Chip-based protein-protein interaction studied by atomic force microscopy

In this article, a technique for accurate direct measurement of protein‐to‐protein interactions before and after the introduction of a drug candidate is developed using atomic force microscopy (AFM). The method is applied to known immunosuppressant drug candidate Echinacea purpurea derived cynarin. T‐cell/CD28 is on‐chip immobilized and B‐cell/CD80 is immobilized on an AFM tip. The difference in unbinding force between these two proteins before and after the introduction of cynarin is measured. The method is described in detail including determination of the loading rates, maximum probability of bindings, and average unbinding forces. At an AFM loading rate of 1.44 × 10⁴ pN/s, binding events were largely reduced from 61 ± 5% to 47 ± 6% after cynarin introduction. Similarly, maximum probability of bindings reduced from 70% to 35% with a blocking effect of about 35% for a fixed contact time of 0.5 s or greater. Furthermore, average unbinding forces were reduced from 61.4 to 38.9 pN with a blocking effect of ～37% as compared with ～9% by SPR. AFM, which can provide accurate quantitative measures, is shown to be a good method for drug screening. The method could be applied to a wider variety of drug candidates with advances in bio‐chip technology and a more comprehensive AFM database of protein‐to‐protein interactions. Biotechnol. Bioeng. 2012; 109: 2460–2467. © 2012 Wiley Periodicals, Inc.     

AFM‐ and NSOM‐based force spectroscopy and distribution analysis of CD69 molecules on human CD4+ T cell membrane

Although CD69 is well known as an early T cell‐activation marker, the possibility that CD69 are distributed as nano‐structures on membrane for immune regulation during T cell activation has not been tested. In this study, nanoscale features of CD69 expression on activated T cells were determined using the atomic force microscopy (AFM) topographic and force‐binding nanotechnology as well as near‐field scanning optical microscopy (NSOM)‐/fluorescence quantum dot (QD)‐based nanosacle imaging. Unstimulated CD4⁺ T cells showed neglectable numbers of membrane CD69 spots binding to the CD69 Ab‐functinalized AFM tip, and no detectable QD‐bound CD69 as examined by NSOM/QD‐based imaging. In contrast, Phytohemagglutinin (PHA)‐activated CD4⁺ T cells expressed CD69, and displayed many force‐binding spots binding to the CD69 Ab‐functionalized AFM tip on about 45% of cell membrane, with mean binding‐rupture forces 276 ± 71 pN. Most CD69 molecules appeared to be expressed as 100–200 nm nanoclusters on the membrane of PHA‐activated CD4⁺ T cells. Meanwhile, NSOM/QD‐based nanoscale imaging showed that CD69 were non‐uniformly distributed as 80–200 nm nanoclusters on cell‐membrane of PHA‐activated CD4⁺ T cells. This study represents the first demonstration of the nano‐biology of CD69 expression during T cell activation. Copyright © 2009 John Wiley & Sons, Ltd.     

Single molecular dynamic interactions between glycophorin A and lectin as probed by atomic force microscopy

Glycophorin A (GpA) is one of the most abundant transmembrane proteins in human erythrocytes and its interaction with lectins has been studied as model systems for erythrocyte related biological processes. We performed a force measurement study using the force mode of atomic force microscopy (AFM) to investigate the single molecular level biophysical mechanisms involved in GpA-lectin interactions. GpA was mounted on a mica surface or natively presented on the erythrocyte membrane and probed with an AFM tip coated with the monomeric but multivalent Psathyrella velutina lectin (PVL) through covalent crosslinkers. A dynamic force spectroscopy study revealed similar interaction properties in both cases, with the unbinding force centering around 60 pN with a weak loading rate dependence. Hence we identified the presence of one energy barrier in the unbinding process. Force profile analysis showed that more than 70% of GpAs are free of cytoskeletal associations in agreement with previous reports.     

