Atomic-force-microscopy imaging and molecular-recognition-force microscopy of recrystallized heterotetramers comprising an S-layer-streptavidin fusion protein.
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Crystallization↗
Biomedical subjects
Ferry Kienberger
Publications and source records attributed to Ferry Kienberger.
Molecular recognition imaging and force spectroscopy of single biomolecules.
In recent years, considerable attention has focused on biological applications of the atomic force microscope (AFM), in particular on high-resolution imaging of individual biological molecules and on the measurement of molecular forces under near-physiological conditions. The detection of intermolecular forces in the piconewton range has paved the way to investigate details on structural parameters of the binding pockets and the energy landscapes of many biomolecular interactions. The capability of AFM to resolve nanometer-sized details, together with its force detection sensitivity, led to the development of molecular recognition imaging. By a combination of topographical imaging with force measurements, receptor sites are localized with nanometer accuracy. Topography and recognition of target molecules are thereby simultaneously mapped. Thus the AFM can identify specific components in a complex biological sample and retain its high resolution in imaging.
Aluminum Silicates↗
Directed assembly of Au nanoparticles onto planar surfaces via multiple hydrogen bonds.
We have developed a new concept to effect nanoparticle binding on surfaces by use of directed, specific molecular interactions. Hamilton-type receptors displaying a binding strength of approximately 10(5) M(-)(1) were covalently fixed onto self-assembled monolayers via Sharpless-type "click" reactions, thus representing an efficient method to control the densities of ligands over a range from low to complete surface coverage. Au nanoparticles covered with the matching barbituric acid receptors bound with high selectivity onto this surface by a self-assembly process mediated by multiple hydrogen bonds. The binding process was investigated with atomic force microscopy. Moderate control of particle density was achieved by controlling the receptor density on the self-assembled monolayer surface. The method opens a general approach to nanoparticle and small object binding onto patterned surfaces.
Journal Article↗
Single molecule studies of antibody-antigen interaction strength versus intra-molecular antigen stability.
We investigated molecular recognition of antibodies to membrane-antigens and extraction of the antigens out of membranes at the single molecule level. Using dynamic force microscopy imaging and enzyme immunoassay, binding of anti-sendai antibodies to sendai-epitopes genetically fused into bacteriorhodopsin molecules from purple membranes were detected under physiological conditions. The antibody/antigen interaction strength of 70-170 pN at loading rates of 2-50 nN/second yielded a barrier width of x = 0.12 nm and a kinetic off-rate (corresponding to the barrier height) of k(off) = 6s(-1), respectively. Bacteriorhodopsin unfolding revealed a characteristic intra-molecular force pattern, in which wild-type and sendai-bacteriorhodopsin molecules were clearly distinguishable in their length distributions, originating from the additional 13 amino acid residues epitope in sendai purple membranes. The inter-molecular antibody/antigen unbinding force was significantly lower than the force required to mechanically extract the binding epitope-containing helix pair out of the membrane and unfold it (126 pN compared to 204 pN at the same loading rate), meeting the expectation that inter-molecular unbinding forces are weaker than intra-molecular unfolding forces responsible for stabilizing native conformations of proteins.
Antibodies↗
Localization of single avidin-biotin interactions using simultaneous topography and molecular recognition imaging.
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Animals↗
Visualization of single receptor molecules bound to human rhinovirus under physiological conditions.
Dynamic force microscopy (DFM) was used to image human rhinovirus HRV2 alone and complexed with single receptor molecules under near physiological conditions. Specific and site-directed immobilization of HRV2 on a model cell membrane resulted in a crystalline arrangement of virus particles with hexagonal symmetry and 35 nm spacing. High-resolution imaging of the virus capsid revealed about 20 resolvable structural features with 3 nm diameters; this finding is in agreement with protrusions seen by cryo-electron microscopy. Binding of receptor molecules to individual virus particles was observed after injection of soluble receptors into the liquid cell. Virus-receptor complexes with zero, one, two, or three attached receptor molecules were resolved. The number of receptor molecules associated to virions increased over time. Occasionally, dissociation of single receptor molecules from viral particles was also observed.
Capsid↗
Dynamic force microscopy for imaging of viruses under physiological conditions.
