Electrochemical Biosensors Based on S-Layer Proteins.

Designing and development of electrochemical biosensors enable molecule sensing and quantification of biochemical compositions with multitudinous benefits such as monitoring, detection, and feedback for medical and biotechnological applications. Integrating bioinspired materials and electrochemical techniques promote specific, rapid, sensitive, and inexpensive biosensing platforms for (e.g., point-of-care testing). The selection of biomaterials to decorate a biosensor surface is a critical issue as it strongly affects selectivity and sensitivity. In this context, smart biomaterials with the intrinsic self-assemble capability like bacterial surface (S-) layer proteins are of paramount importance. Indeed, by forming a crystalline two-dimensional protein lattice on many sensors surfaces and interfaces, the S-layer lattice constitutes an immobilization matrix for small biomolecules and lipid membranes and a patterning structure with unsurpassed spatial distribution for sensing elements and bioreceptors. This review aims to highlight on exploiting S-layer proteins in biosensor technology for various applications ranging from detection of metal ions over small organic compounds to cells. Furthermore, enzymes immobilized on the S-layer proteins allow specific detection of several vital biomolecules. The special features of the S-layer protein lattice as part of the sensor architecture enhances surface functionalization and thus may feature an innovative class of electrochemical biosensors.

Physiology and methane productivity of Methanobacterium thermaggregans.

Accumulation of carbon dioxide (CO2), associated with global temperature rise, and drastically decreasing fossil fuels necessitate the development of improved renewable and sustainable energy production processes. A possible route for CO2 recycling is to employ autotrophic and hydrogenotrophic methanogens for CO2-based biological methane (CH4) production (CO2-BMP). In this study, the physiology and productivity of Methanobacterium thermaggregans was investigated in fed-batch cultivation mode. It is shown that M. thermaggregans can be reproducibly adapted to high agitation speeds for an improved CH4 productivity. Moreover, inoculum size, sulfide feeding, pH, and temperature were optimized. Optimization of growth and CH4 productivity revealed that M. thermaggregans is a slightly alkaliphilic and thermophilic methanogen. Hitherto, it was only possible to grow seven autotrophic, hydrogenotrophic methanogenic strains in fed-batch cultivation mode. Here, we show that after a series of optimization and growth improvement attempts another methanogen, M. thermaggregas could be adapted to be grown in fed-batch cultivation mode to cell densities of up to 1.56 g L-1. Moreover, the CH4 evolution rate (MER) of M. thermaggregans was compared to Methanothermobacter marburgensis, the CO2-BMP model organism. Under optimized cultivation conditions, a maximum MER of 96.1 ± 10.9 mmol L-1 h-1 was obtained with M. thermaggregans-97% of the maximum MER that was obtained utilizing M. marburgensis in a reference experiment. Therefore, M. thermaggregans can be regarded as a CH4 cell factory highly suited to be applicable for CO2-BMP.

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. Moreover, the obtained results did not indicate the formation of functional α-hemolysin pores, but evidence for functional incorporation of gramicidin into this biomimetic architecture is provided.

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. This system provides the ideal tool for the investigation of cellular heterogeneity, as well as a platform for the investigation of rare cell types such as circulating tumour cells.

Exploring surface structures.

The surface layer of Sulfolobus acidocaldarius consists of a flexible but stable outer protein layer that interacts with an inner, membrane-bound protein.

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. Upon coating with wtSbsB, positive charge of emulsomes shifts to a highly negative zeta potential, whereas those coated with rSbsB become charge neutral. This observation is attributed to the presence of a negatively charged glycan, the secondary cell wall polymer (SCWP), which is associated only with wtSbsB. The present study shows for the first time the ability of recombinant and wildtype S-layer proteins to cover the entire surface of emulsomes with its characteristic crystalline lattice. Furthermore, in vitro cell culture studies reveal that S-layer coated emulsomes can be uptaken by human liver carcinoma cells (HepG2) without showing any significant cytotoxicity over a wide range of concentrations. The utilization of S-layer fusion proteins equipped in a nanopatterned fashion by identical or diverse functions may lead to further development of emulsomes in nanomedicine, especially for drug delivery and targeting.

