Single Molecular Force Research Articles

A Chemo-Mechanically Coupled DNA Origami Clamp Capable of Generating Robust Compression Forces.

DNA nanostructures have been utilized to study biological mechanical processes and construct artificial nanosystems. Many application scenarios necessitate nanodevices able to robustly generate large single molecular forces. However, most existing dynamic DNA nanostructures are triggered by probabilistic hybridization reactions between spatially separated DNA strands, which only non-deterministically generate relatively small compression forces (≈0.4 piconewtons (pN)). Here, an intercalator-triggered dynamic DNA origami nanostructure is developed, where large amounts of local binding reactions between intercalators and the nanostructure collectively lead to the robust generation of relatively large compression forces (≈11.2 pN). Biomolecular loads with different stiffnesses, 3, 4, and 6-helix DNA bundles are efficiently bent by the compression forces. This work provides a robust and powerful force-generation tool for building highly chemo-mechanically coupled molecular machines in synthetic nanosystems.

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.

Adhesion dependent cell motility is regulated by integrin-mediated single molecular force

Cells sense surrounding micro-environment through integrin-mediated adhesion and alter their morphology dynamically to adapt to continuously changing external stimuli. Cell migration is one of the most essential features of diverse cellular functions such as wound healing, immune response, and cancer metastasis. Previous studies have shown that cell adhesion and spreading are determined by the integrin-mediated single molecular force. However, how the single molecular force between integrin and ligand determines cell behaviors such as cell differentiation and cell migration remains confusing.

In situ quantitative determination of the intermolecular attraction between amines and a graphene surface using atomic force microscopy

The adsorption of pollutants on carbonaceous environmental media has been widely studied via batch sorption experiments and spectroscopic characterization. However, the molecular interactions between pollutants and interfacial sites on carbonaceous materials have only been indirectly investigated. To comprehend the adsorption mechanisms in situ, we applied atomic force microscopy force spectroscopy (AFM-FS) to quantitatively determine the molecular interactions between typical amines (methylamines and N-methylaniline) and the surface of highly oriented pyrolytic graphite (HOPG), which was supported by the single molecule interaction derived from density functional theory and batch adsorption experiments. This method achieved direct and in situ characterization of the molecular interactions in the adsorption process. The molecular interactions between the amines and the adsorption sites on the graphite surface were affected by pH and peaked at pH 7 due to strong cation-π interactions. When the pH was 11, the attractions were weak due to a lack of cation-π interaction, whereas, when the pH was 3, the competitive occupation of hydronium ions on the surface reduced the attraction between the amines and HOPG. Based on AFM-FS, the single molecule force of methylamine and N-methylaniline on the graphite surface was estimated to be 0.224 nN and 0.153 nN, respectively, which was consistent with density functional theory (DFT) calculations. This study broadens our comprehension of cation-π interactions between amines and electron-rich aromatic compounds at the micro/nanoscale.

Investigation of the interaction between MeCP2 methyl-CpG binding domain and methylated DNA by single molecule force spectroscopy

MeCP2 is an essential transcriptional repressor that mediates transcriptional inhibition by binding to methylated DNA. The binding specificity of MeCP2 protein to methylated DNA was considered to depend on its methyl-CpG binding domain (MBD). In this study, we used atomic force microscope based single-molecular force spectroscopy to investigate the interaction of MeCP2 MBD and methylated DNA. The specific interaction forces of the MeCP2 MBD-methylated DNA complexes were measured for the first time. The dynamics was also investigated by measuring the unbinding force of the complex at different loading rates. In addition, the distribution of unbinding forces and binding probabilities of MeCP2 MBD and different DNA were studied at the same loading rate. It was found that MeCP2 MBD had weak interaction with hemi-methylated and unmethylated DNA compared to methylated DNA. This work revealed the binding characteristics of MeCP2 MBD and methylated DNA at the single-molecule level. It provides a new idea for exploring the molecular mechanism of MeCP2 in regulating methylation signals.

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.

