Prolonged stochastic single ion channel recordings in S-layer protein stabilized lipid bilayer membranes

S-layer proteins are commonly found in bacteria and archaea as two-dimensional monomolecular crystalline arrays as the outermost cell membrane component. These proteins have the unique property that following disruption by chemical agents, monomers of the protein can re-assemble to their original lattice structure. This unique property makes S-layers interesting for utilization in bio-nanotechnological applications. Here, we show that the addition of S-layer proteins to bilayer lipid membranes increases the lifetime and the stability of the bilayer. M2delta ion channels were functionally incorporated into these S-layer stabilized membranes and we were able to record their activity for up to 20 h. Transmission electron microscopy (TEM) was used to visualize the 2D crystalline pattern of the S-layer and the M2delta ion channel characteristics in bilayer lipid membrane's were compared in the presence and absence of S-layers.     

Surface layer protein characterization by small angle x-ray scattering and a fractal mean force concept: from protein structure to nanodisk assemblies

Surface layers (S-layers) are the most commonly observed cell surface structure of prokaryotic organisms. They are made up of proteins that spontaneously self-assemble into functional crystalline lattices in solution, on various solid surfaces, and interfaces. While classical experimental techniques failed to recover a complete structural model of an unmodified S-layer protein, small angle x-ray scattering (SAXS) provides an opportunity to study the structure of S-layer monomers in solution and of self-assembled two-dimensional sheets. For the protein under investigation we recently suggested an atomistic structural model by the use of molecular dynamics simulations. This structural model is now refined on the basis of SAXS data together with a fractal assembly approach. Here we show that a nondiluted critical system of proteins, which crystallize into monomolecular structures, might be analyzed by SAXS if protein-protein interactions are taken into account by relating a fractal local density distribution to a fractal local mean potential, which has to fulfill the Poisson equation. The present work demonstrates an important step into the elucidation of the structure of S-layers and offers a tool to analyze the structure of self-assembling systems in solution by means of SAXS and computer simulations.     

Influence of surface chemistry and protein concentration on the adsorption rate and S-layer crystal formation

Bacterial crystalline surface layers (S-layers) are the outermost envelope of prokaryotic organisms representing the simplest biological membranes developed during evolution. In this context, the bacterial protein SbpA has already shown its intrinsic ability to reassemble on different substrates forming protein crystals of square lattice symmetry. In this work, we present the interaction between the bacterial protein SbpA and five self-assembled monolayers carrying methyl (CH(3)), hydroxyl (OH), carboxylic acid (COOH) and mannose (C(6)H(12)O(6)) as functional groups. Protein adsorption and S-layer formation have been characterized by atomic force microscopy (AFM) while protein adsorption kinetics, mass uptake and the protein layer viscoelastic properties were investigated with quartz crystal microbalance with dissipation monitoring (QCM-D). The results indicate that the protein adsorption rate and crystalline domain area depend on surface chemistry and protein concentration. Furthermore, electrostatic interactions tune different protein rate adsorption and S-layer recrystallization pathways. Electrostatic interactions induce faster adsorption rate than hydrophobic or hydrophilic interactions. Finally, the shear modulus and the viscosity of the recrystallized S-layer on CH(3)C(6)S, CH(3)C(11)S and COOHC(11)S substrates were calculated from QCM-D measurements. Protein-protein interactions seem to play a main role in the mechanical stability of the formed protein (crystal) bilayer.     