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. The present study introduces a novel nanocarrier system, so-called CurcuEmulsomes, where curcumin is encapsulated inside the solid core of emulsomes. RESULTS: CurcuEmulsomes are spherical solid nanoparticles with an average size of 286 nm and a zeta potential of 37 mV. Encapsulation increases the bioavailability of curcumin by up to 10,000 fold corresponding to a concentration of 0.11 mg/mL. Uptaken by HepG2 human liver carcinoma cell line, CurcuEmulsomes show a significantly prolonged biological activity and demonstrated therapeutic efficacy comparable to free curcumin against HepG2 in vitro - with a delay in response, as assessed by cell viability, apoptosis and cell cycle studies. The delay is attributed to the solid character of the nanocarrier prolonging the release of curcumin inside the HepG2 cells. CONCLUSIONS: Incorporation of curcumin into emulsomes results in water-soluble and stable CurcuEmulsome nanoformulations. CurcuEmulsomes do not only successfully facilitate the delivery of curcumin into the cell in vitro, but also enable curcumin to reach its effective concentrations inside the cell. The enhanced solubility of curcumin and the promising in vitro efficacy of CurcuEmulsomes highlight the potential of the system for the delivery of lipophilic drugs. Moreover, high degree of compatibility, prolonged release profile and tailoring properties feature CurcuEmulsomes for further therapeutic applications in vivo.

Intermodal coupling spectroscopy of mechanical modes in microcantilevers.

Atomic force microscopy (AFM) is highly regarded as a lens peering into the next discoveries of nanotechnology. Fundamental research in atomic interactions, molecular reactions, and biological cell behaviour are key focal points, demanding a continuous increase in resolution and sensitivity. While renowned fields such as optomechanics have marched towards outstanding signal-to-noise ratios, these improvements have yet to find a practical way to AFM. As a solution, we investigate here a mechanism in which individual mechanical eigenmodes of a microcantilever couple to one another, mimicking optomechanical techniques to reduce thermal noise. We have a look at the most commonly used modes in AFM, starting with the first two flexural modes of cantilevers and asses the impact of an amplified coupling between them. In the following, we expand our investigation to the sea of eigenmodes available in the same structure and find a maximum coupling of 9.38 × 103 Hz/nm between two torsional modes. Through such findings we aim to expand the field of multifrequency AFM with innumerable possibilities leading to improved signal-to-noise ratios, all accessible with no additional hardware.

Activity of Single Insect Olfactory Receptors Triggered by Airborne Compounds Recorded in Self-Assembled Tethered Lipid Bilayer Nanoarchitectures.

Membrane proteins are among the most difficult to study as they are embedded in the cellular membrane, a complex and fragile environment with limited experimental accessibility. To study membrane proteins outside of these environments, model systems are required that replicate the fundamental properties of the cellular membrane without its complexity. We show here a self-assembled lipid bilayer nanoarchitecture on a solid support that is stable for several days at room temperature and allows the measurement of insect olfactory receptors at the single-channel level. Using an odorant binding protein, we capture airborne ligands and transfer them to an olfactory receptor from Drosophila melanogaster (OR22a) complex embedded in the lipid membrane, reproducing the complete olfaction process in which a ligand is captured from air and transported across an aqueous reservoir by an odorant binding protein and finally triggers a ligand-gated ion channel embedded in a lipid bilayer, providing direct evidence for ligand capture and olfactory receptor triggering facilitated by odorant binding proteins. This model system presents a significantly more user-friendly and robust platform to exploit the extraordinary sensitivity of insect olfaction for biosensing. At the same time, the platform offers a new opportunity for label-free studies of the olfactory signaling pathways of insects, which still have many unanswered questions.

Lipidomics and Comparative Metabolite Excretion Analysis of Methanogenic Archaea Reveal Organism-Specific Adaptations to Varying Temperatures and Substrate Concentrations.

Methanogenic archaea possess diverse metabolic characteristics and are an ecologically and biotechnologically important group of anaerobic microorganisms. Although the scientific and biotechnological value of methanogens is evident with regard to their methane-producing physiology, little is known about their amino acid excretion, and virtually nothing is known about the lipidome at different substrate concentrations and temperatures on a quantitative comparative basis. Here, we present the lipidome and a comprehensive quantitative analysis of proteinogenic amino acid excretion as well as methane, water, and biomass production of the three autotrophic, hydrogenotrophic methanogens Methanothermobacter marburgensis, Methanothermococcus okinawensis, and Methanocaldococcus villosus under varying temperatures and nutrient supplies. The patterns and rates of production of excreted amino acids and the lipidome are unique for each tested methanogen and can be modulated by varying the incubation temperature and substrate concentration, respectively. Furthermore, the temperature had a significant influence on the lipidomes of the different archaea. The water production rate was much higher, as anticipated from the rate of methane production for all studied methanogens. Our results demonstrate the need for quantitative comparative physiological studies connecting intracellular and extracellular constraints of organisms to holistically investigate microbial responses to environmental conditions. IMPORTANCE Biological methane production by methanogenic archaea has been well studied for biotechnological purposes. This study reveals that methanogenic archaea actively modulate their lipid inventory and proteinogenic amino acid excretion pattern in response to environmental changes and the possible utilization of methanogenic archaea as microbial cell factories for the targeted production of lipids and amino acids.

