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Claudia Steinem

Publications and source records attributed to Claudia Steinem.

At least 19 recordsLinked to original sources
PubMed2006

Cooperative adsorption of ezrin on PIP2-containing membranes.

By means of the quartz crystal microbalance (QCM) and scanning force microscopy (SFM), the adsorption of ezrin, a member of the ezrin/radixin/moesin protein family, on l-alpha-phosphatidylinositol-4,5-bisphosphate (PIP(2)) containing solid-supported membranes was investigated. An increase in the PIP(2) content in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes resulted in an increased amount of bound ezrin strongly supporting the crucial role of PIP(2) for ezrin recruitment to membranes. No ezrin adsorption to membranes composed of pure POPC was detected. To characterize the binding process in more detail, the kinetics and reversibility of ezrin adsorption were investigated by the QCM technique, showing that the protein remains partly bound after rinsing with pure buffer, which we suspected to be a result of lateral interactions between the proteins. SFM images revealed the formation of two-dimensional ezrin clusters on PIP(2)-doped POPC membranes. Time-elapsed SFM images show that the growth of protein domains occurs from a few nucleation sites. The QCM data in conjunction with the results obtained by SFM led us to propose that the binding process of ezrin occurs in a positive cooperative manner. When lateral interactions of the proteins on the membrane were taken into account, we were able to simulate the kinetics obtained from time-resolved QCM readouts by employing a model developed by Minton. On the basis of the kinetic analysis, we were also able to reconstruct the adsorption isotherm.

Adsorption↗
PubMed2006

Measuring cell adhesion on RGD-modified, self-assembled PEG monolayers using the quartz crystal microbalance technique.

In this study, the suitability of a flow-through quartz crystal microbalance system for the detection of the adhesion of rMSCs and 3T3-L1 fibroblasts on different surfaces is demonstrated. Frequency shifts for rMSCs of -6.7 mHz x cell(-1) and -2.0 mHz x cell(-1) for 3T3-L1 cells could be detected on non-modified gold sensors, revealing that the frequency shift per cell is comparable to that of a static setup. Modifying the sensor surface with SAMs of thioalkylated omega-amine-terminated PEG derivatives led to cell-adhesion-resistant surfaces. Total frequency shifts of only -20 +/- 7 Hz showed that protein adsorption was also significantly reduced. Attaching 35 pmol x mm(-2) of the GRGDS cell adhesion motif to the SAMs induced specific cell adhesion due to RGD-integrin interactions; the resonance frequency dropped by 3.4 mHz x cell(-1). Furthermore, the kinetics of cell detachment could be determined. The corresponding processes were completed after 10 min for trypsin, and not before 90 min with GRGDS. Moreover, the detectability of cell adhesion was shown to increase after the addition of manganese cations. The total decrease in the resonance frequency was almost 80 Hz in the presence of Mn(2+) (6.4 mHz x cell(-1)). [image: see text] Staining the cytoskeleton of the rMSCs shows that the GRGDS-modified surfaces are almost completely covered with well-spread cells.

Animals↗
PubMed2006

Influence of alpha-hydroxylation of glycolipids on domain formation in lipid monolayers.

By means of fluorescence and scanning force microscopy (SFM), we investigated the phase behavior of lipid monolayers composed of a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, sphingomyelin, and cholesterol (5/2/3) with either alpha-hydroxylated or nonhydroxylated galactocerebroside. Fluorescence images of lipid monolayers at the air-water interface demonstrate that, independent of the lipid mixture, phase separation occurs at low surface pressure up to 4-6 mN m(-1), while an almost homogeneous phase is observed at larger surface pressures. However, by means of SFM of lipid monolayers transferred by the Langmuir-Blodgett technique at around 30 mN m(-1), nanometer-sized domains became discernible in those lipid mixtures that contained galactocerebroside, while, in that without a glycolipid, no such domain formation was visible. Moreover, the alpha-hydroxy group of the galactocerebroside alters the size and the total area of the domains significantly.

Glycolipids↗
PubMed2006

Channel activity of OmpF monitored in nano-BLMs.

Free-standing lipid bilayer membranes can be formed on small apertures (60 nm diameter) on highly ordered porous alumina substrates. The formation process of the membranes on a 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol submonolayer was followed by impedance spectroscopy. After lipid bilayers had thinned, the reconstitution and ionic conducting properties of the outer membrane protein OmpF of E. coli were monitored using single-channel recordings. The characteristic conductance states of the three monomers, fast kinetics, and subconductance states were observed. Blockade of the ion flow as a result of interaction of the antibiotic ampicillin with the protein was verified, indicating the full functionality of the protein channel in nanometer-scale bilayer membranes.