Bionanofabrication of metallic and semiconductor nanoparticle arrays using S-layer protein lattices with different lateral spacings and geometries

Two-dimensional (2-D) surface layer (S-layer) protein lattices isolated from the gram-positive bacterium Deinococcus radiodurans and the acidothermophilic archaeon Sulfolobus acidocaldarius were investigated and compared for their ability to biotemplate the formation of self-assembled, ordered arrays of inorganic nanoparticles (NPs). The NPs employed for these studies included citrate-capped gold NPs and various species of CdSe/ZnS core/shell quantum dots (QDs). The QD nanocrystals were functionalized with different types of thiol ligands (negative- or positive-charged/short- or long-chain length) in order to render them hydrophilic and thus water-soluble. Transmission electron microscopy, Fourier transform analyses, and pair correlation function calculations revealed that ordered nanostructured arrays with a range of spacings (approximately 7-22 nm) and different geometrical arrangements could be fabricated through the use of the two types of S-layers. These results demonstrate that it is possible to exploit the physicochemical/structural diversity of prokaryotic S-layer scaffolds to vary the morphological patterning of nanoscale metallic and semiconductor NP arrays.     

Ultrafiltration membranes prepared from crystalline bacterial cell surface layers as model systems for studying the influence of surface properties on protein adsorption

Crystalline bacterial cell surface layers (S-layers) were used for the preparation of the active filtration layer of ultrafiltration membranes (S-layer ultrafiltration membranes; SUMs). Since the S-layer is uniform in its pore size and morphology and its functional groups are aligned in well-defined positions, the SUMs provide ideal model systems for studying protein adsorption and membrane fouling. Due to the presence of surface-located carboxyl groups the standard SUMs have the net negative charge but exhibit basically a hydrophobic character. In order to change the net charge, the charge density and the accessibility of charged groups of the SUMs as well as their hydrophobicity, free carboxyl groups of the S-layer protein were modified with selected low molecular weight nucleophiles under conditions of preserving the crystalline lattice structure. SUMs with 1.6 to 7 charged or functional groups exposed per nm² of the membrane area were used for adsorption experiments. After solutions of differently sized and charged test proteins were filtered, the relative flux losses of distilled particle free water were measured. The results showed that the adsorption capacity of the SUMs increased with the extent of their hydrophobicity. Test proteins showed their own specific adsorption characteristics, which clearly demonstrated the difficulties in determining parameters controlling the membrane fouling. Independent of the net charge of the test proteins and that of the SUMs, the flux loss of SUMs increased with the increased charge density and an improved accessibility of the charged groups on the S-layer surface. No essential differences in the adsorption characteristics were observed between the zwitterionic SUMs of slightly surplus of free carboxyl groups and the standard SUMs of net negative charge.     

High-affinity tags fused to s-layer proteins probed by atomic force microscopy

Two-dimensional, crystalline bacterial cell surface layers, termed S-layers, are one of the most commonly observed cell surface structures of prokaryotic organisms. In the present study, genetically modified S-layer protein SbpA of Bacillus sphaericus CCM 2177 carrying the short affinity peptide Strep-tag I or Strep-tag II at the C terminus was used to generate a 2D crystalline monomolecular protein lattice on a silicon surface. Because of the genetic modification, the 2D crystals were addressable via Strep-tag through streptavidin molecules. Atomic force microscopy (AFM) was used to investigate the topography of the single-molecules array and the functionality of the fused Strep-tags. In high-resolution imaging under near-physiological conditions, structural details such as protein alignment and spacing were resolved. By applying molecular recognition force microscopy, the Strep-tag moieties were proven to be fully functional and accessible. For this purpose, streptavidin molecules were tethered to AFM tips via approximately 8-nm-long flexible polyethylene glycol (PEG) linkers. These functionalized tips showed specific interactions with 2D protein crystals containing either the Strep-tag I or Strep-tag II, with similar energetic and kinetic behavior in both cases.     

Thin film processing using S-layer proteins: biotemplated assembly of colloidal gold etch masks for fabrication of silicon nanopillar arrays

We explored the bionanofabrication of silicon nanopillar structures using ordered gold nanoparticle arrays generated from microbial surface layer (S-layer) protein templates. The S-layer template used for these thin film processing experiments was isolated from the Gram-positive bacterium Deinococcus radiodurans. In this preliminary work, S-layers preimmobilized onto chemically modified silicon substrates were initially used to template the fabrication of a nanolithographic hard mask pattern comprised of a hexagonally ordered array of 5-nm gold nanoparticles (lattice constant = 18 nm). Significantly, the use of the biotemplated gold nanoparticle mask patterns in an inductively coupled plasma (ICP) etching process successfully yielded silicon nanopillar structures. However, it was found that the resultant nanopillars (8–13 nm wide at the tip, 15–20 nm wide at half-height, 20–30 nm wide at the base, and 60–90 nm tall) appeared to lack any significant degree of translational ordering. The results suggest that further studies are needed in order to elucidate the optimal plasma processing parameters that will lead to the generation of long-range ordered arrays of silicon-based nanostructures using S-layer protein templates.     

