Single Molecular Force Spectroscopy Research Articles

Polyelectrolyte chain conformation matters in macroscopic supramolecular self-assembly.

We demonstrate molecular-conformation-dependent macroscopic supramolecular self-assembly (MSA) driven by electrostatic interactions. Evidence from single molecular force spectroscopy reveals that polyelectrolytes modified on MSA component surfaces make MSA possible with a loop conformation, while those with a flat conformation lead to no assembly, which is attributed to distinct molecular mobility. We believe that this finding is also applicable in fundamental phenomena such as surface adsorption and adhesion regarding polymers.

Heparan sulfate dependent binding of plasmatic von Willebrand factor to blood circulating melanoma cells attenuates metastasis

Heparan sulfate (HS), a highly negatively charged glycosaminoglycan, is ubiquitously present in all tissues and also exposed on the surface of mammalian cells. A plethora of molecules such as growth factors, cytokines or coagulation factors bear HS binding sites. Accordingly, HS controls the communication of cells with their environment and therefore numerous physiological and pathophysiological processes such as cell adhesion, migration, and cancer cell metastasis. In the present work, we found that HS exposed by blood circulating melanoma cells recruited considerable amounts of plasmatic von Willebrand factor (vWF) to the cellular surface. Analyses assisted by super-resolution microscopy indicated that HS and vWF formed a tight molecular complex. Enzymatic removal of HS or genetic engineering of the HS biosynthesis showed that a reduced length of the HS chains or complete lack of HS was associated with significantly reduced vWF encapsulation. In microfluidic experiments, mimicking a tumor-activated vascular system, we found that vWF-HS complexes prevented vascular adhesion. In line with this, single molecular force spectroscopy suggested that the vWF-HS complex promoted the repulsion of circulating cancer cells from the blood vessel wall to counteract metastasis. Experiments in wild type and vWF knockout mice confirmed that the HS-vWF complex at the melanoma cell surface attenuated hematogenous metastasis, whereas melanoma cells lacking HS evade the anti-metastatic recognition by vWF. Analysis of tissue samples obtained from melanoma patients validated that metastatic melanoma cells produce less HS. Transcriptome data further suggest that attenuated expression of HS-related genes correlate with metastases and reduced patients’ survival. In conclusion, we showed that HS-mediated binding of plasmatic vWF to the cellular surface can reduce the hematogenous spread of melanoma. Cancer cells with low HS levels evade vWF recognition and are thus prone to form metastases. Therefore, therapeutic expansion of the cancer cell exposed HS may prevent tumor progression.

Direct electrochemical grafting of crystalline PAEK macromolecule on carbon fiber to enhance the interfacial properties of PEEK/CF composites

Electrochemical grafting of diazonium salts have been applied to improve interfacial properties of carbon fiber and matrix resin. However, these graft modifications are still limited to general-purpose carbon fiber reinforced resin matrix composites such as epoxy. In this research, the direct grafting of Poly (aryl ether ketone) (PAEK) macromolecule on carbon fiber was achieved with mild condition. Firstly, aniline was introduced into Poly (ether ether ketone) (PEEK) as pendent group to improve solubility and amino terminated group was introduced as precursor of diazonium salt. Then the graft was conduct through diazonium salt reduction. After modification, the aniline side group could be hydrolyzed off to recover crystallinity. To further analyze the graft, single molecular-force spectroscopy was introduced to investigate the surface of carbon fiber. Impressively, the extension curve suggested the PAEK macromolecule had been attached to surface through strong interaction. Combined this with XPS data, it was believed that the graft was a success. As a result, the grafted polymer along with the polymer that physically absorbed on carbon fiber in electrolyte can cooperate to strengthen the interface. The interfacial shear strength of the grafted carbon fiber and PEEK increased from 42.27 MPa to 97.33 MPa by 130.26%. The improvement mainly originated from the covalent bonding that connected the carbon fiber and the graft coating layer as well as the great compatibility between the graft coating layer and the matrix resin. These results also demonstrated the possibility to graft high performance polymers on carbon fiber through one-step modifications.

Characterization of Hepatitis C Virus Core Protein Dimerization by Atomic Force Microscopy.