Molecular recognition force spectroscopy study of the dynamic interaction between aptamer GBI‐10 and extracellular matrix protein tenascin‐C on human glioblastoma cell

Molecular recognition force spectroscopy (MR‐FS) was applied to investigate the dynamic interaction between aptamer GBI‐10 and tenascin‐C (TN‐C) on human glioblastoma cell surface at single‐molecule level. The unbinding force between aptamer GBI‐10 and TN‐C was 39 pN at the loading rate of 0.3 nN sec^?1. A series of kinetic parameters concerning interaction process such as the unbinding force f_u, the association rate constant k_on, dissociation rate constant at zero force k_off, and dissociation constant K_D for aptamer GBI‐10/TN‐C complexes were acquired. In addition, the interaction of aptamer GBI‐10 with TN‐C depended on the presence of Mg²⁺. This work demonstrates that MR‐FS can be used as an attractive tool for exploring the interaction forces and dynamic process of aptamer and ligand at the single‐molecule level. As a future perspective, MR‐FS may be used as a potential diagnostic and therapeutic tool by combining with other techniques. Copyright © 2012 John Wiley & Sons, Ltd.     

Characterizing the S‐layer structure and anti‐S‐layer antibody recognition on intact Tannerella forsythia cells by scanning probe microscopy and small angle X‐ray scattering

Tannerella forsythia is among the most potent triggers of periodontal diseases, and approaches to understand underlying mechanisms are currently intensively pursued. A ~22‐nm‐thick, 2D crystalline surface (S‐) layer that completely covers Tannerella forsythia cells is crucially involved in the bacterium–host cross‐talk. The S‐layer is composed of two intercalating glycoproteins (TfsA‐GP, TfsB‐GP) that are aligned into a periodic lattice. To characterize this unique S‐layer structure at the nanometer scale directly on intact T. forsythia cells, three complementary methods, i.e., small‐angle X‐ray scattering (SAXS), atomic force microscopy (AFM), and single‐molecular force spectroscopy (SMFS), were applied. SAXS served as a difference method using signals from wild‐type and S‐layer‐deficient cells for data evaluation, revealing two possible models for the assembly of the glycoproteins. Direct high‐resolution imaging of the outer surface of T. forsythia wild‐type cells by AFM revealed a p4 structure with a lattice constant of ~9.0 nm. In contrast, on mutant cells, no periodic lattice could be visualized. Additionally, SMFS was used to probe specific interaction forces between an anti‐TfsA antibody coupled to the AFM tip and the S‐layer as present on T. forsythia wild‐type and mutant cells, displaying TfsA‐GP alone. Unbinding forces between the antibody and wild‐type cells were greater than with mutant cells. This indicated that the TfsA‐GP is not so strongly attached to the mutant cell surface when the co‐assembling TfsB‐GP is missing. Altogether, the data gained from SAXS, AFM, and SMFS confirm the current model of the S‐layer architecture with two intercalating S‐layer glycoproteins and TfsA‐GP being mainly outwardly oriented. Copyright © 2013 John Wiley & Sons, Ltd.     

Direct immobilization of avidin protein on AFM tip functionalized by acrylic acid vapor at RF plasma

The atomic force microscopy (AFM) has been used as a force sensor to measure unbinding forces of single bound complexes in the nanonewton and piconewton range. Force spectroscopy measurements can be applied to study both intermolecular and intramolecular interactions of complex biological and synthetic macromolecules. Although the AFM has been extensively used as a nano force sensor, the commercially available cantilever is limited to silicon and silicon nitride. Those materials reduce the adhesion sensitivity with specific surface and/or molecule. Here, we functionalized the AFM tip with carboxylic groups by applying acrylic acid (AA) vapor at radio frequency plasma treatment at 100 W for 5 min. This method provides a remarkable sensitivity enhancement on the functional group interaction specificity. The functionalized tip was characterized by scanning electron microscopy. The electron beam high resolution images have not shown significant tip sharpness modification. Silicon wafers (1 0 0)-no treated and functionalized by AA plasma treatment-were characterized by Auger electron spectroscopy to elucidate the silicon surface sputtering and demonstrate functionalization. The Fourier transform-infrared spectroscopy spectrum shows a high absorbance of avidin protein over the silicon surface functionalized by AA plasma treatment.We carried out force spectroscopy assay to measure the unbinding force between the well-established pair biotin-avidin. At pulling speed of 2 μm/s, we measured the unbinding force of 106?±?23 pN, which is in good agreement with the literature, demonstrating the effectiveness of the tip functionalization by AA plasma treatment in biological studies.     