An Amperometric Glucose Sensor Based on Isoporous Crystalline Protein Membranes as Immobilization Matrix

Abstract

S-layer ultrafiltration membranes (SUMs) with an active filtration layer composed of coherent two-dimensional, isoporous protein crystals (S-layers) have been used as matrix for immobilizing monolayers of enzymes. Since S-layers are formed by periodic repetition of identical protein subunits, functional groups are present on the crystalline array in an identical position and orientation. As a consequence monolayers of enzymes can bind in a geometrically well defined way. For the covalent immobilization of enzymes carboxyl groups from the S-layer protein were activated with carbodiimide and allowed to react with amino groups of the enzyme. SUMs were employed as a new type of immobilization matrix for the developement of an amperometric glucose sensor using glucose oxidase (GOD) as the biologically active component. Glucose oxidase covalently bound to the surface of the S-layer protein retained approximately 40% of its activity. The enzyme loaded SUMs were covered with a layer of gold or platinum to function as working electrodes. These sensors yielded high signals (150nA/mm²/mmol glucose), fast response times (10–30s) and a linearity range up to 12 mM glucose. The stability under working conditions was more than 48 hours. There was no loss in activity after a storage period of 6 month.     

A Multistep Enzyme Sensor for Sucrose Based on S-Layer Microparticles As Immobilization Matrix

Abstract

In this paper we report on the construction principle and performance of an amperometric 3-enzyme sensor for sucrose based on crystalline bacterial cell surface layers (S-layers) as immobilization matrix for the biological components.

Isoporous, crystalline surface layers (S-layers) have been identified as outermost cell envelope layer in many bacteria. Since they are composed of identical protein or glycoprotein subunits with functional groups in well defined positions and orientations, they represent ideal matrices for the controlled and reproducible immobilization of functional macromolecules, as required for the development of biosensors. Apart from single enzyme sensors, which were described earlier, a strikingly simple method for the assembly and optimization of multistep systems was developed. For the fabrication of an amperometric sucrose sensor invertase, mutarotase and glucose oxidase were individually immobilized on S-layer fragments isolated from Clostridium thermohydrosulfuricum L111-69 via aspartic acid as spacer molecules. Subsequently, appropriate mixtures of enzyme loaded S-layer fragments were deposited on a microfiltration membrane and finally, the composite multifunctional sensing layer was sputtered with gold in order to establish a good metal contact. Amperometric sucrose measurements based on H₂O₂ oxidation revealed a high signal level (1 μA^?1/cm^2?mmol sucrose), 5 min response time and a linear range up to 30 mM sucrose as the main characteristics of the S-layer sucrose sensor.     

Stability of S-layer proteins for electrochemical nanofabrication

Crystalline cell surface layer proteins (S-layers) can be used in electrochemical fabrication to create nanoscale arrays of metals and oxides on surfaces so long as the proteins maintain their long-range order during processing. We have explored the stability of the HPI layer protein (the S-layer protein from the microorganism Deinococcus radiodurans) adsorbed onto platinum surfaces after immersion in sulfuric acid or sodium hydroxide electrolytes ranging in pH from 0 to 14 over time periods ranging from 1 to 1000 s. Topographic data obtained by atomic force microscopy (AFM) was used to characterize the protein stability, judged by its retention of long-range order after immersion. The compiled data revealed that, under these solution conditions and in this environment, the HPI layer protein has a dose-dependent structural stability “envelope” in the acidic range from 1 < pH < 4. The protein retains its long-range order up to 1000 s from pH 4 to 11, and has a sharp stability edge between pH 12 and 13. Interestingly, the more stringent requirement of stability (i.e., retention of long-range order) defined in the context of electrochemical fabrication for this protein narrowed the window of stability in pH and time when compared to previous stability studies reported for this protein.     