Aluminum Oxide↗
PubMed2006

Mechanical properties of pore-spanning lipid bilayers probed by atomic force microscopy.

We measure the elastic response of a free-standing lipid membrane to a local indentation by using an atomic force microscope. Starting point is a planar gold-coated alumina substrate with a chemisorbed 3-mercaptopropionic acid monolayer displaying circular pores of very well defined and tunable size, over which bilayers composed of N,N,-dimethyl-N,N,-dioctadecylammonium bromide or 1,2-dioleoyl-3-trimethylammonium-propane chloride were spread. Centrally indenting these "nanodrums" with an atomic force microscope tip yields force-indentation curves, which we quantitatively analyze by solving the corresponding shape equations of continuum curvature elasticity. Since the measured response depends in a known way on the system geometry (pore size, tip radius) and on material parameters (bending modulus, lateral tension), this opens the possibility to monitor local elastic properties of lipid membranes in a well-controlled setting.

Computer Simulation↗
PubMed2006

Transport across artificial membranes-an analytical perspective.

Biosensors that make use of transport processes across lipid membranes are very rare even though a stimulus, the binding of a single analyte molecule, can enhance the sensor response manifold if the analyte leads to the transport of more than one ion or molecule across the membrane. Prerequisite for a proper function of such membrane based biosensors is the formation of lipid bilayers attached to a support that allow for the insertion of membrane peptides and proteins in a functional manner. In this review, the current state of the art technologies to obtain lipid membranes on various supports are described. Solid supported membranes on transparent and electrically conducting surfaces, lipid bilayers on micromachined apertures and on porous materials are discussed. The focus lies on the applicability of such membranes for the investigation of transport phenomena across lipid bilayers facilitated by membrane embedded peptides, channel proteins and transporters. Carriers and channel forming peptides, which are easy to handle and rather robust, are used frequently to build up membrane based biosensors. However, channel forming proteins and transporters are more difficult to insert functionally and thus, there are yet only few examples that demonstrate the applicability of such systems as biosensor devices.

Animals↗
PubMed2006

Partially reversible adsorption of annexin A1 on POPC/POPS bilayers investigated by QCM measurements, SFM, and DMC simulations.

The kinetics of annexin A1 binding to solid-supported lipid bilayers consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS; 4:1) has been investigated as a function of the calcium ion concentration in the bulk phase. Quartz crystal microbalance measurements in conjunction with scanning force microscopy, fluorescence microscopy, and computer simulations indicate that at a given Ca2+ concentration annexin A1 adsorbs irreversibly on membrane domains enriched in POPS. By contrast, annexin A1 adsorbs reversibly on the POPC-enriched phase, which is composed of single POPS molecules embedded within a POPC matrix. The overall area occupied by the POPS-enriched phase is controlled by the CaCl2 concentration. Monte Carlo simulations suggest that the area of the POPS-enriched phase increases by a factor of 7 when the Ca2+ concentration is changed from 0.01 to 1 mM.

Adsorption↗
PubMed2005

Phosphatidylserine membrane domain clustering induced by annexin A2/S100A10 heterotetramer.

By means of scanning force and fluorescence microscopy of artificial membranes immobilized on mica surfaces, the lateral organization of the annexin A2/S100A10 heterotetramer (annexin A2t) and its influence on the lateral organization of the lipids within the membrane have been elucidated. Planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) were prepared on atomically flat mica surfaces by the spreading of unilamellar vesicles. Fluorescence images of fluorescently labeled annexin A2t and scanning force microscopy images of nonlabeled protein bound to POPC/POPS bilayers show the formation of micrometer-sized lateral protein domains in the presence of 1 mM CaCl2. By means of scanning force microscopy, not only protein domains became discernible but also small membrane domains, which were attributed to POPS-enriched areas. A depletion of these POPS domains was observed in the vicinity of annexin A2t protein domains. These results indicate that annexin A2t is a peripheral membrane-binding complex capable of inducing lipid segregation.

Annexin A2↗
PubMed2005

Photocurrents generated by bacteriorhodopsin adsorbed on nano-black lipid membranes.

