The Effect of Nanosecond, High-Voltage Electric Pulses on the Shape and Permeability of Polymersome GUVs.

Polymersomes, vesicles composed of block copolymers, are promising candidates as membrane alternatives and functional containers, e.g., as potential carriers for functional molecules because of their stability and tunable membrane properties. In the scope of possible use for membrane protein delivery to cells by electrofusion, we investigated the cytotoxicity of such polymersomes as well as the effects of nanosecond electric pulses with variable repetition rate on the shape and permeability of polymersomes in buffers with different conductivities. The polymersomes did not show cytotoxic effects to CHO and B16-F1 cells in vitro in concentrations up to 250 µg/mL (for 48 h) or 1.35 mg/mL (for 60 min), which renders them suitable for interacting with living cells. We observed a significant effect of the pulse repetition rate on electrodeformation of the polymersomes. The electrodeformation was most pronounced in low conductivity buffer, which is favorable for performing electrofusion with cells. However, despite more pronounced deformation at higher pulse repetition rate, the electroporation performance of polymersomes was unaffected and remained in similar ranges both at 10 Hz and 10 kHz. This phenomenon is possibly due to the higher stability and rigidity of polymer vesicles, compared to liposomes, and can serve as an advantage (or disadvantage) depending on the aim in employing polymersomes such as stable membrane alternative architectures or drug vehicles.

Functional Cell Adhesion Receptors (Integrins) in Polymeric Architectures.

Integrins, as transmembrane heterodimeric receptors, have important functions in cell adhesion, migration, proliferation, survival apoptosis and signal transduction, in many physio- as well as pathophysiological settings. Characterisation of integrins and their ligand/antagonist binding is notoriously difficult, due to high integrin redundancy and ubiquity. Bypassing the intrinsic difficulties of cell-based integrin expression, purification and reconstitution, we present for the first time the synthesis of a heterodimeric integrin receptor and its assembly into a block-copolymeric membrane mimic. We present comprehensive data to demonstrate the synthesis of functionally active integrin αv ß3, generated by in vitro membrane-assisted protein synthesis (iMAPS). This work represents the first step towards a robust and adaptable polymer-based platform for characterisation of integrin-ligand interactions.

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.

Synthesis and Functional Reconstitution of Light-Harvesting Complex II into Polymeric Membrane Architectures.

One of most important processes in nature is the harvesting and dissipation of solar energy with the help of light-harvesting complex II (LHCII). This protein, along with its associated pigments, is the main solar-energy collector in higher plants. We aimed to generate stable, highly controllable, and sustainable polymer-based membrane systems containing LHCII-pigment complexes ready for light harvesting. LHCII was produced by cell-free protein synthesis based on wheat-germ extract, and the successful integration of LHCII and its pigments into different membrane architectures was monitored. The unidirectionality of LHCII insertion was investigated by protease digestion assays. Fluorescence measurements indicated chlorophyll integration in the presence of LHCII in spherical as well as planar bilayer architectures. Surface plasmon enhanced fluorescence spectroscopy (SPFS) was used to reveal energy transfer from chlorophyll b to chlorophyll a, which indicates native folding of the LHCII proteins.

Biomimetic membrane platform containing hERG potassium channel and its application to drug screening.

The hERG (human ether-à-go-go-related gene) potassium channel has been extensively studied by both academia and industry because of its relation to inherited or drug-induced long QT syndrome (LQTS). Unpredicted hERG and drug interaction affecting channel activity is of main concern for drug discovery. Although there are several methods to test hERG and drug interaction, it is still necessary to develop some efficient and economic ways to probe hERG and drug interactions. To contribute this aim, we have developed a biomimetic lipid membrane platform into which the hERG channel can be folded. Expression and integration of the hERG channel was achieved using a cell-free (CF) expression system. The folding of hERG in the biomimetic membrane system was investigated using Surface Plasmon Enhanced Fluorescence Spectroscopy (SPFS) and Imaging Surface Plasmon Resonance (iSPR). In addition, the hERG channel folded into our biomimetic membrane platform was used for probing the channel and drug interactions through fluorescence polarization (FP) assay. Our results suggest that the biomimetic system employed is capable of detecting the interaction between hERG and different channel blockers at varied concentrations. We believe that our current approach could be applied to other membrane proteins for drug screening or other protein-related interactions.