Dynamic force microscopy (DFM) allows imaging of the structure and the assessment of the function of biological specimens in their physiological environment. In DFM, the cantilever is oscillated at a given frequency and touches the sample only at the end of its downward movement. Accordingly, the problem of lateral forces displacing or even destroying bio-molecules is virtually inexistent as the contact time and friction forces are reduced. Here, we describe the use of DFM in studies of human rhinovirus serotype 2 (HRV2) weakly adhering to mica surfaces. The capsid of HRV2 was reproducibly imaged without any displacement of the virus. Release of the genomic RNA from the virions was initiated by exposure to low pH buffer and snapshots of the extrusion process were obtained. In the following, the technical details of previous DFM investigations of HRV2 are summarized.
Journal Article↗
Following single antibody binding to purple membranes in real time.
Antibody binding to surface antigens in membranes is the primary event in the specific immune defence of vertebrates. Here we used force microscopy to study the dynamics of antibody recognition of mutant purple membranes from Halobacterium salinarum containing a genetically appended anti-Sendai recognition epitope. Ligation of individual anti-Sendai antibodies to their antigenic epitopes was observed over time. Their increase in number within a small selected area revealed an apparent kinetic on-rate. The membrane-bound antibodies showed many different conformations that ranged from globular to V- and Y-like shapes. The maximum distance of two Fab fragments of the same antibody was observed to be approximately 18 nm, indicating an overall strong intrinsic flexibility of the antibody hinge region. Fab fragments of bound anti-Sendai antibodies were allocated to antigenic sites of the purple membrane, allowing the identification and localization of individual recognition epitopes on the surface of purple membranes.
Antibodies, Monoclonal↗
Monitoring of glass derivatization with pulsed force mode atomic force microscopy.
Non-specific adsorption of proteins at solid/liquid interfaces is a major problem in the use of synthetic biomaterials and in ultrasensitive detection methods. Grafting surfaces with a dense layer of poly(ethylene glycol) (PEG) or other polymers is a most widely used strategy to solve this task. While such modified surfaces have been characterized by their ability to resist protein adsorption, the polymer layers themselves have rarely been studied in fine detail. Atomic force microscopy (AFM) using the pulsed force mode (PFM), is an ideal technique to investigate structural features and physiochemical properties of surfaces because topology and adhesion are simultaneously detected with high lateral resolution. In the present study, PFM-AFM was applied to thoroughly characterize different stages of glass derivatization, up to the formation of a dense PEG layer. Lateral inhomogeneities in topology and/or adhesion were observed at all stages before PEG attachment. The covalently bound PEG, however, was seen to form a densely packed monolayer with maximal thickness, smooth surface, and weak adhesion. Thus, PFM-AFM appears to be a valuable tool for the characterization of protein-repelling surfaces in solution.
Adhesiveness↗
Hydrodynamic damping of a magnetically oscillated cantilever close to a surface.
We studied the frequency response of a magnetically driven atomic force microscope (AFM) cantilever close to a sample surface in liquids. Amplitude-frequency (tuning) curves showed pronounced differences in dependence on the tip-sample separation (from 1 to 50 microm), with significant shifts of the resonance peak. A model was developed in which the cantilever was described in a full shape manner and the hydrodynamic forces acting on the cantilever were approximately calculated. The slight inclination of the cantilever to the surface (alpha approximately 15 degrees) leads to a force profile along the cantilever. Therefore, the mathematical problem can be strictly solved only numerically. For an approximate analytical solution, the hydrodynamic force profile was approximated by a constant force along the cantilever for large separations and by a point force acting on the tip of the cantilever for small separations. The theoretical results calculated within this model agreed well with the experimental data and allowed to determine the cantilever mass in liquid M*, the joint mass at the tip end m*t, and the coefficient of viscous interaction of the cantilever with free liquid, gamma(infinity).
Journal Article↗
Monitoring RNA release from human rhinovirus by dynamic force microscopy.
Human rhinoviruses were imaged under physiological conditions by dynamic force microscopy. Topographical images revealed various polygonal areas on the surfaces of the 30-nm viral particles. RNA release was initiated by exposure to a low-pH buffer. The lengths of the RNAs that were released but still connected to the virus capsid varied between 40 and 330 nm, whereas RNA molecules that were completely released from the virus were observed with lengths up to 1 micro m. Fork-like structure elements with 30-nm extensions were sometimes resolved at one end of the RNA molecules. They possibly correspond to the characteristic multi-stem-loop conformation, the internal ribosomal entry site, located at the 5' region of the genome. This study demonstrates that dynamic force microscopy can be used to study viral RNA release in situ under physiological conditions.