Purple membranes were adsorbed on freestanding lipid bilayers, termed nano-black lipid membranes (nano-BLMs), suspending the pores of porous alumina substrates with average pore diameters of 280 nm. Nano-BLMs were obtained by first coating the upper surface of the highly ordered porous alumina substrates with a thin gold layer followed by chemisorption of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol and subsequent addition of a droplet of 1,2-diphytanoyl-sn-glycero-3-phosphocholine and octadecylamine dissolved in n-decane onto the hydrophobic submonolayer. By means of impedance spectroscopy, the quality of the nano-BLMs was verified. The electrical parameters confirm the formation of single lipid bilayers with high membrane resistances covering the porous matrix. Adsorption of purple membranes on the nano-BLMs was followed by recording the photocurrents generated by bacteriorhodopsin upon continuous light illumination. The membrane system exhibits a very high long-term stability with the advantage that not only transient but also stationary currents are recordable. By adding the proton ionophore carbonyl cyanide-m-chlorophenylhydrazone the conductivity of the nano-BLMs increases, resulting in a higher stationary current, which proves that proton conductance occurs across the nano-BLMs.

Adsorption↗
PubMed2005

Controlling association of vesicle embedded peptides by alteration of the physical state of the lipid matrix.

We report here the reversible association of a designed peptide embedded in a lipid membrane through a stimulus-sensitive trigger that changes the physical state of the bilayer matrix. A peptide designed with the classical 4-3 heptad repeat of coiled coils, equipped with leucine residues at all canonical interface positions, TH1, was rendered membrane soluble by replacement of all exterior residues with randomly selected hydrophobic amino acids. Insertion of TH1 into large unilamellar phosphatidylcholine vesicles was followed by monitoring tryptophan fluorescence. Peptide insertion was observed when the lipids were in the liquid-crystalline state [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)] but not when they were in the crystalline phase [1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)]. Formation of a trimeric alpha-helical bundle in lipid bilayers was followed by fluorescence resonance energy transfer. Global fit analysis revealed a monomer--trimer equilibrium with a dissociation constant of around 10(-5) [corrected] MF(2). A lipid mixture composed of DPPC and POPC exhibiting a phase transition at 34 degrees C between a crystalline/liquid-crystalline coexistence region and a completely miscible liquid-crystalline phase was used to control the formation of the trimeric peptide bundle. TH1 is phase excluded in crystalline DPPC domains below 34 degrees C, leading to a larger number of trimers. However, when the DPPC domains are dispersed at temperatures above 34 degrees C, the number of trimers is reduced.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2005

Impedance and QCM analysis of the protein resistance of self-assembled PEGylated alkanethiol layers on gold.

In this study, we describe the formation and characterization of self-assembled layers of undecanthiol with poly(ethylene glycol) (PEG) moieties of 350Da (PEG350-undecanthiol) and 2000Da (PEG2000-undecanthiol) on gold surfaces. The functionalized surfaces were investigated by means of electrical impedance spectroscopy, which allows calculating the surface coverage from the obtained capacitance values of the formed layers. For both, PEG350-undecanthiol and PEG2000-undecanthiol layers, a surface coverage well above 90% was obtained. Protein resistance of those layers was investigated using the quartz crystal microbalance (QCM) technique, which enables one to monitor protein adsorption label free and in a time resolved manner. The change in resonance frequency of the quartz plate was monitored upon addition of fetal bovine serum indicating that PEG-functionalized surfaces are partly protein resistant compared to hydroxyundecanthiol- and non-functionalized gold surfaces. From QCM experiments, where only a single protein component was added to the PEG-functionalized gold surface, we conclude that the surfaces are fully resistant against serum albumins, while the main protein components that adsorb are globulins. A kinetic analysis reveals that PEG modified gold surfaces do not only significantly diminish the overall amount of bound protein but also significantly slows down the adsorption process.

Adsorption↗
PubMed2005

Label-free detection of protein-ligand interactions by the quartz crystal microbalance.