In vitro expressed GPCR inserted in polymersome membranes for ligand-binding studies.

The dopamine receptor D2 (DRD2), a G-protein coupled receptor is expressed into PBd(22)-PEO(13) and PMOXA(20)-PDMS(54)-PMOXA(20) block copolymer vesicles. The conformational integrity of the receptor is confirmed by antibody- and ligand-binding assays. Replacement of bound dopamine is demonstrated on surface-immobilized polymersomes, thus making this a promising platform for drug screening.

Cell-free synthesis of cytochrome bo(3) ubiquinol oxidase in artificial membranes.

The analysis of membrane proteins is notoriously difficult because isolation and detergent-mediated reconstitution often results in compromising the protein structure and function. We introduce a novel strategy of combining a cell-free expression method for synthesis of a protein species coping with one of the most important obstacles in membrane protein research-preserving the structural-functional integrity of a membrane protein species and providing a stable matrix for application of analytical tools to characterize the membrane protein of interest. We address integration and subsequent characterization of the cytochrome bo(3) ubiquinol oxidase (Cyt-bo(3)) from de novo synthesis without the effort of conventional cell culture, isolation, and purification procedures. The experimental output supports our idea of a suitable platform for in vitro protein synthesis and functional integration into a membrane-mimicking structure. We show the compatibility of different concepts of in vitro synthesis toward biosensor applicability by the example of Cyt-bo(3) protein expression. Our results obtained from in vitro synthesized proteins displayed similar behavior to proteins isolated from the cellular context. Overall, our approach is suitable for the in vitro expression of "complex" protein species such as Cyt-bo(3), which can be reproducible and stably synthesized and preserved in robust, synthetic planar membrane architecture.

Selective deposition and self-assembly of triblock copolymers into matrix arrays for membrane protein production.

To improve the stability of cell membrane mimics, there has been growing interest in the use of block copolymers. Here, we present an easy approach to create an array of planar polymeric matrices capable of hosting membrane proteins. The array of polymeric matrices was formed by the selective deposition of triblock copolymers onto an array of hydrophilic islands situated within a hydrophobic background. The thickness of these matrices corresponds to the length of a single polymer chain. These polymeric matrices were used to host cell-free expressed membrane proteins, and offers a prototype from which a membrane protein array can be created for diagnostics or drug discovery purposes.

Purification and structural characterization of the voltage-sensor domain of the hERG potassium channel.

The hERG (human ether à go-go related gene) potassium channel is a voltage-gated potassium channel playing important roles in the heart by controlling the rapid delayed rectifier potassium current. The hERG protein contains a voltage-sensor domain (VSD) that is important for sensing voltage changes across the membrane. Mutations in this domain contribute to serious heart diseases. To study the structure of the VSD, it was over-expressed in Escherichia coli and purified into detergent micelles. Lyso-myristoyl phosphatidylglycerol (LMPG) was shown to be a suitable detergent for VSD purification and folding. Secondary structural analysis using circular dichroism (CD) spectroscopy indicated that the purified VSD in LMPG micelles adopted α-helical structures. Purified VSD in LMPG micelles produced dispersed cross-peaks in a (15)N-HSQC spectrum. Backbone resonance assignments for residues from transmembrane segments S3 and S4 of VSD also confirmed the presence of α-helical structures in this domain. Our results demonstrated that structure of VSD can be investigated using NMR spectroscopy.

Conformation and topology of amyloid beta-protein adsorbed on a tethered artificial membrane probed by surface plasmon field-enhanced fluorescence spectroscopy.

