Distribution and stability of membrane proteins in lipid membranes on solid supports

Bacteriorhodopsin and the nicotinic acetylcholine receptor were biotinylated and reconstituted in lipidic membranes on silicon supports by fusion with proteoliposomes. The presence and distribution of the proteins were studied by binding with streptavidin. Radio-labelled streptavidin was employed for quantifying the amounts of protein remaining in the supported membranes after storage in buffer. The proteins within the membranes remained bound to the surface for weeks. The biological activity of reconstituted unlabelled receptor upon storage showed stability in membranes formed on silicon supports and a reduced stability when formed onto lipid monolayer covered supports. Atomic force microscopy studies on preparations in liquid showed bilayer structures but also attached, partly fused liposomes and membrane particles. In air, the surface was smoother and contained less of liposomes and more of stacked lipid layers. Preparations labelled with streptavidin conjugated to colloidal gold and imaged in air showed the proteins individually distributed, with no protein-rich patches or protein aggregates.     

Filtration performance of microporous ceramic supports

The use of inorganic membranes in pollution treatment is actually limited by the cost of such membranes. Advantages of inorganic membranes are their chemical, thermal and pH properties. The purpose of this work was the development of microporous ceramic materials based on clay for liquid waste processing. The supports or ceramic filters having various compositions were prepared and thermally treated at 1100 °C. The results show that, at the temperature studied, porosity varied according to the support composition from 12% for the double-layered (ceramic) support to 47% for the activated carbon- filled support with a mean pore diameter between 0.8 and 1.3 μm, respectively. Volumes of 5 l of distilled water were filtered tangentially for 3 h under an applied pressure of 3.5 and 5.5 bar. The retention of tubular supports prepared was tested with molecules of varying size (Evans blue, NaCl and Sacharose). The study of the liquid filtration and flow through these supports showed that the retention rate depends on support composition and pore diameter, and solute molecular weight. The S1 support (mixture of barbotine and 1% (w/w) activated carbon) gave a flux for distilled water of 68 L/m² h while the double-layered support resulted in a flux of 8 L/m² h for the same solution at the pressure of 3.5 bar. At a pressure of 5.5 bar an increase in the distilled water flux through the various supports was observed. It was significant for the S1 support (230 L/m h).     

Atomic Force Microscopy of Biological Membranes

Atomic force microscopy (AFM) is an ideal method to study the surface topography of biological membranes. It allows membranes that are adsorbed to flat solid supports to be raster-scanned in physiological solutions with an atomically sharp tip. Therefore, AFM is capable of observing biological molecular machines at work. In addition, the tip can be tethered to the end of a single membrane protein, and forces acting on the tip upon its retraction indicate barriers that occur during the process of protein unfolding. Here we discuss the fundamental limitations of AFM determined by the properties of cantilevers, present aspects of sample preparation, and review results achieved on reconstituted and native biological membranes.     

Immobilized artificial membrane chromatography: supports composed of membrane lipids

Cell membranes provide an environment for several types of molecular processes and we are attempting to mimic the cell membranes' environment on a chromatography solid support. Chromatography solid supports utilizing lecithin as the bonded phase were synthesized and the HPLC behavior of hydrophilic peptides evaluated. A diC14 lecithin containing a terminal carboxy group on the C2 fatty acid chain was amidated with the surface amines of Nucleosil-300 (7NH2) silica particles. Based on elemental analysis, lecithin was coupled to Nucleosil-300 (7NH2) at a surface density near that of lecithin found in biological membranes and this novel chromatographic support material is denoted as Nucleosil-lecithin, the prototype immobilized artificial membrane. Infrared difference spectra of Nucleosil-lecithin minus Nucleosil-300 (7NH2) clearly showed amide I (1653.1 cm-1) and amide II (1550.9 cm-1) bands, giving direct spectroscopic evidence for the amide linkage. Spectral deconvolution resolved two peaks for the amide I band, and three peaks for the amide II band. This demonstrates lecithin interchain amide hydrogen bonding and/or hydrogen bonds between the lecithin amide link and unreacted silica surface amines. Nucleosil-lecithin as a solid phase mimics membranes and can be used to study the interactions of biomolecules with membranes. Our primary objective is to develop HPLC methods for studying the interaction between cell membranes and peptide sequences found near the interfaces of cell membranes. A frequency distribution of amino acids bracketing approximately 400 transmembrane peptide sequences showed Cys to be the least frequently occurring amino acid at this putative interfacial membrane region. Hydrophilic peptide analogs bearing Cys were used as model compounds to test Nucleosil-lecithin solid supports. Small peptides, six to eight amino acids in length, containing Cys bind approximately 2X tighter to Nucleosil-lecithin compared to identical peptides without the Cys residue. Thus, Cys at the interface of cells may stabilize protein-lipid interactions.     

