Single molecule recognition of protein binding epitopes in brush border membranes by force microscopy

Sidedness and accessibility of protein epitopes in intact brush border membrane vesicles were analyzed by detecting single molecule interaction forces using molecular recognition force microscopy in aqueous physiological solutions. Frequent antibody-antigen recognition events were observed with a force microscopy tip carrying an antibody directed against the periplasmically located gamma-glutamyltrans- peptidase, suggesting a right side out orientation of the vesicles. Phlorizin attached to the tips bound to NA+/D-glucose cotransporter molecules present in the vesicles. The recognition was sodium dependent and inhibited by free phlorizin and D-glucose, and revealed an apparent K(D) of 0.2 microM. Binding events were also observed with an antibody directed against the epitope aa603-aa630 close to the C terminus of the transporter. In the presence of phlorizin the probability of antibody binding was reduced but the most probable unbinding force f(u) = 100 pN remained unchanged. In the presence of D-glucose and sodium, however, both the binding probability and the most probable binding force (f(u) = 50 pN) were lower than in its absence. These studies demonstrate that molecular recognition force microscopy is a versatile tool to probe orientation and conformational changes of epitopes of membrane components during binding and trans-membrane transport.     

Atomic force microscopy imaging and single molecule recognition force spectroscopy of coat proteins on the surface of Bacillus subtilis spore

Coat assembly in Bacillus subtilis serves as a tractable model for the study of the self-assembly process of biological structures and has a significant potential for use in nano-biotechnological applications. In the present study, the morphology of B. subtilis spores was investigated by magnetically driven dynamic force microscopy (MAC mode atomic force microscopy) under physiological conditions. B. subtilis spores appeared as prolate structures, with a length of 0.6-3 microm and a width of about 0.5-2 microm. The spore surface was mainly covered with bump-like structures with diameters ranging from 8 to 70 nm. Besides topographical explorations, single molecule recognition force spectroscopy (SMRFS) was used to characterize the spore coat protein CotA. This protein was specifically recognized by a polyclonal antibody directed against CotA (anti-CotA), the antibody being covalently tethered to the AFM tip via a polyethylene glycol linker. The unbinding force between CotA and anti-CotA was determined as 55 +/- 2 pN. From the high-binding probability of more than 20% in force-distance cycles it is concluded that CotA locates in the outer surface of B. subtilis spores.     

Imaging and determining friction forces of specific interactions between human IgG and rat anti-human IgG

Covalently immobilized rat anti-human immunoglobulin (IgG) monolayers on thiol-modified gold substrates and human IgG linked with the tips were fabricated using the self-assembled monolayer method, and interactions between these systems were studied by friction force microscopy (FFM). In addition to observation of distinct nanostructures of protein monolayers due to recognition events, FFM also quantified the friction force due to protein–protein-specific interactions. The average friction force due to interactions between the antigen functionalized tip and the antibody monolayer was determined as 200–250 pN, significantly greater than that between either the bare tip and the antibody monolayer (0–50 pN), or the blocked antigen tip and the antibody monolayer (50–100 pN), indicative of antigen/antibody-specific interactions. These results, taken together, suggest that FFM is not only capable of tracking recognition events, but also quantifying the friction force due to specific interactions between biological molecules, such as antigen and antibody.     

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.     

A physical approach to reduce nonspecific adhesion in molecular recognition atomic force microscopy.

Atomic force microscopy is one of the few techniques that allow analysis of biological recognition processes at the single-molecule level. A major limitation of this approach is the nonspecific interaction between the force sensor and substrate. We have modeled the nonspecific interaction by looking at the interaction potential between a conical Si3N4 tip with a spherical end face and a mica surface in solution, using DLVO (Derjaguin, Landau, Verwey, Overbeek) theory and numerical calculations. Insertion of the tip-sample potential in a simulation of an approach-retract cycle of the cantilever gives the well-known force-distance curve. Simulating a force-distance curve at low salt concentration predicts a discrete hopping of the tip, caused by thermal fluctuations. This hopping behavior was observed experimentally and gave rise to a novel approach to making measurements in adhesion mode that essentially works in the repulsive regime. The distance between tip and sample will still be small enough to allow spacer-involved specific interactions, and the percentage of nonspecific interactions of the bare tip with the mica is minimized. We have validated this physical model by imaging intercellular adhesion molecule 1 (ICAM-1) antigen with a tip functionalized with anti-ICAM-1 antibody. The measurement demonstrated that a significant decrease in the number of nonspecific interactions was realized, and the topographical image quality and the specific bonding capability of the tip were not affected.     