Progressive depositions of cerebral amyloid are primary neuropathologic features of Alzheimer's disease (AD). The amyloid is composed of a 39-42 amino acid peptide called the amyloid beta-protein (Abeta). Repeated investigation suggests that the conformational transition of Abeta from alpha-helix or random coil to beta-sheet structure plays a key role in the inappropriate accumulation of cerebral amyloid plaques. In this manuscript, we describe a fluorescence-based immunoassay technology to investigate the conformation and topology of Abeta peptides interacting with peptide-tethered planar lipid bilayers. Dual monoclonal antibodies (mAbs) labelled with fluorophores were employed to recognise a linear N- and a beta-sheet C-terminus of Abeta peptides on the model membrane, respectively. Kinetics of antibody-Abeta binding were determined by surface plasmon field-enhanced fluorescence spectroscopy (SPFS). The conformational transition of Abeta by melatonin, a defined beta-sheet breaker, was probed using paired monoclonal antibodies. The Abeta interaction with the membrane was evaluated by carefully analyzing the change in kinetic/affinity parameters in the presence or absence of melatonin. These results show that SPFS can be used to examine conformational transition of Abeta on an artificial membrane, providing a novel and versatile platform for conveniently monitoring protein-membrane interaction and screening for new beta-sheet breakers.

The effect of fluid flow on selective protein adsorption on polystyrene-block-poly(methyl methacrylate) copolymers.

The effect of fluid flow on protein patterning and the stability of the adsorbed protein array templated by polystyrene-block-poly(methyl methacrylate) (PS-b-PMMA) has been investigated. Protein nanoarrays can be formed on a PS-b-PMMA copolymer surface by physisorption and rinsing with an open flow. The protein arrays inherit the original hexagonal pattern generated by the phase separation of PS-b-PMMA with the proteins only residing on the PS domains. Subsequent analysis of the protein array stability under confined flow field reveals that the array integrity is strongly dependent on the flow velocity and the duration time. The interplay between the intermolecular forces and the hydrodynamic shear force has been discussed in detail. A simplified model has been proposed to explain the site-selective adsorption and protein migration under the shear of fluid flow. This study provides valuable information on the formation mechanism and stability of physisorbed protein nanopatterns under conditions concerning biosensing applications. It also represents an explorative study of protein adsorption under hydrodynamic flow conditions, which may assist in better designs of fluidic devices relevant to protein studies.

Differential tumor biological role of the tumor suppressor KAI1 and its splice variant in human breast cancer cells.

The tetraspanin and tumor suppressor KAI1 is downregulated or lost in many cancers which correlates with poor prognosis. KAI1 acts via physical/functional crosstalk with other membrane receptors. Also, a splice variant of KAI1 (KAI1-SP) has been identified indicative of poor prognosis. We here characterized differential effects of the two KAI1 variants on tumor biological events involving integrin (αvß3) and/or epidermal growth factor receptor (EGF-R). In MDA-MB-231 and -435 breast cancer cells, differential effects were documented on the expression levels of the tumor biologically relevant integrin αvß3 which colocalized with KAI1-WT but not with KAI1-SP. Cellular motility was assessed by video image processing, including motion detection and vector analysis for the quantification and visualization of cell motion parameters. In MDA-MB-231 cells, KAI1-SP provoked a quicker wound gap closure and higher closure rates than KAI1-WT, also reflected by different velocities and average motion amplitudes of singular cells. KAI1-SP induced highest cell motion adjacent to the wound gap borders, whereas in MDA-MB-435 cells a comparable induction of both KAI1 variants was noticed. Moreover, while KAI1-WT reduced cell growth, KAI1-SP significantly increased it going along with a pronounced EGF-R upregulation. KAI1-SP-induced cell migration and proliferation was accompanied by the activation of the focal adhesion and Src kinase. Our findings suggest that splicing of KAI1 does not only abrogate its tumor suppressive functions, but even more, promotes tumor biological effects in favor of cancer progression and metastasis.

Development of a Multifunctional Nanobiointerface Based on Self-Assembled Fusion-Protein rSbpA/ZZ for Blood Cell Enrichment and Phenotyping.

We present a multifunctional nanobiointerface for blood cell capture and phenotyping applications that features both excellent antifouling properties and high antibody activity. Multifunctionality is accomplished by modifying polymeric materials using self-assembled S-layer fusion-protein rSbpA/ZZ to immobilize high density antibodies at the two protein A binding sites of the rSbpA/ZZ nanolattice structure. Controlled orientation and alignment of the antibodies reduced antibody consumption 100-fold and increased cell capture efficiency 4-fold over standard methodologies. Cell analysis in complex samples was made possible by the remarkable antifouling properties of the rSbpA domain, while at the same time reducing unspecific binding and forgoing tedious blocking procedures. An automated microfluidic in situ cell analysis platform for isolation and phenotyping of primary peripheral blood mononuclear cells was developed as practical application. Results obtained using our automated microfluidic cell analysis platform showed that the multifunctional nanobiointerface can discriminate among T helper and cytotoxic T cells, and thymocytes. Additionally, on-chip cell capture under flow conditions using a high affinity CD 3 selective nanobiointerface preferentially isolated cells with strong surface marker expression. This means that our dynamic microfluidic cell purification method allows the enrichment of 773 CD 8 positive cytotoxic T cells out of a total blood cell population of 7728 PBMCs, which is an increase in cell enrichment of 8-fold with a purity of 85%.