Probing interactions between human lung adenocarcinoma A549 cell and its aptamers at single‐molecule resolution

Because cell‐specific aptamers have high potential for biomedical applications, investigation of the interaction between cell and its aptamers may be of key importance for an improved understanding of biochemical processes. Herein, the interaction between human lung adenocarcinoma A549 cell and its four aptamers was explored using single‐molecule force spectroscopy (SMFS). The values of the unbinding force varied from 117.1 to 171.0 pN at the loading rate of 1.8 × 10⁵ pN/s. Based on the dependence of singe molecule force on the atomic force microscopy loading rate, the corresponding kinetic parameters were obtained. The results revealed two activation barriers and two transient states in the unbinding process of aptamer/cell interaction. More importantly, the binding sites on A549 cells with its four aptamers were defined to be different using SMFS and flow cytometry. This work demonstrated that SMFS can be used as a powerful tool for exploring the aptamer/cell binding behavior at the single‐molecule level, and may provide valuable information for the design and application of aptamer probes. Copyright © 2014 John Wiley & Sons, Ltd.     

Measurement of interaction force between nanoarrayed integrin alphavbeta3 and immobilized vitronectin on the cantilever tip

Protein nanoarrays containing integrin alphavbeta3 or BSA were fabricated on ProLinker-coated Au surface by dip-pen nanolithography (DPN). An atomic force microscope (AFM) tip coated with ProLinker was modified by vitronectin. We measured the interaction force between nanoarrayed integrin alphavbeta3 or BSA and immobilized vitronectin on the cantilever tip by employing tethering-unbinding method. The unbinding force between integrin alphavbeta3 and vitronectin (1087+/-62 pN) was much higher than that of between BSA and vitronectin (643+/-74 pN). These results demonstrate that one can distinguish a specific protein interaction from non-specific interactions by means of force measurement on the molecular interactions between the nanoarrayed protein and its interacting protein on the AFM tip.     

MAPPING CELL WALL POLYSACCHARIDES OF LIVING MICROBIAL CELLS USING ATOMIC FORCE MICROSCOPY

Functionalized atomic force microscope tips were used to sense specific forces of interaction between ligand—receptor pairs and to map the positions of polysaccharides on a living microbial cell surface. Gold-coated tips were functionalized with concanavalin A using a cross-linker with a spacer arm of 15.6Å. It was possible to measure the binding force between concanavalin A and mannan polymers on the yeast (Saccharomyces cerevisiae) cell surface. This force ranged from 75 to 200pN. The shape of the force curve indicated that the polymers were pulled away from the cell surface for a fairly long distance that sometimes reached several hundred nanometres. The distribution of mannan on the cell surface was mapped by carrying out the force measurement in the force volume mode of atomic force microscopy (AFM). During the measurement, the maximum cantilever deflection after contact between the tip and the sample was kept constant at 10nm using trigger mode to keep the pressing force on the sample surface as gently as possible at a force of 180pN. This regime was used to minimize the non-specific adhesion between the tip and the cell surface. Specific molecular recognition events took place on specific areas of the cell surface that could be interpreted as reflecting a non-uniform distribution of mannan on the cell surface.     

Analysis of DNA and Zinc finger interactions using mechanical force spectroscopy

The unbinding force of Zif268-DNA complex has been studied by atomic force microscopy (AFM). DNA and Zif268 were covalently immobilized on the surfaces of an AFM tip and glass substrate, respectively. Confocal microscopy was used to confirm the successful immobilization of DNA. Because of the complexity of the protein-DNA interaction, parallel experiments were designed to discriminate specific interactions. For such experiments, a typical unbinding force of a single Zif268-DNA complex (approx 550 pN at 40 nN/s force loading rate) was evaluated.     