A new, simple method for linking of antibodies to atomic force microscopy tips

Functionalization of atomic force microscope (AFM) tips with bioligands converts them into monomolecular biosensors which can detect complementary receptor molecules on the sample surface. Flexible PEG tethers are preferred because the bioligand can freely reorient and locally palpate the sample surface while the AFM tip is moved along. In a well-established coupling scheme [Hinterdorfer et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 3477-3481], a heterobifunctional PEG linker is used to tether thiol-containing bioligands to amino-functionalized AFM tips. Since antibodies contain no free thiol residues, prederivatization with N-succinimidyl 3-(acetylthio)propionate (SATP) is needed which causes a relatively high demand for antibody. The present study offers a convenient alternative with minimal protein consumption (e.g., 5 microg of protein in 50 microL of buffer) and no prederivatization, using a new heterobifunctional cross-linker that has two different amino-reactive functions. One end is an activated carboxyl (N-hydroxysuccinimide ester) which is much faster to react with the amino groups of the tips than the benzaldehyde function on its other end. The reactivity of the latter is sufficient, however, to covalently bind lysine residues of proteins via Schiff base formation. The method has been critically examined, using biotinylated IgG as bioligand on the tip and mica-bound avidin as complementary receptor. These experiments were well reproduced on amino-functionalized silicon nitride chips where the number of specifically bound IgG molecules (approximately 2000 per microm2) was estimated from the amount of specifically bound ExtrAvidin-peroxidase conjugate. For a bioscientific application, human rhinovirus particles were tethered to the tip, very-low-density lipoprotein receptor fragments were tethered to mica, and the specific interaction was studied by force microscopy.     

Site localization of membrane-bound proteins on whole cell level using atomic force microscopy

This study presents molecular recognition method, which is based on specific force measurements between modified AFM (atomic force microscopy) tip and mammalian cell. The presented method allows recognition of specific cell surface proteins and receptor sites by nanometer accuracy level. Here we demonstrate specific recognition of membrane-bound Osteopontin (OPN) sites on preosteogenic cell membrane. By merging specific force detection map of the proteins and topography image of the cell, we create a new image (recognition image), which demonstrates the exact locations of the proteins relative to the cell membrane. The recognition results indicate the strong affinity between the modified tip and the target molecules, therefore, it enables the use of an AFM as a remarkable nanoscale tracking tool on the whole cell level.     

Detecting CD20-Rituximab specific interactions on lymphoma cells using atomic force microscopy

Elucidating the underlying mechanisms of cell physiology is currently an important research topic in life sciences. Atomic force microscopy methods can be used to investigate these molecular mechanisms. In this study, single-molecule force spectroscopy was used to explore the specific recognition between the CD20 antigen and anti-CD20 antibody Rituximab on B lymphoma cells under near-physiological conditions. The CD20-Rituximab specific binding force was measured through tip functionalization. Distribution of CD20 on the B lymphoma cells was visualized three-dimensionally. In addition, the relationship between the intramolecular force and the molecular extension of the CD20-Rituximab complex was analyzed under an external force. These results facilitate further investigation of the mechanism of Rituximab’s anti-cancer effect.     

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.     

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.     

Hydration force in the atomic force microscope: A computational study.