In vivo detection of membrane protein expression using surface plasmon enhanced fluorescence spectroscopy (SPFS).

Surface plasmon enhanced fluorescence spectroscopy (SPFS) was applied for the detection of expression and functional incorporation of integral membrane proteins into plasma membranes of living cells in real time. A vesicular stomatitis virus (VSV) tagged mutant of photoreceptor bovine rhodopsin was generated for high level expression with the semliki forest virus (SFV) system. Adherent baby hamster kidney (BHK-21) cells were cultivated on fibronectin-coated gold surfaces and infected with genetically engineered virus driving the expression of rhodopsin. Using premixed fluorescently (Alexa Fluor 647) labeled anti-mouse secondary antibody and monoclonal anti-VSV primary antibody, expression of rhodopsin in BHK-21 cells was monitored by SPFS. Fluorescence enhancement by surface plasmons occurs exclusively in the close vicinity of the gold surface. Thus, only the Alexa Fluor 647 labeled antibodies binding to the VSV-tag at rhodopsin molecules exposed on the cell surface experienced fluorescence enhancement, whereas, unbound antibody molecules in the bulk solution were negligibly excited. With this novel technique, we successfully recorded an increase of fluorescence with proceeding rhodopsin expression. Thus, we were able to observe the incorporation of heterologously expressed rhodopsin in the plasma membrane of living cells in real time using a relatively simple and rapid method. We confirmed our results by comparison with conventional wide field fluorescence microscopy.

Surface plasmon field-enhanced fluorescence spectroscopy in PCR product analysis by peptide nucleic acid probes.

Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) was recently developed for PCR product analysis, which allowed for real-time monitoring of hybridization processes and for the detection of trace amounts of PCR products, with a detection limit of 100 fmol on the peptide nucleic acid (PNA) probe surface, and 500 fmol on the DNA probe surface. By selectively labeling the strands of PCR-amplified DNA, it was shown that the heat denaturation process in combination with the application of low-salt condition substantially reduced the interference from the antisense strands and thus simplified the surface hybridization. Furthermore, SPFS was demonstrated to be capable of quantitatively discriminating the difference induced by single nucleotide substitution, even within one minute of contact time.

Traceability of fluorescent engineered nanomaterials and their fate in complex liquid waste matrices.

The number of products containing engineered nanomaterials (ENMs) has increased due to their high industrial relevance as well as their use in diverse consumer products. At the end of their life cycle ENMs might be released to the environment and therefore concerns arise regarding their environmental impact. In order to track their fate upon disposal, it is crucial to establish methods to trace ENMs in complex environmental samples and to differentiate them from naturally-occurring nanoparticles. The goal of this study was to distinctively trace ENMs by (non-invasive) detection methods. For this, fluorescent ENMs, namely quantum dots (QDs), were distinctively traced in complex aqueous matrices, and were still detectable after a period of two months using fluorescence spectroscopy. In particular, two water-dispersible QD-species, namely CdTe/CdS QDs with N-acetyl-l-cysteine as capping agent (NAC-QDs) and surfactant-stabilized CdSe/ZnS QDs (Brij(®)58-QDs), were synthesized to examine their environmental fate during disposal as well as their potential interaction with naturally-occurring substances present in landfill leachates. When QDs were spiked into a leachate from an old landfill site, alteration processes, such as sorption, aggregation, agglomeration, and interactions with dissolved organic carbon (DOC), led to modifications of the optical properties of QDs. The spectral signatures of NAC-QDs deteriorated depending on residence time and storage temperature, while Brij(®)58-QDs retained their photoluminescence fingerprints, indicating their high colloidal stability. The observed change in photoluminescence intensity was mainly caused by DOC-interaction and association with complexing agents, such as fulvic or humic acids, typically present in mature landfill leachates. For both QD-species, the results also indicated that pH of the leachate had no significant impact on their optical properties. As a result, the unique spectroscopic fingerprints of QDs, specifically surfactant-stabilized QDs, allowed distinctive tracing in complex aqueous waste matrices in order to study their long-term behavior and ultimate fate.