Rhizobial Surface Biopolymers and their Interaction with Lectin Measured by Atomic Force Microscopy

Summary Atomic force microscopy (AFM) was used to measure the morphology and physicochemical properties of rhizobia and to probe cell-surface polymers with tips modified with soybean agglutinin (SBA). AFM measurements of the length, width, and height of Sinorhizobium fredii CCRC15769 were 1580±450, 870±70, and 270±50 nm, respectively (means±SD). Different AFM image modes revealed the morphology, adhesion, viscoelasticity, and surface roughness of rhizobia in air using the tapping operation. Force–distance relationships between SBA-terminated AFM probes and Bradyrhizobium japonicum USDA110 were recorded and the retraction curves showed an unbinding force of 106±48 pN with a loading rate of 1 nN/s in PBS containing 0.1 mM Mn²⁺ and 0.1 mM Ca²⁺ (pH 7.2). The technique of AFM provides information about the morphology and molecular interaction forces of rhizobia under physiological conditions.     

Atomic force microscopy study of the antigen‐antibody binding force on patient cancer cells based on ROR1 fluorescence recognition

Knowledge of drug–target interaction is critical to our understanding of drug action and can help design better drugs. Due to the lack of adequate single‐molecule techniques, the information of individual interactions between ligand‐receptors is scarce until the advent of atomic force microscopy (AFM) that can be used to directly measure the individual ligand‐receptor forces under near‐physiological conditions by linking ligands onto the surface of the AFM tip and then obtaining force curves on cells. Most of the current AFM single‐molecule force spectroscopy experiments were performed on cells grown in vitro (cell lines) that are quite different from the human cells in vivo. From the view of clinical practice, investigating the drug–target interactions directly on the patient cancer cells will bring more valuable knowledge that may potentially serve as an important parameter in personalized treatment. Here, we demonstrate the capability of AFM to measure the binding force between target (CD20) and drug (rituximab, an anti‐CD20 monoclonal antibody targeted drug) directly on lymphoma patient cancer cells under the assistance of ROR1 fluorescence recognition. ROR1 is a receptor expressed on some B‐cell lymphomas but not on normal cells. First, B‐cell lymphoma Raji cells (a cell line) were used for ROR1 fluorescence labeling and subsequent measurement of CD20‐rituximab binding force. The results showed that Raji cells expressed ROR1, and the labeling of ROR1 did not influence the measurement of CD20‐rituximab binding force. Then the established experimental procedures were performed on the pathological samples prepared from the bone marrow of a follicular lymphoma patient. Cancer cells were recognized by ROR1 fluorescence. Under the guidance of fluorescence, with the use of a rituximab‐conjugated tip, the cellular topography was visualized by using AFM imaging and the CD20‐Rituximab binding force was measured by single‐molecule force spectroscopy. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterizing the effect of polymyxin B antibiotics to lipopolysaccharide on Escherichia coli surface using atomic force microscopy

Lipopolysaccharide (LPS) on gram‐negative bacterial outer membranes is the first target for antimicrobial agents, due to their spatial proximity to outer environments of microorganisms. To develop antibacterial compounds with high specificity for LPS binding, the understanding of the molecular nature and their mode of recognition is of key importance. In this study, atomic force microscopy (AFM) and single molecular force spectroscopy were used to characterize the effects of antibiotic polymyxin B (PMB) to the bacterial membrane at the nanoscale. Isolated LPS layer and the intact bacterial membrane were examined with respect to morphological changes at different concentrations of PMB. Our results revealed that 3 hours of 10 μg/mL of PMB exposure caused the highest roughness changes on intact bacterial surfaces, arising from the direct binding of PMB to LPS on the bacterial membrane. Single molecular force spectroscopy was used to probe specific interaction forces between the isolated LPS layer and PMB coupled to the AFM tip. A short range interaction regime mediated by electrostatic forces was visible. Unbinding forces between isolated LPS and PMB were about 30 pN at a retraction velocity of 500 nm/s. We further investigated the effects of the polycationic peptide PMB on bacterial outer membranes and monitored its influences on the deterioration of the bacterial membrane structure. Polymyxin B binding led to rougher appearances and wrinkles on the outer membranes surface, which may finally lead to lethal membrane damage of bacteria. Our studies indicate the potential of AFM for applications in pathogen recognition and nano‐resolution approaches in microbiology.     