Using a hard sphere model and numerical calculations, the effect of the hydration force between a conical tip and a flat surface in the atomic force microscope (AFM) is examined. The numerical results show that the hydration force remains oscillatory, even down to a tip apex of a single water molecule, but its lateral extent is limited to a size of a few water molecules. In general, the contribution of the hydration force is relatively small, but, given the small imaging force ( approximately 0.1 nN) typically used for biological specimens, a layer of water molecules is likely to remain "bound" to the specimen surface. This water layer, between the tip and specimen, could act as a "lubricant" to reduce lateral force, and thus could be one of the reasons for the remarkably high resolution achieved with contact-mode AFM. To disrupt this layer, and to have a true tip-sample contact, a probe force of several nanonewtons would be required. The numerical results also show that the ultimate apex of the tip will determine the magnitude of the hydration force, but that the averaged hydration pressure is independent of the radius of curvature. This latter conclusion suggests that there should be no penalty for the use of sharper tips if hydration force is the dominant interaction between the tip and the specimen, which might be realizable under certain conditions. Furthermore, the calculated hydration energy near the specimen surface compares well with experimentally determined values with an atomic force microscope, providing further support to the validity of these calculations.     

AFM functional imaging on vascular endothelial cells

Vascular endothelial (VE)-cadherin is predominantly responsible for the mechanical linkage between endothelial cells, where VE-cadherin molecules are clustered and linked through their cytoplasmic domain to the actin-based cytoskeleton. Clustering and linkage of VE-cadherin to actin filaments is a dynamic process and changes according to the functional state of the cells. Here nano-mapping of VE-cadherin was performed using simultaneous topography and recognition imaging (TREC) technique onto microvascular endothelial cells from mouse myocardium (MyEnd). The recognition maps revealed prominent 'dark' spots (domains or clusters) with the sizes from 10 to 250 nm. These spots arose from a decrease of oscillation amplitude during specific binding between VE-cadherin cis-dimers. They were assigned to characteristic structures of the topography images. After treatment with nocodazole so as to depolymerize microtubules, VE-cadherin domains with a typical ellipsoidal form were still found to be collocalized with cytoskeletal filaments supporting the hypothesis that VE-cadherin is linked to actin filaments. Compared to other conventional techniques such as immunochemistry or single molecule optical microscopy, TREC represents an alternative method to quickly obtain the local distribution of receptors on cell surface with an unprecedented lateral resolution of several nanometers.     

Nano-scale dynamic recognition imaging on vascular endothelial cells

Combination of high-resolution atomic force microscope topography imaging with single molecule force spectroscopy provides a unique possibility for the detection of specific molecular recognition events. The identification and localization of specific receptor binding sites on complex heterogeneous biosurfaces such as cells and membranes are of particular interest in this context. Here simultaneous topography and recognition imaging (TREC) was applied to gently fixed microvascular endothelial cells from mouse myocardium (MyEnd) to identify binding sites of vascular endothelial (VE)-cadherin, known to play a crucial role in calcium-dependent, homophilic cell-to-cell adhesion. TREC images were acquired with magnetically oscillating atomic-force microscope tips functionalized with a recombinant VE-cadherin-Fc cis-dimer. The recognition images revealed single molecular binding sites and prominent, irregularly shaped dark spots (domains) with sizes ranging from 10 to 100 nm. These domains arose from a decrease of the oscillation amplitude during specific binding between active VE-cadherin cis-dimers. The VE-cadherin clusters were subsequently assigned to topography features. TREC represents an exquisite method to quickly obtain the local distribution of receptors on cellular surface with an unprecedented lateral resolution of 5 nm.     

Extraction of membrane proteins from a living cell surface using the atomic force microscope and covalent crosslinkers

The force curve mode of the atomic force microscope (AFM) was applied to extract intrinsic membrane proteins from the surface of live cells using AFM tips modified by amino reactive bifunctional covalent crosslinkers. The modified AFM tips were individually brought into brief contact with the living cell surface to form covalent bonds with cell surface molecules. The force curves recorded during the detachment process from the cell surface were often characterized by an extension of a few hundred nanometers followed mostly by a single step jump to the zero force level. Collection and analysis of the final rupture force revealed that the most frequent force values (of the force) were in the range of 0.4–0.6 nN. The observed rupture force most likely represented extraction events of intrinsic membrane proteins from the cell membrane because the rupture force of a covalent crosslinking system was expected to be significantly larger than 1.0 nN, and the separation force of noncovalent ligand-receptor pairs to be less than 0.2 nN, under similar experimental conditions. The transfer of cell surface proteins to the AFM tip was verified by recording characteristic force curves of protein stretching between the AFM tips used on the cell surface and a silicon surface modified with amino reactive bifunctional crosslinkers. This method will be a useful addition to bionanotechnological research for the application of AFM.     