Formation of a gold superlattice on an S-layer with square lattice symmetry

This work describes a new strategy in which a crystalline bacterial cell surface layer (S-layer) composed of a monolayer of a single protein species was used as periodic nanometric template in the nucleation of ordered arrays of gold nanoparticles. A square superlattice of uniform 4 to 5 nm sized gold particles with 12.8 nm repeat distance was fabricated by exposing the S-layer lattice of Bacillus sphaericus CCM2177, in which thiol groups had been introduced before, to a tetrachloroauric(III) acid solution. Transmission electron microscopical studies showed that the gold nanoparticles were formed in the pore region during electron irradiation of an initially grainy gold coating covering the whole S-layer lattice. The shape of the gold particles resembled the morphology of the pore region of the square S-layer lattice. By electron diffraction and energy dispersive X-ray analysis the crystallites were identified as gold (Au(0)). Electron diffraction patterns revealed that the gold nanoparticles were crystalline but in the long range order not crystallographically aligned. It is postulated that S-layers will allow the fabrication of a wide range of inorganic nanocrystal superlattice arrays.     

Analysis of the Interaction Between Bacillus coagulans and Bacillus thuringiensis S-layers and Calcium Ions by XRD,Light Microscopy,and FTIR

S-layer is a self-assemble regularly crystalline surface that covers major cell wall component of many bacteria and archaea and exhibits a high metal-binding capacity. We have studied the effect of the calcium ions and type of solid support (glass or mica) on the structure of the S-layers from Bacillus coagulans HN-68 and Bacillus thuringiensis MH14 upon simple methods based on light microscopy and AFM. Furthermore, the Fourier transform infrared spectroscopy (FTIR) study is indicated that the calcium–S-layer interaction occurred mainly through the carboxylate groups of the side chains of aspartic acid (Asp) and glutamic acid (Glu) and nitrogen atoms of Lys, Asn, and histidine (His) amino acids and N–H groups of the peptide backbone. Studied FTIR revealed that inner faces of S-layer are mainly negative, and outer faces of S-layer are mainly positive. Probably, calcium ions with positive charges bound to the carboxyl groups of Glu and Asp. Accordingly, calcium ions are anchored in the space between the inner faces of S-layer with negative charge and the surface of mica with negative charge. This leads to regular arrangement of the S-layer subunits.     

Nanoengineered polymeric S-layers based capsules with targeting activity

Nanostructured polymeric capsules are regarded as highly promising systems with different potential applications ranging from drug delivery, biosensing and artificial cells. To fully exploit this potential, it is required to produce bio-activated stable and biocompatible capsules. To this purpose, in present work we proposed the combination of the layer-by-layer self assembly method with bacterial S-layer technology to fabricate stable and biocompatible polymeric capsules having a well defined arrangement of functional groups allowing the covalent attachment of antibody molecules. Hollow microcapsules were obtained by the layer-by-layer self assembly of oppositely charged polyelectrolytes onto colloidal particles, followed by removal of the cores at acidic pH. S-layers were crystallized onto the shell of the obtained capsules. Quartz crystal microbalance was used to characterize the crystallization process onto planar surfaces. S-layer containing capsules were investigated by atomic force microscopy. Immunoenzymatic tests were performed to assess the effective modification of the S-layer with antibody molecules both on planar surfaces and on hollow capsules. Fluorescent microscopy was employed to visualize the presence of the antibody molecules onto the capsule shell and immunological tests used to assess the bioactivity of the immobilized antibodies. Finally, the in vitro cytotoxicity of fabricated S-layer containing capsules was studied. The obtained results demonstrated the possibility to fabricate bio-activated S-layer containing capsules with improved features in terms of biocompatibility.     