Dimerization of core protein is a crucial step in the formation of the hepatitis C virus (HCV) nucleocapsid, and inhibition of dimer formation is regarded as an attractive approach to design anti-HCV drugs. In this work, we developed the atomic force microscopy based single molecular force spectroscopy (AFM-SMFS) method for the characterization of core protein dimerization with the advantages of small amount of sample consumption and no need of labeling. Interaction force of the core protein with its antibody or aptamer was analyzed to investigate its stoichiometry and binding property. The two specific binding forces were detected due to the probing of dimeric and monomeric core protein, respectively. Moreover, the binding property of protein dimer was different from the monomer. Our work offers a new approach to study the dimerization of core protein, as well as other proteins, and to screen the HCV candidate inhibitors.

Probing Amyloid β and the Antibody Interaction Using Atomic Force Microscopy.

Amyloid β (Aβ) peptide is considered to be the critical causative factor in the pathogenesis of Alzheimer's disease (AD) because the hydrophilic molecules accumulated outside of the neural cells and results in the formation of highly toxicity amyloid plaque. In this study, we probed the interaction between Aβ and the antibody using atomic force microscopy (AFM). We compared two kinds of antibodies which are the antibody for Aβ 1-42 (antibody42) and the antibody for Aβ 1-16 (antibody16). To detect the interaction between Aβ and the antibodies, the single molecular force spectroscopy was carried out using Aβ modified glass substrate and the antibodies modified AFM probes. In the results, the single Aβ-antibody42 dissociation constant was estimated to be 5.2 × 10-3 s-1 and the single Aβ-antibody16 dissociation constant was 2.8×10-2 s-1. The Aβ-antibody42 showed 5.3 times longer bond life time compare with Aβ-antibody16. It suggested that antibody42 is better choice for the Aβ sensor development.

Interaction of von Willebrand factor domains with collagen investigated by single molecule force spectroscopy.

von Willebrand factor (VWF) is a huge multimeric protein that plays a key role in primary hemostasis. Sites for collagen binding, an initial event of hemostasis, are located in the VWF-domains A1 and A3. In this study, we investigated single molecule interactions between collagen surfaces and wild type VWF A1A2A3 domain constructs, as well as clinically relevant VWF A3 domain point mutations, such as p.Ser1731Thr, p.Gln1734His, and p.His1786Arg. For this, we utilized atomic force microscopy based single molecular force spectroscopy. The p.Ser1731Thr mutant had no impact on the VWF-collagen type III and VI interactions, while the p.Gln1734His and p.His1786Arg mutants showed a slight increase in bond stability to collagen type III. This effect probably arises from additional hydrogen bonds that come along with the introduction of these mutations. Using the same mutants, but collagen type VI as a binding partner, resulted in a significant increase in bond stability. VWF domain A1 was reported to be essential for the interaction with collagen type VI and thus our findings strengthen the hypothesis that the VWF A1 domain can compensate for mutations in the VWF A3 domain. Additionally, our data suggest that the mutations could even stabilize the interaction between VWF and collagen without shear. VWF-collagen interactions seem to be an important system in which defective interactions between one VWF domain and one type of collagen can be compensated by alternative binding events.

Characterizing the effect of polymyxin B antibiotics to lipopolysaccharide on Escherichiacoli 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 3hours of 10μg/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 30pN at a retraction velocity of 500nm/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.

Characterization of Inter- and Intramolecular Interactions of Amyloid Fibrils by AFM-Based Single-Molecule Force Spectroscopy

Amyloids are fibrous protein aggregates defined by shared specific structural features. Abnormal accumulation of amyloid in organs leads to amyloidosis, which results in various neurodegenerative diseases. Atomic force microscopy (AFM) has proven to be an excellent tool investigating amyloids; it has been extensively utilized to characterize its morphology, assembly process, and mechanical properties. This review summarizes studies which applied AFM to detect the inter- and intramolecular interactions of amyloid fibrils and classified the influencing factors of amyloid’s nanomechanics in detail. The characteristics of amyloid fibrils driven by inter- and intramolecular interactions, including various morphologies of amyloid fibrils, self-assembly process, and the aggregating pathway, are described. Successful examples where AFM provided abundant information about inter- and intramolecular interactions of amyloid fibrils in different environments are presented. Direct force measurement of intra- or intermolecular interactions utilizing an AFM-based tool, single-molecular force spectroscopy (SMFS), is introduced. Some mechanical information such as elasticity, adhesiveness, and strength was obtained by stretching amyloid fibrils. This review helps researchers in understanding the mechanism of amyloidogenesis and exploring the properties of amyloid using AFM techniques.