Microneme secretion in Coccidia: confocal laser scanning and electron microscope study of Sarcocystis muris in cell culture.

A monoclonal antibody (mcab) raised against a subcellular fraction of Sarcocystis muris cystozoites was used to localize microneme antigens before, during and after invasion of cultured cells. The mcab recognized a 20 and 22 kDa protein under reducing and non-reducing conditions on Western blots and localized an antigen in cystozoites in the apical part of the parasites. Confocal laser scanning microscopy of invading cystozoites revealed the secretion of a microneme antigen at the apical tip of the parasite. The secreted microneme antigen was attached to the host cell surface at the invasion site and spread along the surface of the infected cells. Electron microscopy using immunogold labeling showed that the microneme antigen was distributed in patches on the surface of infected cells and present on infected cells more than 60 min post-infection. The function of microneme antigens during parasite-host cell interactions is discussed.     

Nanostructures and molecular force bases of a highly sensitive capacitive immunosensor

While biosensors have been constructed using various strategies, there is no report describing nanostructures of antibody-immobilized electrode interface in an immunosensor. Here, atomic force microscopy (AFM) and electrochemistry analyses were employed to construct and characterize the nanostructures and electrochemistry of biosensing surface that was created by a sequential self-assembling of bioactive aminobenzenthiol oligomer (o-ABT), glutareldehyde and anti-transferrin (anti-Tf) antibody on the electrode gold surface. Under AFM, a complete coverage of bioactive o-ABT interface could be achieved by anti-Tf antibody at an optimal concentration. The anti-Tf antibody immobilized on electrode surface of the immunosensor exhibited globular-shape topography with some degree of aggregation. Extensive force-curve analysis allowed mapping the functional spots of the anti-Tf immunosensor. Surprisingly, although immunosensing surface was fully covered by anti-Tf antibodies at the optimal concentration, only about 52% of coated anti-Tf antibody molecules (spots) on the electrode surface were able to specifically capture or bind Tf antigen under AFM. Despite limited functional spots, however, the anti-Tf immunosensor was highly specific and sensitive for sensitizing Tf antigen in solution. The anti-Tf molecules on the immunosensor exhibited a greater molecular force bound to holo-Tf (iron-containing form of Tf) than that to apo-Tf (iron-absent form of Tf). Consistently, the anti-Tf immunosensor had a greater electrochemical capacity to sensitize apo-Tf than holo-Tf, supporting the molecular force-based finding by AFM. Thus, the present study elucidated the nanostructures and molecular force bases for the immunosensing capacity of a highly sensitive capacitive immunosensor.     

Visualization of the exocytosis/endocytosis secretory cycle in cultured adrenal chromaffin cells

Cultured bovine adrenal medullary chromaffin cells were stimulated to secrete catecholamines by addition of veratridine or nicotine. The formation of an exocytotic pit exposes a major secretory granule membrane antigen, the enzyme dopamine beta-hydroxylase, to the external medium. By including antiserum to this enzyme in the medium, we were able to visualize sites of exocytosis by decoration of bound antibody using a fluorescent second antibody. Internalization of this antibody- antigen complex was then followed in chase experiments: approximately half the surface complex was internalized in 15-30 min. In other experiments, secretion was triggered in the absence of antiserum, and surface enzyme was revealed by binding antibodies at various times after secretion had been halted by an antagonist. Surface patches of antigen remained discrete from the bulk of the plasma membrane for at least 30 min, although a substantial proportion of the antigen was internalized within this time. Cell surface concanavalin A receptors were internalized at a roughly similar rate, suggesting that mechanisms may be similar. After internalization, chromaffin granule membranes fused to larger structures, possibly lysosomes, and were transported over a few hours to the perinuclear region of the cell.     

