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 t…
No Label Required: Protein Binding at Membrane Interfaces Visualized through Colloid Phase Transitions
Quantification of the Raf-C1 Interaction With Solid-Supported Bilayers
By use of the quartz crystal microbalance technique, the interaction of the Raf-Ras binding domain (RafRBD) and the cysteine-rich domain Raf-C1 with lipids was quantified by using solid-supported bilayers immobilized on gold electrodes deposited on 5 MHz quartz plates. Solid-supported lipid bilayers were composed of an initial octanethiol monolayer chemisorbed on gold and a physisorbed phospholipid monolayer varying in its lipid composition as the outermost layer. The integrity of bilayer preparation was monitored by impedance spectroscopy. For binding experiments, a protein construct comprising the RafRBD and Raf-C1 linked to the maltose binding protein and a His tag, termed MBP-Raf-C1, wa…
Quartz Crystal Microbalance for Bioanalytical Applications
The quartz crystal microbalance (QCM) was first introduced as a mass sensor in gas phase and in vacuum. Since oscillator circuits capable of exciting shear vibrations of quartz resonators under liquid load have been developed, the QCM became accepted as a new powerful technique to monitor adsorption processes at solid/liquid interfaces in chemical and biological research rendering the method an attractive low-cost alternative for bioanalytic applications. In the last decade, adsorption of biomolecules on functionalized surfaces turned out to be one of the paramount applications of piezoelectric transducers comprising the interaction of DNA and RNA with complementary strands, specific recogn…
Biochemical Applications of Solid Supported Membranes on Gold Surfaces: Quartz Crystal Microbalance and Impedance Analysis
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 propor…
Viscoelasticity of pore-spanning polymer membranes derived from giant polymersomes
We show how the viscoelastic properties of membranes formed from poly(butadiene)-block-poly(ethylene oxide) (PB130-b-PEO66) block copolymers can be locally accessed by atomic force microscopy. Polymer membranes are spread on microstructured porous silicon substrates from PB130-b-PEO66 vesicles by decreasing the osmotic pressure of the solution. Local viscoelastic properties of the pore-spanning polymer membranes were obtained from site-specific indentation experiments. Elastic moduli of these membranes were in the order of few MPa, while the elastic moduli of cross-linked membranes considerably increased to few GPa. Furthermore, the energy dissipation and velocity dependence of the hysteres…
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 are…
Formation of irreversibly bound annexin A1 protein domains on POPC/POPS solid supported membranes
AbstractThe specific interaction of annexin A1 with phospholipid bilayers is scrutinized by means of scanning force and fluorescence microscopy, quartz crystal microbalance, ellipsometry, and modeled by dynamic Monte Carlo simulations. It was found that POPC/POPS bilayers exhibit phase separation in POPC- and POPS-enriched domains as a function of Ca2+ concentration. Annexin A1 interacts with POPC/POPS bilayers by forming irreversibly bound protein domains with monolayer thickness on POPS-enriched nanodomains, while the attachment of proteins to the POPC-enriched regions is fully reversible. A thorough kinetic analysis of the process reveals that both, the binding constant of annexin A1 at …
Scanning Force Microscopy of Artificial Membranes
Visualization of biological membranes by scanning force microscopy (SFM) has tremendously improved the current understanding of protein ‐ lipid interactions under physiological conditions. SFM is the only tool to directly image processes on surfaces in aqueous solution at molecular resolution. Besides being a supportive means to confirm results on lipid phases and domains obtained from fluorescence spectroscopy, calorimetry, and X-ray crystallography, SFM has contributed distinct aspects on the formation of 2D crystals of various membrane-confined proteins and morphological changes of membranes due to the interaction of peptides and proteins. This review will focus on recent results in SFM …
Biosensors, Porous Silicon
Biosensors consist of a biologically active layer that responding to an analyte in solution and a powerful transducer that transforms and amplifies the reaction into a measurable signal. Biosensors can constantly measure the presence, absence, or concentration of specific organic or inorganic substances in short response time and ultimately at low cost. They are used commercially in health care, biotechnological process control, agriculture, veterinary medicine, defense, and environmental pollution monitoring. A common requirement of all of these applications is on-site chemical information—preferably in real time—on some dynamic or rapidly evolving process. Most biosensors are based on mol…
Mechanical Properties of Pore-Spanning Lipid Bilayers Probed by Atomic Force Microscopy
AbstractWe 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 respon…
Adhesion of liposomes: a quartz crystal microbalance study
Three different systems are presented, exploring the adhesion of liposomes mediated by electrostatic and lipid–protein interactions as well as molecular recognition of ligand receptor pairs. Liposomes are frequently used to gain insight into the complicated processes involving adhesion and subsequent events such as fusion and fission mainly triggered by specific proteins. We combined liposome technology with the quartz crystal microbalance (QCM) technique as a powerful tool to study the hidden interface between the membrane and functionalized surface. Electrostatic attraction and molecular recognition were employed to bind liposomes to the functionalized quartz crystal. The QCM was used to …
Energy landscapes of ligand-receptor couples probed by dynamic force spectroscopy.
