattices that present functional multimeric enzymes on their surface, thereby guaranteeing most accurate spatial distribution and orientation, as well as maximal effectiveness and stability of these enzymes. For proof of concept, a tetrameric and a trimeric extremozyme are chosen for the construction of S-layer/extremozyme fusion proteins. By using a flexible peptide linker, either one monomer of the tetrameric xylose isomerase XylA from the thermophilic Thermoanaerobacterium strain JW/SL-YS 489 or, in another approach, one monomer of the trimeric carbonic anhydrase from the methanogenic archaeon Methanosarcina thermophila are genetically linked to one monomer of the S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177.

S-layer coated emulsomes as potential nanocarriers.

The present study introduces a novel nanocarrier system comprising lipidic emulsomes and S-layer (fusion) proteins as functionalizing tools coating the surface. Emulsomes composed of a solid tripalmitin core and a phospholipid shell are created reproducibly with an average diameter of approximately 300 nm using temperature-controlled extrusion steps. Both wildtype (wt) and recombinant (r) S-layer protein SbsB of Geobacillus stearothermophilus PV72/p2 are capable of forming coherent crystalline envelope structures with oblique (p1) lattice symmetry, as evidenced by transmission electron microscopy.

Nanotechnology with s-layer proteins.

Nanosciences are distinguished by the cross-fertilization of biology, chemistry, material sciences, and solid-state physics and hence open up a great variety of new opportunities for innovation. The technological utilization of self-assembly systems, wherein molecules spontaneously associate under equilibrium conditions into reproducible supramolecular aggregates, is one key challenge in nanosciences for life and nonlife science applications. The attractiveness of such processes is due to their ability to build uniform, ultrasmall functional units and the possibility to exploit such structures at meso- and macroscopic scale very frequently by newly developed techniques and methods.