Size-selective separations of biological macromolecules on mesocylinder silica arrays

In order to control the design functionality of mesocylinder filters for molecular sieving of proteins, we fabricated tight mesocylinder silica nanotube (NT) arrays as promising filter candidates for size-exclusion separation of high-concentration macromolecules, such as insulin (INS), α-amylase (AMY), β-lactoglobulin (β-LG), and myosin (MYO) proteins. In this study, hexagonal mesocylinder structures were fabricated successfully inside anodic alumina membrane (AAM) nanochannels using a variety of cationic and nonionic surfactants as templates. The systematic design of the nanofilters was based on densely patterned polar silane coupling agents ("linkers") onto the AAM nanochannels, leading to the fabrication of mesocylinder silica arrays with vertical alignment and open surfaces of top-bottom ends inside AAM. Further surface coating of silica NTs hybrid AAM with hydrophobic agents facilitated the production of extremely robust constructed sequences of membranes without the formation of air gaps among NT arrays. The fabricated membranes with impermeable coated layers, robust surfaces, and uniformly multidirectional cylinder pores in nanoscale sizes rapidly separate large quantities of proteins within seconds. Meanwhile, comprehensive factors that affect the performance of the molecular transport, diffusivity, and filtration rate through nanofilter membranes were discussed. The mesocylinder filters of macromolecules show promise for the efficient separation and molecular transport of large molecular weight and size as well as concentrations of proteins.     

Electrochemistry of nano-scale bacterial surface protein layers on gold

The mechanism of the recrystallization of nano-scale bacterial surface protein layers (S-layers) on solid substrates is of fundamental interest in the understanding and engineering of biomembranes and e.g. biosensors. In this context, the influence of the charging state of the substrate had to be clarified. Therefore, the electrochemical behaviour of the S-layers on gold electrodes has been investigated by in-situ electrochemical quartz microbalance (EQMB) measurements, scanning force microscopy (SFM) and small-spot X-ray photoelectron spectroscopy (SS-XPS) of potentiostatically emersed substrates. It was shown that the negatively charged bonding sites of the S-layer units (e.g. carboxylates) can bond with positively charged Au surface atoms in the positively charged electrochemical double layer region positive of the point of zero charge ( approximately -0.8 V vs. saturated mercury-mercurous sulphate electrode). Surface conditions in other potential regions decelerated the recrystallization and fixation of S-layers. Time-resolved in-situ and ex-situ measurements demonstrated that two-dimensional S-layer crystal formation on gold electrodes can occur within few minutes in contrast to hours common in self-assembled monolayer (SAM) generation. These results proved that the recrystallization and fixation of 2D-crystalline S-layers on an electronic conductor can be influenced and controlled by direct electrochemical manipulation.     

Structural control of surface layer proteins at electrified interfaces investigated by in situ Fourier transform infrared spectroscopy

In situ Fourier Transform Infrared (FTIR) Spectroscopy complemented by Electrochemical Quartz Microbalance (EQMB) investigations allowed a detailed insight into the influence of the electrode potential on competing adsorption processes and bonding mechanisms of buffer ions and S-layer protein molecules of Lysinibacillus sphaericus CCM2177 at an electrified liquid/gold interface. The S-layer proteins adsorb on gold polarized positively of the point of zero charge by displacing perchlorate anions in the Helmholtz plane by their carboxylate groups. This is indicated by an increase of the peptide and carboxylate infrared absorption signals accompanied by a decrease of the perchlorate signal. S-layers interlinked laterally with Ca(2+) ions, positive of the point of zero charge, resulted in the formation of a crystalline layer participating in the Helmholtz layer. In contrast to the absence of the Ca(2+)-linkers, S-layers remain structurally intact also in the negative polarization domain where the Helmholtz layer is solely sustained by mainly solvated cations without participation of the negatively charged protein carboxylate functions.     

Cation‐Transporting Peptides: Scaffolds for Functionalized Pores?