Influence of the nature of coupling agents on insulin adsorption on supports grafted with sialic acid for high-performance affinity chromatography

Porous silica exhibits excellent mechanical properties for use as a stationary phase for high-performance liquid chromatography. However, negative surface charges make it unusable in its native state. For this reason, silica beads are coated with dextran polymers carrying a calculated amount of diethylaminoethyl groups. Both the minimization of non-specific interactions and the hydrophilic character of such supports allow their functionalization with biospecific ligands and finally their use in high-performance affinity chromatography of biological products. The use of these modified supports in high-performance affinity chromatography requires a better understanding of various characteristics of stationary phases. For this purpose, several techniques were utilized, in particular, size-exclusion chromatography and adsorption of radiolabelled albumin. These methods provided complementary information on the structure of these supports. Coated silica-based supports were functionalized with sialic acid by means of different coupling agents. The affinity of these supports for insulin was determined by the establishment of adsorption isotherms and by high-performance affinity chromatography, to evidence the relationships between structural characteristics of the supports and their separation properties. The study of interactions between these supports and insulin allowed us to show the importance of the coupling method on the performances of supports in affinity chromatography.     

Scanning tunnelling microscopy in biotechnology.

The scanning tunnelling microscope (STM) is capable of atomic resolution of highly conductive materials. Whether biological molecules can be visualized to the same extent remains an open question, but remarkable progress in the past year confirms the possibility of seeing the fine structure of nucleic acids, proteins, membranes and viruses, and provides evidence that their dynamic interactions can be monitored under conditions approximating to those of the native environment.     

Solid supports for combinatorial chemistry

Over the past year, numerous techniques have been used to study the resins commonly utilised in solid-phase synthesis to allow a greater understanding of the chemical nature and the physical properties of the supports. In addition, to overcome some of the drawbacks of existing materials, several new resins and new methods of handling solid supports have been developed. New methodologies have also been introduced to simplify the preparation of solid supports.     

Recent developments in affinity chromatography supports

The development and wide usage of affinity chromatography depends upon the ready availability of support materials and facile methods for attaching ligands and proteins to them. This paper reviews recent developments in affinity chromatographic supports, details some of the underlying technology in the synthesis of microaffinity chromatographic supports and attempts to answer the question ‘Are more affinity chromatographic supports needed?’     

Magnetic supports for immobilized enzymes and bioaffinity adsorbents

Magnetic separation of immobilized enzymes or bioaffinity adsorbents allows their selective recovery from liquors containing other suspended solids, and gives easier handling of large numbers of samples in analysis. Non-porous magnetic supports seem to be more resistant to fouling, diffusional limitation and attrition than conventional porous supports. A variety of magnetic powders and linkage methods have been used in the preparation of supports, though it is not clear how many of these have the required properties for potential applications (including linkage stability, particle size and magnetic properties). Non-porous magnetic supports are attractive for use in liquors containing fouling materials or suspended solids, either for bioaffinity adsorbents or for immobilized enzymes acting on small molecular substrates. Magnetic supports also offer considerable advantages in analysis based on bioaffinity interactions, such as immunoassay.     