On the molecular interaction between albumin and ibuprofen: An AFM and QCM-D study

The adsorption of proteins on surfaces often results in a change of their structural behavior and consequently, a loss of bioactivity. One experimental method to study interactions on a molecular level is single molecular force spectroscopy that permits to measure forces down to the pico-newton range.In this work, the binding force between human serum albumin (HSA), covalently immobilized on glutaraldehyde modified gold substrates, and ibuprofen sodium salt was studied by means of single molecular force spectroscopy. First of all, a protocol was established to functionalize atomic force microscopy (AFM) tips with ibuprofen. The immobilization protocol was additionally tested by quartz crystal microbalance with dissipation (QCM-D) and contact angle measurements. AFM was used to characterize the adsorption of HSA on gold substrates, which lead to a packed monolayer of thickness slightly lower than the reported value in solution.Finally, single molecule spectroscopy results were used to characterize the binding force between albumin and ibuprofen and calculate the distance of the transition state (0.6nm) and the dissociation rate constant (0.055s−1). The results might indicate that part of the adsorbed protein still preserves its functionality upon adsorption.

Characterizing the Cell Surface Structure and Antibody Recognition Forces on Intact Microbial Cells using Scanning Probe Microscopy

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 crystallinesurface (S‑) layer that completely covers T. 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, atomic force microscopy (AFM) based topography imaging and single-molecular force spectroscopy (SMFS) were applied. 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 S-layer 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 less strongly attached to the mutant cell surface when the co-assembling TfsB-GP is missing. Altogether, our data confirm the current model of the S‑layer architecture with two intercalating S-layer glycoproteins and TfsA-GP mainly outwardly orientated. This study was funded by a Marie Curie International Incoming Fellowship of the 7th European Community Framework Program (to Y. J. O.). Financial support came from the Austrian Science Fund FWF projects P20605-B12 and P24317-B22 (to C.S.).

Single Molecule Force Spectroscopy Reveals the Molecular Mechanical Anisotropy of the FeS4 Metal Center in Rubredoxin

Mechanical anisotropy is an important feature of materials. Depending on the pulling direction, a material can exhibit very different mechanical properties. Mechanical anisotropy on the microscopic scale has also been observed for individual elastomeric proteins. Depending upon the pulling direction, a protein can unfold via different mechanical unfolding pathways and exhibit different mechanical stability. However, it remains to be demonstrated if the concept of mechanical anisotropy can be extended to molecular scale for small molecular objects containing only a few chemical bonds. Here, we choose the iron-sulfur center FeS4 in rubredoxin as a model system to demonstrate the molecular level mechanical anisotropy. We used single molecular force spectroscopy to investigate the mechanical rupture of the FeS4 center along different pulling directions. The FeS4 cluster is a simple molecular object with defined three-dimensional structure, where a ferric ion and four coordinating cysteinyl ligands are arranged into a distorted tetrahedral geometry. Mutating two specific residues in rubredoxin to cysteines provides anchoring points that enable us to stretch and rupture the FeS4 center along five distinct and precisely controlled directions. Our results showed that mechanical stability as well as the rupture mechanism and kinetics of the FeS4 center are strongly dependent upon the direction along which it is stretched, suggesting that the very small FeS4 center exhibits considerable mechanical anisotropy. It is likely that structural asymmetry in the FeS4 cluster and the modulation of the local environment due to partial unfolding of rubredoxin are responsible for the observed mechanical anisotropy. Our results suggest that mechanical anisotropy is a universal feature for any asymmetrical three-dimensional structure, even down to a molecular scale, and such mechanical anisotropy can be potentially utilized to control the mechanochemical reactivity of molecular objects.

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.