Robust mechanobiological behavior emerges in heterogeneous myosin systems

Biological complexity presents challenges for understanding natural phenomenon and engineering new technologies, particularly in systems with molecular heterogeneity. Such complexity is present in myosin motor protein systems, and computational modeling is essential for determining how collective myosin interactions produce emergent system behavior. We develop a computational approach for altering myosin isoform parameters and their collective organization, and support predictions with in vitro experiments of motility assays with α-actinins as molecular force sensors. The computational approach models variations in single myosin molecular structure, system organization, and force stimuli to predict system behavior for filament velocity, energy consumption, and robustness. Robustness is the range of forces where a filament is expected to have continuous velocity and depends on used myosin system energy. Myosin systems are shown to have highly nonlinear behavior across force conditions that may be exploited at a systems level by combining slow and fast myosin isoforms heterogeneously. Results suggest some heterogeneous systems have lower energy use near stall conditions and greater energy consumption when unloaded, therefore promoting robustness. These heterogeneous system capabilities are unique in comparison with homogenous systems and potentially advantageous for high performance bionanotechnologies. Findings open doors at the intersections of mechanics and biology, particularly for understanding and treating myosin-related diseases and developing approaches for motor molecule-based technologies.

Playing a tug of war with integrin and Notch receptors

It is now widely appreciated that cancer cells and stem cells can change their cell fate (differentiation, metastasis, etc.) depending on their mechanical environment. Mechanical sensing is likely to be initiated by individual membrane receptor proteins that are in direct contact with the mechanical environment. In order to understand how molecular level mechanical events trigger a cellular response, we need to examine the forces applied across individual cellular proteins during mechanical signaling.To define the single molecular forces required to activate signaling through a ligand‐receptor bond, we developed the tension gauge tether (TGT) approach in which the ligand is immobilized to a surface through a rupturable tether before receptor engagement. TGT serves as an autonomous gauge to restrict the receptor‐ligand tension. Using a range of tethers with tunable tension tolerances, we showed that cells apply a peak tension of about 40 picoNewtons (pN) to single integrin‐ligand bonds during initial adhesion. This tension value was consistent across different cell types. We then presented a mixture of weak and strong TGTs (12 pN and 54 pN nominal tension tolerance) to the cells in order to better mimic native cellular environments that are likely heterogeneous. Surprisingly, just one or two strong TGTs were sufficient to allow the cells to adhere as long as there is a high density of weak tethers. Therefore, mechanical tugging through one or two integrins is enough to convince the cell that the substrate is stiff enough for adhesion. Even more surprisingly, when we presented a mixture of two different weak TGTs (12 pN and 23 pN), cells adhered to the surface despite the fact that individually neither of the two tethers could support cell adhesion. Furthermore, we found that the 23 pN tether can exert its influence even at very high dilution, down to as few as two tethers per cell. This phenomenon of ‘ultrasensitivity in differential force sensing through integrin’ can be explained by a model where simultaneous rupture of weaker bonds exerts a transient high tension across a single integrin to activate that integrin.Notch signaling, involved in development and tissue homeostasis, is activated at the cell‐cell interface through ligand‐receptor interactions. Previous studies have implicated mechanical forces in the activation of Notch receptor upon binding to its ligand. In our original TGT paper, we showed that Notch signaling is activated even for the weakest TGT (12 pN), suggesting that if Notch activation requires force, it must be less than 12 pN. Subsequently, the Blacklow lab show that the exposure of a Notch cleavage site required for its activation occurs at a tension of ~5 pN39, consistent with our estimate of a 12 pN upper limit. We recently developed LTGT (a low tension gauge tether) utilizing the low unbinding force of ~ 4 pN between single‐stranded (ss)DNA and E. coli ssDNA binding protein (SSB) and showed that Notch activation requires forces between 4 and 12 pN.

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.

Probing the role of metal cations on the aggregation behavior of amyloid β-peptide at a single molecule level by AFM

With the development of nanotechnology, understanding of intermolecular interactions on a single molecule level by atomic force spectroscopy (AFM) has played an important role in molecular biology and biomedical science. In recent years, some research suggested that the presence of metal cations is an important regulator in the processes of misfolding and aggregation of the amyloid β-protein (Aβ), which may be an important etiological factor of Alzheimer’s disease. However, the knowledge on the principle of interactions between Aβ and metal cations at the single molecule level is still poor understood. In this paper, the amyloid β-protein (Aβ) was fabricated on substrate of mixed thiol-modified gold nanoparticles using self-assembled monolayer method and the adhesion force in the longitudinal direction between metal cations and Aβ42 were investigated by AFM. The role of metal ions on Aβ aggregation is discussed from the perspective of single molecular force. The force results showed that the specific adhesion force Fi and the nonspecific force F0 between a single Aβ–Aβ pair in control experiment were calculated as 42 ± 3 and 80 pN, respectively. However, Fi between a single Aβ–Aβ pair in the presence of Cu2+, Zn2+, Ca2+ and Al3+ increased dramatically to 84 ± 6, 89 ± 3, 73 ± 5, 95 ± 5 pN successively, which indicated that unbinding between Aβ proteins is accelerated in the presence of metal cations. What is more, the imaging results showed that substoichiometric copper cations accelerate the formation of fibrils within 3 days. The combined atomic force spectroscopy and imaging analysis indicate that metal cations play a role in promoting the aggregating behavior of Aβ42.