A novel synthetic peptide polymer with cyclic RGD motifs supports serum-free attachment of anchorage-dependent cells

Cell adhesivity is a basic biological principle, which provides mechanisms for construction of multicellular organisms, tissue genesis, migration and individual cell survival. In vivo, the cell adhesive environment is provided by extracellular matrix molecules, neighboring cell surfaces and soluble factors delivered either by tissue cells or by blood circulation. The exact molecular composition of the microenvironment of a cell is not properly understood. The nondefined molecular composition of "native" adhesive components hinders their application when defined culture conditions are necessary, as, for an example, growing human cells for further clinical application. Applying large, substrate-coating molecules as backbones for carrying specific adhesive peptide motifs provides a relatively cheap, reproducible, and chemically defined group of synthetic adhesion molecules. Here, we report on the design, synthesis, and testing of a novel cyclic RGD-containing coating material, which promotes initial attachment, spreading, survival, and proliferation of a number of different cell types. The potent adhesive polypeptide-brush, composed of poly[Lys(DL-Ala(m))] branched chain polypeptide (AK) and multiple copies of cyclic(arginyl-glycyl-aspartyl-D-phenylalanyl-cysteine) pentapeptide prevents anoikis and supports cell attachment in the absence of serum or other biological additives. The defined conditions for cell maintenance make this material a promising candidate for coating artificial cell substrates even for therapeutic applications.     

Trout gill cells in primary culture on solid and permeable supports

Trout gill cells in primary culture on solid and permeable supports were compared. Cultures were carried out by directly seeding cells on each support after gill dissociation. Most of the cell types present in culture were similar, regardless of culture support (pavement cells, mucous cells (3–4%), but no mitochondria-rich cells). However, insertion of mucous cells in cultured epithelium on permeable support presented a morphology more similar to gills in situ. Gene expression of ion transporters and hormonal receptors indicated similar mRNA levels in both systems. Cortisol inhibited cell proliferation on both supports and maintained or increased the total cell number on solid and permeable membranes, respectively. This inhibition of mitosis associated with an increase or maintenance of total gill cells suggests that cortisol reduced cell degeneration. In the presence of cortisol, transepithelial resistance of cultured gill cells on permeable membranes was increased and maintained for a longer time in culture. In conclusion, gill cells in primary culture on permeable support present: (i) a morphology more similar to epithelium in situ; and (ii) specific responses to cortisol treatment. New findings and differences with previous studies on primary cultures of trout gill cells on permeable membrane are discussed.     

Trout gill cells in primary culture on solid and permeable supports.

Trout gill cells in primary culture on solid and permeable supports were compared. Cultures were carried out by directly seeding cells on each support after gill dissociation. Most of the cell types present in culture were similar, regardless of culture support (pavement cells, mucous cells (3-4%), but no mitochondria-rich cells). However, insertion of mucous cells in cultured epithelium on permeable support presented a morphology more similar to gills in situ. Gene expression of ion transporters and hormonal receptors indicated similar mRNA levels in both systems. Cortisol inhibited cell proliferation on both supports and maintained or increased the total cell number on solid and permeable membranes, respectively. This inhibition of mitosis associated with an increase or maintenance of total gill cells suggests that cortisol reduced cell degeneration. In the presence of cortisol, transepithelial resistance of cultured gill cells on permeable membranes was increased and maintained for a longer time in culture. In conclusion, gill cells in primary culture on permeable support present: (i) a morphology more similar to epithelium in situ; and (ii) specific responses to cortisol treatment. New findings and differences with previous studies on primary cultures of trout gill cells on permeable membrane are discussed.     