Protein pores that selectively transport ions across membranes are among nature’s most efficient machines. The selectivity of these pores can be exploited for ion sensing and water purification. Since it is difficult to reconstitute membrane proteins in their active form for practical applications it is desirable to develop robust synthetic compounds that selectively transport ions across cell membranes. One can envision tuning the selectivity of pores by incorporating functional groups inside the pore. Readily accessible octapeptides containing (aminomethyl)benzoic acid and alanine are reported here that preferentially transport cations over halides across the lipid bilayer. Ion transport is hypothesized through pores formed by stable assemblies of the peptides. The aromatic ring(s) appear to be proximal to the pore and could be potentially utilized for functionalizing the pore interior.     

S-layer templated bioinspired synthesis of silica

The current understanding of the molecular mechanisms involved in the bioinspired formation of silica structures laid foundation for investigating the potential of the S-layer protein SbpA from Lysinibacillus sphaericus CCM 2177 as catalyst, template and scaffold for the generation of novel silica architectures. SbpA reassembles into monomolecular lattices with square (p4) lattice symmetry and a lattice constant of 13.1 nm. Silica layers on the S-layer lattice were formed using tetramethoxysilane (TMOS) and visualized by transmission electron microscopy. In situ quartz crystal microbalance with dissipation monitoring (QCM-D) measurements showed the adsorption of silica in dependence on the presence of phosphate in the silicate solution and on the preceding chemical modification of the S-layer. An increased amount of precipitated silica could be observed when K₂HPO₄/KH₂PO₄ was present in the solution (pH 7.2). Further on, independent of the presence of phosphate the silica deposition was higher on S-layer lattices upon activation of their carboxyl groups with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) compared to native S-layers or EDC treated S-layers when the activated carboxyl groups were blocked with ethylene diamine (EDA). Fourier transform infrared attenuated total reflectance (FTIR-ATR) spectroscopy revealed the formation of an amorphous silica gel (SiO₂)_x·yH₂O on the S-layer. The silica surface concentrations on the S-layer was 4 × 10^?9 to 2 × 10^?8 mol cm^?2 depending on the modification of the protein layer and corresponded to 4–21 monolayers of SiO₂.     

Bilayer lipid membrane formation on a chemically modified S-layer lattice

The present paper describes the generation of a biomimetic model lipid membrane on bacterial surface (S-)layer which covered the entire surface of various sensors. The S-layer lattice allows one to be independent from the underlying solid material and provides a biological surface and anchoring structure for lipid membranes. S-layer proteins were chemically modified via binding of two amine-terminated phospholipids. Subsequently, a bimolecular lipid membrane anchored to the previously generated viscoelastic lipid monolayer was generated by the rapid solvent exchange technique. Characterization of the intermediate (monolayer) and final membrane structures (bilayer) was performed by imaging, surface-sensitive, and electrochemical techniques. This bilayer lipid membrane generated on an S-layer lattice revealed a thickness of ～6 nm and constitutes a stable supported model membrane system with highly isolating properties showing a membrane resistance of 8.5 MΩ × cm(2).     

Redesigning of Microbial Cell Surface and Its Application to Whole-Cell Biocatalysis and Biosensors

Microbial cell surface display technology can redesign cell surfaces with functional proteins and peptides to endow cells some unique features. Foreign peptides or proteins are transported out of cells and immobilized on cell surface by fusing with anchoring proteins, which is an effective solution to avoid substance transfer limitation, enzyme purification, and enzyme instability. As the most frequently used prokaryotic and eukaryotic protein surface display system, bacterial and yeast surface display systems have been widely applied in vaccine, biocatalysis, biosensor, bioadsorption, and polypeptide library screening. In this review of bacterial and yeast surface display systems, different cell surface display mechanisms and their applications in biocatalysis as well as biosensors are described with their strengths and shortcomings. In addition to single enzyme display systems, multi-enzyme co-display systems are presented here. Finally, future developments based on our and other previous reports are discussed.