Single molecular dissection of the ligand binding property of epidermal growth factor receptor

The understanding of ligand binding interactions is an important component of understanding the fundamental mechanism of receptor function. In this study, the binding abilities of EGF and TGF-α to EGFR on human bladder cancer (T24) cells were investigated by single molecular force spectroscopy (SMFS) based on atomic force microscopy (AFM). By approaching the specifically functionalized AFM tips to the T24 cell surface and subsequent retraction, specific unbinding events of the EGF/EGFR complexes and TGF-α/EGFR complexes were investigated. Further, the unbinding forces and the kinetic off rate constants that govern the bond stabilities were calculated through varying the external mechanic forces applied. Meanwhile, the distances from the energy minimum to the transition states along the separation paths of the EGF/EGFR complexes and TGF-α/EGFR complexes were deduced. This study at single-molecule level may enrich our understanding of the ligand binding properties of EGFR and provide some new information to the development of improved EGFR inhibitors. In addition, the results present new insight into the study of the energy landscape of the dissociation of ligand-EGFR system.

Probing Binding Pocket of Serotonin Transporter by Single Molecular Force Spectroscopy on Living Cells

The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). The interaction energies involved in binding of such compounds to the transporter are unknown. Here, we used atomic force microscopy (AFM) to probe single molecular interactions between the serotonin transporter and MFZ2-12 (a potent cocaine analog) in living CHOK1 cells. For the AFM measurements, MFZ2-12 was immobilized on AFM tips by using a heterobifunctional cross-linker. By varying the pulling velocity in force distance cycles drug-transporter complexes were ruptured at different force loadings allowing for mapping of the interaction energy landscape. We derived chemical rate constants from these recordings and compared them with those inferred from inhibition of transport and ligand binding: koff values were in good agreement with those derived from uptake experiments; in contrast, the kon values were scaled down when determined by AFM. Our observations generated new insights into the energy landscape of the interaction between SERT and inhibitors. They thus provide a useful framework for molecular dynamics simulations by exploring the range of forces and energies that operate during the binding reaction.

Biological Atomic Force Microscopic Imaging and Force Spectroscopy of Protein Constructs as Potential Anti-Malaria Vaccines

Atomic force microscope (AFM) is quickly becoming a general nano-characterization tool for biomedical samples. Here we have used AFM imaging and single molecular force spectroscopy to characterize leading malaria vaccine candidates that are various recombinant forms of Plasmodium falciparum circumsporozoite protein (Plassmeyer, M., et al., 2009. J. Bio. Chem., 284:26951-63)). We have achieved high-resolution AFM single molecule imaging and force spectroscopy of these important antigens under various buffer and surface deposition conditions to better understand the recombinant protein constructs. The protein folding domains and tertiary structure features observed from AFM imaging and deduced from AFM force spectroscopy are closely correlated with accurate computational structural predictions derived from the primary sequences and bulk solution characterizations. Bio-AFM characterizations can discriminate alternative forms of recombinant proteins according to their oligomerization and surface presentations. Our results showcase AFM as an insightful tool of nanomedicine for in-depth understanding of single proteins and protein-protein interactions.

A comparative molecular force spectroscopy study of homophilic JAM‐A interactions and JAM‐A interactions with reovirus attachment protein σ1

JAM-A belongs to a family of immunoglobulin-like proteins called junctional adhesion molecules (JAMs) that localize at epithelial and endothelial intercellular tight junctions. JAM-A is also expressed on dendritic cells, neutrophils, and platelets. Homophilic JAM-A interactions play an important role in regulating paracellular permeability and leukocyte transmigration across epithelial monolayers and endothelial cell junctions, respectively. In addition, JAM-A is a receptor for the reovirus attachment protein, sigma1. In this study, we used single molecular force spectroscopy to compare the kinetics of JAM-A interactions with itself and sigma1. A chimeric murine JAM-A/Fc fusion protein and the purified sigma1 head domain were used to probe murine L929 cells, which express JAM-A and are susceptible to reovirus infection. The bond half-life (t(1/2)) of homophilic JAM-A interactions was found to be shorter (k(off)(o) = 0.688 +/- 0.349 s(-1)) than that of sigma1/JAM-A interactions (k(off)(o) = 0.067 +/- 0.041 s(-1)). These results are in accordance with the physiological functions of JAM-A and sigma1. A short bond lifetime imparts a highly dynamic nature to homophilic JAM-A interactions for regulating tight junction permeability while stable interactions between sigma1 and JAM-A likely anchor the virus to the cell surface and facilitate viral entry.