Quantitative Analysis of Core Lipid Production in Methanothermobacter marburgensis at Different Scales.

Archaeal lipids have a high biotechnological potential, caused by their high resistance to oxidative stress, extreme pH values and temperatures, as well as their ability to withstand phospholipases. Further, methanogens, a specific group of archaea, are already well-established in the field of biotechnology because of their ability to use carbon dioxide and molecular hydrogen or organic substrates. In this study, we show the potential of the model organism Methanothermobacter marburgensis to act both as a carbon dioxide based biological methane producer and as a potential supplier of archaeal lipids. Different cultivation settings were tested to gain an insight into the optimal conditions to produce specific core lipids. The study shows that up-scaling at a constant particle number (n/n = const.) seems to be a promising approach. Further optimizations regarding the length and number of the incubation periods and the ratio of the interaction area to the total liquid volume are necessary for scaling these settings for industrial purposes.

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. In the present study, fragments of the cell envelope from the hyperthermophilic acidophilic archaeon Saccharolobus solfataricus P2 (SSO) have been isolated by two different methods and characterized. The organization of the fragments and the molecular sieving properties have been elucidated by transmission electron microscopy and by determining the retention efficiency of proteins varying in size, respectively. The porosity of the archaeal S-layer fragments was determined to be 45%. S-layer fragments of SSO showed a retention efficiency of up to 100% for proteins having a molecular mass of ≥ 66 kDa. Moreover, the extraction costs for SSO fragments have been reduced by more than 80% compared to conventional methods, which makes the use of these archaeal S-layer material economically attractive.

Functional incorporation of the insect odorant receptor coreceptor in tethered lipid bilayer nanoarchitectures.

Membrane proteins are among the most important drug targets. To improve drug design, it is critical to study membrane proteins. However, due to the myriad roles fulfilled by the cellular membrane, it is a highly complex environment and challenging to study. Tethered membranes reproduce the basic physicochemical properties of the cellular membrane without its inherent complexity. The high electrical resistance and stability makes them ideal to study membrane proteins, particularly ion channels. However, due to the close proximity of the membrane to the support and the reduced fluidity and high packing density, they are unsuitable to study larger membrane proteins. We present here a tethered membrane system which adresses these challenges, allowing the functional reconstitution of the odorant receptor coreceptor from Drosophila melanogaster, a tetrameric ionotropic receptor was incorporated and its sensitivity to various ligands was examined via electrochemical impedance spectroscopy and atomic force microscopy.

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. The functional groups on the surface and in the pores of the S-layer protein lattice are accessible for chemical modifications and for binding functional molecules in very precise fashion. S-layer ultrafiltration membranes (SUMs) can be produced by depositing S-layer fragments as a coherent (multi)layer on microfiltration membranes. After inter- and intramolecular crosslinking of the composite structure, the chemical and thermal resistance of these membranes was shown to be comparable to polyamide membranes. Chemical modification and/or specific binding of differently sized molecules allow the tuning of the surface properties and molecular sieving characteristics of SUMs. SUMs can be utilized as matrices for the controlled immobilization of functional biomolecules (e.g., ligands, enzymes, antibodies, and antigens) as required for many applications (e.g., biosensors, diagnostics, enzyme- and affinity-membranes). Finally, SUM represent unique supporting structures for stabilizing functional lipid membranes at meso- and macroscopic scale.

Archaea Biotechnology.

Archaea are a domain of prokaryotic organisms with intriguing physiological characteristics and ecological importance. In Microbial Biotechnology, archaea are historically overshadowed by bacteria and eukaryotes in terms of public awareness, industrial application, and scientific studies, although their biochemical and physiological properties show a vast potential for a wide range of biotechnological applications. Today, the majority of microbial cell factories utilized for the production of value-added and high value compounds on an industrial scale are bacterial, fungal or algae based. Nevertheless, archaea are becoming ever more relevant for biotechnology as their cultivation and genetic systems improve. Some of the main advantages of archaeal cell factories are the ability to cultivate many of these often extremophilic organisms under non-sterile conditions, and to utilize inexpensive feedstocks often toxic to other microorganisms, thus drastically reducing cultivation costs. Currently, the only commercially available products of archaeal cell factories are bacterioruberin, squalene, bacteriorhodopsin and diether-/tetraether-lipids, all of which are produced utilizing halophiles. Other archaeal products, such as carotenoids and biohydrogen, as well as polyhydroxyalkanoates and methane are in early to advanced development stages, respectively. The aim of this review is to provide an overview of the current state of Archaea Biotechnology by describing the actual state of research and development as well as the industrial utilization of archaeal cell factories, their role and their potential in the future of sustainable bioprocessing, and to illustrate their physiological and biotechnological potential.