Playing a dominant role in many biochemical processes are the dynamic properties of molecular linkages; examples include cell adhesion, enzyme-catalyzed reactions, and molecular recognition by antibodies. Dynamic force spectroscopy, namely separating molecular bonds under external force ramps has rapidly become a powerful tool to study the rugged energy landscape of noncovalent ligand-receptor bonds. The picture shows a surface and tip-bound pair being pulled apart and the derived potential energy diagram.
ChemInform Abstract: Scanning Force Microscopy of Artificial Membranes
Visualization of biological membranes by scanning force microscopy (SFM) has tremendously improved the current understanding of protein ‐ lipid interactions under physiological conditions. SFM is the only tool to directly image processes on surfaces in aqueous solution at molecular resolution. Besides being a supportive means to confirm results on lipid phases and domains obtained from fluorescence spectroscopy, calorimetry, and X-ray crystallography, SFM has contributed distinct aspects on the formation of 2D crystals of various membrane-confined proteins and morphological changes of membranes due to the interaction of peptides and proteins. This review will focus on recent results in SFM …
Modellmembranen auf Oberflächen. Verankert und doch mobil
Membranmodelle, die auf Festkorperoberflachen verankert sind, ermoglichen es, elektrische Verfahren sowie oberflachenanalytische und abbildende Methoden einzusetzen. So kann mit Membranen auf Oberflachen die laterale Organisation von Lipiden und Proteinen bis in den Nanometerbereich hinein visualisiert werden. Gleichzeitig sind diese Membranen sehr stabil und damit attraktiv fur verschiedene Bereiche der Biosensorik. Durch eine individuelle Adressierung kleinster mikrometergroser Membranareale mit verschiedenen Lipidkompositionen ist es moglich geworden, die Bindung von Proteinen an funktionale Membranen auf kleinstem Raum zu quantifizieren und zu parallelisieren. Fur die Untersuchung von T…
Biochemical Applications of Solid Supported Membranes on Gold Surfaces: Quartz Crystal Microbalance and Impedance Analysis
Since their inception in 1985 by Tamm and McConnell [1], solid supported lipid bilayers have been widely used as model systems for cellular membranes [2]. They have been applied in fundamental and applied studies of lipid assemblies on surfaces, to study the structure of membranes and membrane dynamics, lipidreceptor-interactions and electrochemical properties of membranes [3-5]. Several attempts have been made to apply solid supported membranes (SSM) in biosensor devices [6]. Planar lipid membranes can be formed on various surfaces, i.e. glass, silicon, mica or metal surfaces such as platinum or gold. Surface attachment of the lipids is typically achieved following two different strategies…
Phase Transition of Individually Addressable Microstructured Membranes Visualized by Imaging Ellipsometry
The phase transition of individually addressable microstructured lipid bilayers was investigated by means of imaging ellipsometry. Microstructured bilayers were created on silicon substrates by micromolding in capillaries, and the thermotropic behavior of various saturated diacyl phosphatidylcholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipentadecoyl-sn-glycero-3-phosphocholine, and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)) bilayers as well as DMPC/cholesterol membranes was determined by measuring the area expansion and thickness of the bilayer as a function of temperature. We found an increase in the main phase transition temperature T(M) of 2-6 degrees C and a substa…