Capsid↗
Imaging morphological details and pathological differences of red blood cells using tapping-mode AFM.
The surface topography of red blood cells (RBCs) was investigated under near-physiological conditions using atomic force microscopy (AFM). An immobilization protocol was established where RBCs are coupled via molecular bonds of the membrane glycoproteins to wheat germ agglutinin (WGA), which is covalently and flexibly tethered to the support. This results in a tight but non-invasive attachment of the cells. Using tapping-mode AFM, which is known as gentle imaging mode and therefore most appropriate for soft biological samples like erythrocytes, it was possible to resolve membrane skeleton structures without major distortions or deformations of the cell surface. Significant differences in the morphology of RBCs from healthy humans and patients with systemic lupus erythematosus (SLE) were observed on topographical images. The surface of RBCs from SLE patients showed characteristic circular-shaped holes with approx. 200 nm in diameter under physiological conditions, a possible morphological correlate to previously published changes in the SLE erythrocyte membrane.
Erythrocyte Membrane↗
Simultaneous topography and recognition imaging using force microscopy.
We present a method for simultaneously recording topography images and localizing specific binding sites with nm positional accuracy by combining dynamic force microscopy with single molecule recognition force spectroscopy. For this we used lysozyme adsorbed to mica, the functionality of which was characterized by enzyme immunoassays. The topography and recognition images were acquired using tips that were magnetically oscillated during scanning and contained antibodies directed against lysozyme. For cantilevers with low Q-factor (approximately 1 in liquid) driven at frequencies below resonance, the surface contact only affected the downward deflections (minima) of the oscillations, whereas binding of the antibody on the tip to lysozyme on the surface only affected the upwards deflections (maxima) of the oscillations. The recognition signals were therefore well separated from the topographic signals, both in space (Delta z approximately 5 nm) and time (approximately 0.1 ms). Topography and recognition images were simultaneously recorded using a specially designed electronic circuit with which the maxima (U(up)) and the minima (U(down)) of each sinusoidal cantilever deflection period were depicted. U(down) was used for driving the feedback loop to record the height (topography) image, and U(up) provided the data for the recognition image.
Aluminum Silicates↗
A molecular switch between alternative conformational states in the complex of Ran and importin beta1.
Several million macromolecules are exchanged each minute between the nucleus and cytoplasm by receptor-mediated transport. Most of this traffic is controlled by the small GTPase Ran, which regulates assembly and disassembly of the receptor-cargo complexes in the appropriate cellular compartment. Here we applied dynamic force spectroscopy to study the interaction of Ran with the nuclear import receptor importin beta1 (impbeta) at the single-molecule level. We found that the complex alternates between two distinct conformational states of different adhesion strength. The application of an external mechanical force shifts equilibrium toward one of these states by decreasing the height of the interstate activation energy barrier. The other state can be stabilized by a functional Ran mutant that increases this barrier. These results support a model whereby functional control of Ran-impbeta is achieved by a population shift between pre-existing alternative conformations.
Guanosine Diphosphate↗
Dynamic force microscopy imaging of native membranes.
We employed magnetic ACmode atomic force microscopy (MACmode AFM) as a novel dynamic force microscopy method to image surfaces of biological membranes in their native environments. The lateral resolution achieved under optimized imaging conditions was in the nanometer range, even when the sample was only weakly attached to the support. Purple membranes (PM) from Halobacterium salinarum were used as a test standard for topographical imaging. The hexagonal arrangement of the bacteriorhodopsin trimers on the cytoplasmic side of PM was resolved with 1.5nm lateral accuracy, a resolution similar to images obtained in contact and tapping-mode AFM. Human rhinovirus 2 (HRV2) particles were attached to mica surfaces via nonspecific interactions. The capsid structure and 2nm sized protein loops of HRV2 were routinely obtained without any displacement of the virus. Globular and filamentous structures on living and fixed endothelial cells were observed with a resolution of 5-20nm. These examples show that MACmode AFM is a favorable method in studying the topography of soft and weakly attached biological samples with high resolution under physiological conditions.
Capsid↗