Phosphorylation and degradation of ribosomes in starved Tetrahymena pyriformis.

We have characterized an embryonic antigen on the surface of chick erythrocytes using immunochemical electron microscopy. An indirect surface labeling technique (hemocyanin conjugated to goat antirabbit IgG and specific antisera prepared in rabbits) revealed that the antigenic sites, at hatching, nearly saturate the surface of erythrocytes with hemocyanin markers. The number of antigenic sites gradually decreases with age, and the antigen can no longer be detected at 7 months. Further, the antigen has been detected on the very earliest primitive erythrocytes which form in the extra-embryonic mesenchyme before circulation begins. The embryonic antigen appears to be firmly associated with the erythrocyte surface and cannot be removed by extensive washing either with phosphate-buffered saline or with EDTA. Labeling unfixed cells at 37 °C produces clustering of the surface markers, suggesting that the antigen is associated with a membrane component which is fairly free to move in the plane of the membrane. In addition, the erythrocytes from newly hatched chicks were found to agglutinate more readily with several different lectins, particularly Concanavalin A (ConA), than did the erythrocytes from adults. Three times more ConA is bound to chick erythrocytes than to adult erythrocytes, as estimated by electron microscopy. Although this difference in lectin binding suggests that the ConA-binding sites might be related to the embryonic antigen, the sugars known to block lectin-induced hemagglutination had no blocking effect on antiserum-induced agglutination or on antibody binding, as visualized by the electron microscope technique. Also, ConA binding was not inhibited by treatment of the chick erythrocytes with the specific antiserum.     

Anti-rabbit immunoglobulin G detection in complex medium by PM-RAIRS and QCM Influence of the antibody immobilisation method

Two antibody immobilisation procedures were compared to set up an immunosensor for goat anti-rabbit immunoglobulin (anti-rIgG), i.e. rIgG covalently bound or immobilised via affinity to protein A (PrA). In both cases, the first layer of protein was covalently bound to a mixed self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) and mercaptohexanol (C6OH) on a gold surface. The elaboration of the sensitive surfaces, as well as their selectivity and sensitivity were studied step by step by polarization modulation-reflection absorption infra-red spectroscopy (PM-RAIRS) and quartz crystal microbalance (QCM) with impedance measurement. QCM measurements showed that the viscoelastic properties of the antibody layer were markedly modified during the antigen recognition when the antibody was bound by affinity to PrA. The specific detection of antigen within a complex medium was assessed by PM-RAIRS thanks to the grafting of cobalt-carbonyl probes. Affinity constants between the immobilised rIgG and the anti-rIgG were determined from PM-RAIRS analysis.     

A general and efficient cantilever functionalization technique for AFM molecular recognition studies

Atomic force microscopy (AFM) is a versatile technique for the investigation of noncovalent molecular associations between ligand–substrate pairs. Surface modification of silicon nitride AFM cantilevers is most commonly achieved using organic trialkoxysilanes. However, susceptibility of the Si? O bond to hydrolysis and formation of polymeric aggregates diminishes attractiveness of this method for AFM studies. Attachment techniques that facilitate immobilization of a wide variety of organic and biological molecules via the stable Si? C bond on silicon nitride cantilevers would be of great value to the field of molecular recognition force spectroscopy. Here, we report (1) the formation of stable, highly oriented monolayers on the tip of silicon nitride cantilevers and (2) demonstrate their utility in the investigation of noncovalent protein–ligand interactions using molecular recognition force spectroscopy. The monolayers are formed through hydrosilylation of hydrogen‐terminated silicon nitride AFM probes using a protected α‐amino‐ω‐alkene. This approach facilitates the subsequent conjugation of biomolecules. The resulting biomolecules are bound to the tip by a strong Si? C bond, completely uniform with regard to both epitope density and substrate orientation, and highly suitable for force microscopy studies. We show that this attachment technique can be used to measure the unbinding profiles of tip‐immobilized lactose and surface‐immobilized galectin‐3. Overall, the proposed technique is general, operationally simple, and can be expanded to anchor a wide variety of epitopes to a silicon nitride cantilever using a stable Si? C bond. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 761–765, 2012.