Formation of planar hybrid lipid/polymer membranes anchored to an S-layer protein lattice by vesicle binding and rupture.

Exploitation of biomolecular and biomimetic components on solid surfaces gain increasing importance for the design of stable functional platforms. The present study performed by quartz crystal microbalance with dissipation monitoring (QCM-D) reports on the formation of planar hybrid lipid/polymer membranes anchored to a crystalline surface (S-) layer protein lattice. In this approach, hybrid lipid/polymer vesicles were chemically bound to the S-layer protein lattice. Subsequently, to form a hybrid planar layer rupture and fusion was triggered either by (1) ß- diketone - europium ion complex formation or (2) successive application of calcium ions, lowering the pH from 9 to 4, and the detergent CHAPS. As determined by QCM-D, method 1 resulted for a polymer content of 5% in a planar membrane with some imbedded intact vesicles, whereas method 2 succeeded in planar hybrid membranes with a polymer content of even up to 70%. These results provide evidence for the effective formation of planar lipid/polymer membranes varying in their composition on an S-layer protein lattice.

Formation and characteristics of mixed lipid/polymer membranes on a crystalline surface-layer protein lattice.

The implementation of self-assembled biomolecules on solid materials, in particular, sensor and electrode surfaces, gains increasing importance for the design of stable functional platforms, bioinspired materials, and biosensors. The present study reports on the formation of a planar hybrid lipid/polymer membrane on a crystalline surface layer protein (SLP) lattice. The latter acts as a connecting layer linking the biomolecules to the inorganic base plate. In this approach, chemically bound lipids provided hydrophobic anchoring moieties for the hybrid lipid/polymer membrane on the recrystallized SLP lattice. The rapid solvent exchange technique was the method of choice to generate the planar hybrid lipid/polymer membrane on the SLP lattice. The formation process and completeness of the latter were investigated by quartz crystal microbalance with dissipation monitoring and by an enzymatic assay using the protease subtilisin A, respectively. The present data provide evidence for the formation of a hybrid lipid/polymer membrane on an S-layer lattice with a diblock copolymer content of 30%. The hybrid lipid/polymer showed a higher stiffness compared to the pure lipid bilayer. Most interestingly, both the pure and hybrid membrane prevented the proteolytic degradation of the underlying S-layer protein by the action of subtilisin A. Hence, these results provide evidence for the formation of defect-free membranes anchored to the S-layer lattice.

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. Moreover, newly developed techniques and methods open up the possibility to exploit these structures at meso- and macroscopic scale. An immense significance at innovative approaches for the self-assembly of supramolecular structures and devices with dimensions of a few to tens of nanometers constitutes the utilization of crystalline bacterial cell surface proteins. The latter have proven to be particularly suited as building blocks in a molecular construction kit comprising of all major classes of biological molecules. The controlled immobilization of biomolecules in an ordered fashion on solid substrates and their directed confinement in definite areas of nanometer dimensions are key requirements for many applications including the development of bioanalytical sensors, biochips, molecular electronics, biocompatible surfaces, and signal processing between functional membranes, cells, and integrated circuits.

Vienna Graph Clustering.

This paper serves as a user guide to the Vienna graph clustering framework. We review our general memetic algorithm, VieClus, to tackle the graph clustering problem. A key component of our contribution are natural recombine operators that employ ensemble clusterings as well as multi-level techniques. Lastly, we combine these techniques with a scalable communication protocol, producing a system that is able to compute high-quality solutions in a short amount of time. After giving a description of the algorithms employed, we establish the connection of the graph clustering problem to protein-protein interaction networks and moreover give a description on how the software can be used, what file formats are expected, and how this can be used to find functional groups in protein-protein interaction networks.

Composite S-layer lipid structures.

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 how S-layer proteins, lipids, and polysaccharides may be used as basic building blocks for the assembly of S-layer supported lipid membranes. These biomimetic membrane systems are distinguished by a nanopatterned fluidity, enhanced stability and longevity and thus, provide a dedicated reconstitution matrix for membrane-active peptides and transmembrane proteins. Exciting areas for application of composite S-layer membrane systems concern sensor systems involving specific membrane functions.