Effect of negative hydro-aero ions on the structure and functional properties of mitochondria

Studies were carried out of the physical mechanism of biological effect of natural stimulant of the living activity--light negative hydroaeroions. The latter are a hydroxyl separated from water ions and designated here as electrically noncompensated hydroxyl OH-. Unique effect of OH-, i.e. fast restoration and prevention of fine damage of native mitochondria (NM) at storage was revealed on mitochondria preparations by specific procedure, providing preservation of the ability of self-organization--formation of aggregated and denser packing--native mitochondria. This effect of OH- is conditioned by increased formation of native mitochondria aggregates and is concerned with better ADP phosphorylation and Ca2- release. The effect of the air current OH- is produced by water action presaturated with OH-. OH- induces in water cooperative structure formation preserved for many days, which is indicated by an increase of its heat capacity and decrease of surface tension. The principal factor in OH- effect both on mitochondria and water seems to be a decrease of local positive changes. It is of more advantage in NM water suspensions to order (structurate) NM with disordering water, which explains formation of NM stable aggregates. OH- effect supports the main function of the organism struggling with proton excess which destroys biological self-organization. Strengthened structural self-organization under the effect of OH- found in NM seems to be of universal importance for biological macromolecules and membranes and is a primary effect of OH- on the living objects.     

Nonporous magnetic materials as enzyme supports: studies with immobilized chymotrypsin.

Chymotrypsin has been immobilized to several nonporous magnetic materials. Nickel particles were considered to be most suitable as immobilized enzyme supports. Chymotrypsin immobilized to nonporous magnetic supports was not fouled significantly by either whole milk or clarified yeast homogenate. AE-cellulose-chymotrypsin was rapidly fouled by both these materials and chymotrypsin immobilized to acrylic-based ion exchangers was slowly fouled. Immobilized enzyme activity was found to be inversely proportional to particle diameter for nonporous rock magnetic particles. Immobilization by adsorption and then glutaraldehyde crosslinking was used to produce controlled amounts of chymotrypsin on the particles. Esterolytic activity increased with enzyme loading but caseinolytic activity did not increase. Chymotrypsin is inhibited by metal ions from the magnetic supports. It is partially protected by use of a preliminary protein coating and may be reactivated by incubation with EDTA or BSA.     

Highly robust lipid membranes on crystalline S-layer supports investigated by electrochemical impedance spectroscopy

In the present work, S-layer supported lipid membranes formed by a modified Langmuir-Blodgett technique were investigated by electrochemical impedance spectroscopy (EIS). Basically two intermediate hydrophilic supports for phospholipid- (DPhyPC) and bipolar tetraetherlipid- (MPL from Thermoplasma acidophilum) membranes have been applied: first, the S-layer protein SbpA isolated from Bacillus sphaericus CCM 2177 recrystallized onto a gold electrode; and second, as a reference support, an S-layer ultrafiltration membrane (SUM), which consists of a microfiltration membrane (MFM) with deposited S-layer carrying cell wall fragments. The electrochemical properties and the stability of DPhyPC and MPL membranes were found to depend on the used support. The specific capacitances were 0.53 and 0.69 microF/cm(2) for DPhyPC bilayers and 0.75 and 0.77 microF/cm(2) for MPL monolayers resting on SbpA and SUM, respectively. Membrane resistances of up to 80 mega Ohm cm(2) were observed for DPhyPC bilayers on SbpA. In addition, membranes supported by SbpA exhibited a remarkable long-term robustness of up to 2 days. The membrane functionality could be demonstrated by reconstitution of membrane-active peptides such as valinomycin and alamethicin. The present results recommend S-layer-supported lipid membranes as promising structures for membrane protein-based biosensor technology.     

Non-porous magnetic supports for cell immobilization

A new method for covering magnetic particles with a stable non-porous layer of a material like zeolite or activated carbon was used for the preparation of support materials with good properties for the immobilization of yeast Saccharomyces cerevisiae cells. The immobilized cells can be used in batch and continuous alcoholic fermentation. A productivity of 35.6 g ethanol/l · h was reached. The adsorption isotherms of the immobilized yeast cells were determined. Yeast cell immobilization on non-porous magnetic supports obeyed the Langmuir isotherm equation. Satisfactory results were obtained also from repeated batch fermentations with fixed cells on supports additionally treated with glutaraldehyde or by simple adsorption.     

Immobilization of biocatalysts with poly(vinyl alcohol) supports.