RNA DNA discrimination by the antitermination protein NusB.

The regulation of ribosomal RNA biosynthesis in Escherichia coli by antitermination requires binding of NusB protein to a dodecamer sequence designated boxA on the nascent RNA. The affinity of NusB protein for boxA RNA exceeds that for the homologous DNA segment by more than three orders of magnitude as shown by surface plasmon resonance measurements. DNA RNA discrimination by NusB protein was shown to involve methyl groups (i.e. discrimination of uracil versus thymine) and 2' hydroxyl groups (i.e. discrimination of ribose versus deoxyribose side-chains) in the RNA motif. Ligand perturbation experiments monitored by 1H15N correlation NMR experiments identified amide NH groups whose chemical shifts are affected selectively by ribose/deoxyribose exchange in the 5' and the central part of the dodecameric boxA motif respectively. The impact of structural modification of the boxA motif on the affinity for NusB protein as observed by 1H15N heterocorrelation was analysed by a generic algorithm.

Photocontrol of cell adhesion processes: model studies with cyclic azobenzene-RGD peptides.

A photoresponsive integrin ligand was synthesized by backbone-cyclization of a heptapeptide containing the integrin binding motif Arg-Gly-Asp (RGD) with 4-(aminomethyl)phenylazobenzoic acid (AMPB). Surface plasmon enhanced fluorescence spectroscopy showed that binding of the azobenzene peptide to alpha(v)beta(3) integrin depends on the photoisomeric state of the peptide chromophore. The higher affinity of the trans isomer could be rationalized by comparing the NMR conformations of the cis and trans isomers with the recently solved X-ray structure of a cyclic RGD-pentapeptide bound to integrin.

Current limitations and challenges in nanowaste detection, characterisation and monitoring.

Engineered nanomaterials (ENMs) are already extensively used in diverse consumer products. Along the life cycle of a nano-enabled product, ENMs can be released and subsequently accumulate in the environment. Material flow models also indicate that a variety of ENMs may accumulate in waste streams. Therefore, a new type of waste, so-called nanowaste, is generated when end-of-life ENMs and nano-enabled products are disposed of. In terms of the precautionary principle, environmental monitoring of end-of-life ENMs is crucial to allow assessment of the potential impact of nanowaste on our ecosystem. Trace analysis and quantification of nanoparticulate species is very challenging because of the variety of ENM types that are used in products and low concentrations of nanowaste expected in complex environmental media. In the framework of this paper, challenges in nanowaste characterisation and appropriate analytical techniques which can be applied to nanowaste analysis are summarised. Recent case studies focussing on the characterisation of ENMs in waste streams are discussed. Most studies aim to investigate the fate of nanowaste during incineration, particularly considering aerosol measurements; whereas, detailed studies focusing on the potential release of nanowaste during waste recycling processes are currently not available. In terms of suitable analytical methods, separation techniques coupled to spectrometry-based methods are promising tools to detect nanowaste and determine particle size distribution in liquid waste samples. Standardised leaching protocols can be applied to generate soluble fractions stemming from solid wastes, while micro- and ultrafiltration can be used to enrich nanoparticulate species. Imaging techniques combined with X-ray-based methods are powerful tools for determining particle size, morphology and screening elemental composition. However, quantification of nanowaste is currently hampered due to the problem to differentiate engineered from naturally-occurring nanoparticles. A promising approach to face these challenges in nanowaste characterisation might be the application of nanotracers with unique optical properties, elemental or isotopic fingerprints. At present, there is also a need to develop and standardise analytical protocols regarding nanowaste sampling, separation and quantification. In general, more experimental studies are needed to examine the fate and transport of ENMs in waste streams and to deduce transfer coefficients, respectively to develop reliable material flow models.