In recent years the quartz crystal microbalance (QCM) has been accepted as a powerful technique to monitor adsorption processes at interfaces in different chemical and biological research areas. In the last decade, the investigation of adsorption of biomolecules on functionalized surfaces turned out to be one of the paramount applications of the QCM comprising the interaction of nucleic acids, specific molecular recognition of protein-receptor couples, and antigen-antibody reactions realized in immunosensors. The advantage of the QCM technique is that it allows for a label free detection of molecules. This is a result of the fact that the frequency response of the quartz resonator is proportional to the increase in thickness of the adsorbed layer. However, in recent years it became more and more evident that quartz resonators used in fluids are more than mere mass or thickness sensors. The sensor response is also influenced by viscoelastic properties of the adhered biomaterial, surface charges of adsorbed molecules and surface roughness. These phenomena have been used to get new insights in the adhesion process of living cells and to understand their response to pharmacological substances by determining morphological changes of the cells. In this chapter we describe a protocol to explore the kinetics and thermodynamics of specific interactions of different proteins such as lectins and annexins with their ligands using receptor bearing solid supported lipid bilayers.

Animals↗
PubMed2004

Channel activity of a viral transmembrane peptide in micro-BLMs: Vpu(1-32) from HIV-1.

We report for the first time on pore-suspending lipid bilayers, which we call micro-black lipid membranes (micro-BLMs), based on a highly ordered macroporous silicon array. Micro-BLMs were established by first functionalizing the backside porous silicon surface with gold and then chemisorbing 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol followed by spreading 1,2-diphytanoyl-sn-glycero-3-phosphocholine dissolved in n-decane. Impedance spectroscopy revealed the formation of single lipid bilayers confirmed by a mean specific capacitance of 0.6 +/- 0.2 microF/cm2. Membrane resistances were in the G omega-regime and beyond. The potential of the system for single channel recordings was demonstrated by inserting the transmembrane domain of the HIV-1 accessory peptide Vpu(1-32), which forms helix bundles with characteristic opening states. We elucidated different amilorides as potential drugs to inhibit channel activity of Vpu.

Amiloride↗
PubMed2004

Scrutiny of annexin A1 mediated membrane-membrane interaction by means of a thickness shear mode resonator and computer simulations.

The dissipational quartz crystal microbalance (D-QCM) technology was applied to monitor the adsorption of vesicles to membrane-bound annexin A1 by simultaneously reading out the shifts in resonance frequency and dissipation. Solid-supported membranes (SSMs) composed of a chemisorbed octanethiol monolayer and a physisorbed 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine monolayer were immobilized on the gold electrode of a 5 MHz quartz plate. Adsorption and desorption of annexin A1 to the SSM was followed by means of the QCM technique. After nonbound annexin A1 was removed from solution, the second membrane binding was monitored by the D-QCM technique, which allowed distinguishing between adsorbed and ruptured vesicles. The results show that vesicles stay always intact independent of the amount of bound annexin and the vesicle and buffer composition. It was shown that the vesicle adsorption process to membrane-bound annexin A1 is fully irreversible and is mediated by two-dimensional annexin clusters. For N-terminally truncated annexin A1, a decrease in the amount of bound vesicles was observed, which might be the result of fewer binding sites presented by the annexin A1 core. Supported by computer simulations, the results demonstrate that the vesicle adsorption process is electrostatically driven, but compared to those of sole electrostatic binding, the rate constants of adsorption are 1-2 orders of magnitude smaller, indicating the presence of a potential barrier.

Adsorption↗
PubMed2004

Impedance analysis and single-channel recordings on nano-black lipid membranes based on porous alumina.

Ordered porous alumina substrates with pore diameters of 55 and 280 nm, respectively, were produced and utilized as a support to prepare membranes suspending the pores of the material. Highly ordered porous alumina was prepared by an anodization process followed by dissolution of the remaining aluminum and alumina at the backside of the pores. The dissolution process of Al(2)O(3) at the backside of the pores was monitored by electrical impedance spectroscopy ensuring the desired sieve-like structure of the porous alumina. One side of the porous material with an area of 7 mm(2) was coated with a thin gold layer followed by chemisorption of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol. The hydrophobic monolayer on top of the upper surface was a prerequisite for the formation of suspending membranes, termed nano-black lipid membranes (nano-BLMs). The formation process, and long-term and mechanical stability of the nano-BLMs were followed by electrical impedance spectroscopy indicating the formation of lipid bilayers with typical specific membrane capacitances of (0.65 +/- 0.2) micro F/cm(2) and membrane resistances of up to 1.6 x 10(8) Omega cm(2). These high membrane resistances allowed for single-channel recordings. Gramicidin as well as alamethicin was successfully inserted into the nano-BLMs exhibiting characteristic conductance states.

Adsorption↗