Force‐induced globule–coil transition in laminin binding protein and its role for viral–cell membrane fusion

The specific interactions of the pairs laminin binding protein (LBP)–purified tick‐borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single‐molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves‐based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVβ3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus–cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus–cell membrane fusion, SMDFS data reveal the existence of a force‐induced (effective already for forces as small as 30–70 pN) sharp globule–coil transition for LBP and LBP–fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first‐order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus–cell membrane fusion process. Copyright © 2014 John Wiley & Sons, Ltd.     

Direct force measurement of single DNA–peptide interactions using atomic force microscopy

The selective interactions between DNA and miniature (39 residues) engineered peptide were directly measured at the single‐molecule level by using atomic force microscopy. This peptide (p007) contains an α‐helical recognition site similar to leucine zipper GCN4 and specifically recognizes the ATGAC sequence in the DNA with nanomolar affinity. The average rupture force was 42.1 pN, which is similar to the unbinding forces of the digoxigenin–antidigoxigenin complex, one of the strongest interactions in biological systems. The single linear fit of the rupture forces versus the logarithm of pulling rates showed a single energy barrier with a transition state located at 0.74 nm from the bound state. The smaller k_off compared with that of other similar systems was presumably due to the increased stability of the helical structure by putative folding residues in p007. This strong sequence‐specific DNA–peptide interaction has a potential to be utilized to prepare well‐defined mechanically stable DNA–protein hybrid nanostructures. Copyright © 2013 John Wiley & Sons, Ltd.     

Temporal analysis of vascular smooth muscle cell elasticity and adhesion reveals oscillation waveforms that differ with aging

A spectral analysis approach was developed for detailed study of time‐resolved, dynamic changes in vascular smooth muscle cell (VSMC) elasticity and adhesion to identify differences in VSMC from young and aged monkeys. Atomic force microscopy (AFM) was used to measure Young’s modulus of elasticity and adhesion as assessed by fibronectin (FN) or anti‐beta 1 integrin interaction with the VSMC surface. Measurements demonstrated that VSMC cells from old vs. young monkeys had increased elasticity (21.6 kPa vs. 3.5 kPa or a 612% increase in elastic modulus) and adhesion (86 pN vs. 43 pN or a 200% increase in unbinding force). Spectral analysis identified three major frequency components in the temporal oscillation patterns for elasticity (ranging from 1.7 × 10^?3 to 1.9 × 10^?2 Hz in old and 8.4 × 10^?4 to 1.5 × 10^?2 Hz in young) and showed that the amplitude of oscillation was larger (P < 0.05) in old than in young at all frequencies. It was also observed that patterns of oscillation in the adhesion data were similar to the elasticity waveforms. Cell stiffness was reduced and the oscillations were inhibited by treatment with cytochalasin D, ML7 or blebbistatin indicating the involvement of actin–myosin‐driven processes. In conclusion, these data demonstrate the efficacy of time‐resolved analysis of AFM cell elasticity and adhesion measurements and that it provides a uniquely sensitive method to detect real‐time functional differences in biomechanical and adhesive properties of cells. The oscillatory behavior suggests that mechanisms governing elasticity and adhesion are coupled and affected differentially during aging, which may link these events to changes in vascular stiffness.     