Two polymer materials, poly(vinyl alcohol) (PVA) superfine fibers and photocrosslinkable PVA bearing styrylpyridinium groups, have been developed to immobilize biocatalysts. The former has a large surface consisting of relatively large-size pores and the fibers can immobilize a large amount of biocatalyst on their surface by ionic interaction. The latter entraps many kinds of biocatalysts by cyclodimerization caused by visible light irradiation. The biocatalysts on/in these supports maintain high activity and thermal stability. These materials can easily be formed into various shapes suitable for various applications. A new bioreactor system was constructed for evaluating a variety of biocatalysts and supports.     

Highly robust lipid membranes on crystalline S-layer supports investigated by electrochemical impedance spectroscopy

In the present work, S-layer supported lipid membranes formed by a modified Langmuir-Blodgett technique were investigated by electrochemical impedance spectroscopy (EIS). Basically two intermediate hydrophilic supports for phospholipid- (DPhyPC) and bipolar tetraetherlipid- (MPL from Thermoplasma acidophilum) membranes have been applied: First, the S-layer protein SbpA isolated from Bacillus sphaericus CCM 2177 recrystallized onto a gold electrode; and second, as a reference support, an S-layer ultrafiltration membrane (SUM), which consists of a microfiltration membrane (MFM) with deposited S-layer carrying cell wall fragments. The electrochemical properties and the stability of DPhyPC and MPL membranes were found to depend on the used support. The specific capacitances were 0.53 and 0.69 μF/cm² for DPhyPC bilayers and 0.75 and 0.77 μF/cm² for MPL monolayers resting on SbpA and SUM, respectively. Membrane resistances of up to 80 MΩ cm² were observed for DPhyPC bilayers on SbpA. In addition, membranes supported by SbpA exhibited a remarkable long-term robustness of up to 2 days. The membrane functionality could be demonstrated by reconstitution of membrane-active peptides such as valinomycin and alamethicin. The present results recommend S-layer-supported lipid membranes as promising structures for membrane protein-based biosensor technology.     

Cyclotide-membrane interactions: defining factors of membrane binding, depletion and disruption

The cyclotide family of plant-derived peptides is defined by a cyclic backbone and three disulfide bonds locked into a cyclic cystine knot. They display a diverse range of biological activities, many of which have been linked to an ability to target biological membranes. In the current work, we show that membrane binding and disrupting properties of prototypic cyclotides are dependent on lipid composition, using neutral (zwitterionic) membranes with or without cholesterol and/or anionic lipids. Cycloviolacin O2 (cyO2) caused potent membrane disruption, and showed selectivity towards anionic membranes, whereas kalata B1 and kalata B2 cyclotides were significantly less lytic towards all tested model membranes. To investigate the role of the charged amino acids of cyO2 in the membrane selectivity, these were neutralized using chemical modifications. In contrast to previous studies on the cytotoxic and antimicrobial effects of these derivatives, the Glu6 methyl ester of cyO2 was more potent than the native peptide. However, using membranes of Escherichia coli lipids gave the opposite result: the activity of the native peptide increased 50-fold. By using a combination of ellipsometry and LC-MS, we demonstrated that this unusual membrane specificity is due to native cyO2 extracting preferentially phosphatidylethanolamine-lipids from the membrane, i.e., PE-C16:0/cyC17:0 and PE-C16:0/C18:1.     

Tryptophan supports interaction of transmembrane helices

Interactions of transmembrane helices play an important role in folding and oligomerization of integral membrane proteins. The interfacial residues of these helices frequently correspond to heptad repeat motifs. In order to uncover novel mechanisms underlying these interactions, we randomised a heptad repeat pattern with a complete set of amino acids. Those sequences that were capable of high-affinity self-interaction upon integration into bacterial inner membranes were selected by means of the POSSYCCAT system. A comparison between selected and non-selected sequences reveals that high-affinity sequences were strongly enriched in tryptophan residues that accumulated at specific positions of the heptad motif. Mutation of Trp in selected clones significantly reduced self-interaction of the transmembrane segments without affecting their efficiency of membrane integration. Conversely, grafting Trp onto artificial transmembrane segments strongly enhanced their interaction. We conclude that tryptophan supports interaction of transmembrane segments.