THE STRUCTURE AND NANOMECHANICAL PROPERTIES OF THE ADHESIVE MUCILAGE THAT MEDIATES DIATOM‐SUBSTRATUM ADHESION AND MOTILITY1

We investigated the adhesive mucilage and mechanism of cell‐substratum adhesion of two benthic raphid diatoms, the marine species Craspedostauros australis E. J. Cox and the freshwater species Pinnularia viridis (Nitzsch) Ehrenberg. SEM images of P. viridis and C. australis cells revealed the presence of multistranded tethers that appear to arise along the raphe openings and extend for a considerable distance from the cell before forming a “holdfast‐like” attachment with the substratum. We propose that the tethers result from the elongation/stretching of composite adhesive mucilage strands secreted from raphes during the onset of cell adhesion and reorientation. Atomic force microscopy (AFM) force measurements reveal that the adhesive strands originating from the nondriving raphe of live C. australis and P. viridis are highly extensible and accumulate to form tethers. During force measurements tethers can be chemically stained and are seen to extend between the cantilever tip and a cell during elongation and relaxation. In most cases, AFM force measurements recorded an interaction with a number of adhesive strands that are secreted from the raphe. The force curves of C. australis and P. viridis revealed a sawtooth pattern, suggesting the successive unbinding of modular domains when the adhesive strands were placed under stress. In addition, we applied the “fly‐fishing” technique that allowed the cantilever, suspended a distance above the cell, to interact with single adhesive strands protruding from the raphe. These force curves revealed sawtooth patterns, although the binding forces recorded were in the range for single molecule interactions.     

AFM force measurements of the gp120-sCD4 and gp120 or CD4 antigen-antibody interactions

Soluble CD4 (sCD4), anti-CD4 antibody, and anti-gp120 antibody have long been regarded as entry inhibitors in human immunodeficiency virus (HIV) therapy. However, the interactions between these HIV entry inhibitors and corresponding target molecules are still poorly understood. In this study, atomic force microscopy (AFM) was utilized to investigate the interaction forces among them. We found that the unbinding forces of sCD4–gp120 interaction, CD4 antigen–antibody interaction, and gp120 antigen–antibody interaction were 25.45 ± 20.46, 51.22 ± 34.64, and 89.87 ± 44.63 pN, respectively, which may provide important mechanical information for understanding the effects of viral entry inhibitors on HIV infection. Moreover, we found that the functionalization of an interaction pair on AFM tip or substrate significantly influenced the results, implying that we must perform AFM force measurement and analyze the data with more caution.     

Dynamic response of HPV16/anti-HPV16 pairs with unbinding events studied by atomic force microscopy

This paper proposes an effective approach to distinguish whether samples include Human Papilloma virus type-16 (HPV16) by Atomic force microscopy (AFM). AFM is an important instrument in nanobiotechnology field. At first we identified the HPV16 by Polymerase chain reaction (PCR) analysis and Western blotting from specimen of the HPV patient (E12) and the normal (C2), and then we used an AFM to observe the surface ultrastructure by tapping mode and to measure the unbinding force between HPV16 coupled to an AFM tip and anti-HPV16 L1 coated on the substrate surface by contact mode. The experimental results by tapping mode show that the size of a single HPV viron was similar to its SEM image from the previous literatures; moreover, based on the purposed methods and the analysis, two obvious findings that we can determine whether or not the subject is a HPV patient can be derived from the results; one is based on the distribution of unbinding forces, and the other is based on the distribution of the stiffness. Furthermore, the proposed method could be a useful technique for further investigating the potential role among subtypes of HPVs in the oncogenesis of human cervical cancer.     

Direct Force Measurement of the Interaction Between Liposome and the C2A Domain of Synaptotagmin I using Atomic Force Microscopy

The binding force between a liposome and the C2A domain of synaptotagmin I was determined by an atomic force microscopy (AFM). Liposomes were immobilized on the surface of the L1 sensor chip and the C2A domains, which recognize phosphatidylserine, were chemically conjugated onto a gold-coated cantilever tip. The average interaction force between the C2A domain and the liposome was 306 (±57) pN while the force between untreated cantilever and the liposome was 58 (±16) pN. This work helps understand the physicochemical interactions between proteins and lipid vesicles for the design of high affinity protein probes against the apoptotic cell surface. Revisions requested 13 December 2005; Revisions received 9 January 2006