Defining Single Molecular Forces Required for Notch Activation Using Nano Yoyo.

Notch signaling, involved in development and tissue homeostasis, is activated at the cell-cell interface through ligand-receptor interactions. Previous studies have implicated mechanical forces in the activation of Notch receptor upon binding to its ligand. Here we aimed to determine the single molecular force required for Notch activation by developing a novel low tension gauge tether (LTGT). LTGT utilizes the low unbinding force between single-stranded DNA (ssDNA) and Escherichia coli ssDNA binding protein (SSB) (∼4 pN dissociation force at 500 nm/s pulling rate). The ssDNA wraps around SSB and, upon application of force, unspools from SSB, much like the unspooling of a yoyo. One end of this nano yoyo is attached to the surface though SSB, while the other end presents a ligand. A Notch receptor, upon binding to its ligand, is believed to undergo force-induced conformational changes required for activating downstream signaling. If the required force for such activation is larger than 4 pN, ssDNA will unspool from SSB, and downstream signaling will not be activated. Using these LTGTs, in combination with the previously reported TGTs that rupture double-stranded DNA at defined forces, we demonstrate that Notch activation requires forces between 4 and 12 pN, assuming an in vivo loading rate of 60 pN/s. Taken together, our study provides a direct link between single-molecular forces and Notch activation.

Single molecular force sensitivity and threshold for the activation of B cell receptors

Abstract B lymphocytes show remarkable mechanosensing capability by utilizing the surface expressed B cell receptors (BCRs) to sense antigens. However, the sensitivity and threshold for the activation of BCRs resulting from the stimulation by mechanical forces are unknown. Here we systemically addressed this question using a ds-DNA based tension gauge tether (TGT) system serving as a predefined single molecular force gauge ranging from 12 to 56 pN to restrict BCR and antigen tension. Utilizing total internal reflection fluorescence microscopy, we observed that IgM-BCR activation is sensitive to single molecular forces. We observed an obvious multi-threshold effect with a low-end 12–16 pN force which triggered a weak activation, whereas a 23–43 pN force initiated a medium-level activation, and a high-end 50–56 pN force produced a strong activation. Such patterned dependence on mechanical forces with multi-threshold effects did not strictly rely on myosin IIA, dynein and integrin. However, the break-through of the high-end but not the medium-level and low-end mechanical force threshold in IgM-BCR activation required myosin IIA and the inside-out activation of integrin. Surprisingly, we found that the activation of the isotype-switched IgG-BCR or IgE-BCR only requires an extremely low threshold of less than 12 pN, providing an explanation for their rapid activation in response to antigen stimulation. Mechanistically, we found that the cytoplasmic tail of the IgG-BCR heavy chain is both required and sufficient to account for the low mechanical force threshold to activate the IgG-BCR. These results defined the single molecular force sensitivity and threshold that are required to activate different isotyped BCRs.

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.

Single molecular force across single integrins dictates cell spreading.

Cells' ability to sense and interpret mechanical signals from the extracellular milieu modulates the degree of cell spreading. Yet how cells detect such signals and activate downstream signaling at the molecular level remain elusive. Herein, we utilize tension gauge tether (TGT) platform to investigate the underlying molecular mechanism of cell spreading. Our data from both differentiated cells of cancerous and non-cancerous origin show that for the same stiff underlying glass substrates and for same ligand density it is the molecular forces across single integrins that ultimately determine cell spreading responses. Furthermore, by decoupling molecular stiffness and molecular tension we demonstrate that molecular stiffness has little influence on cell spreading. Our data provide strong evidence that links molecular forces at the cell-substrate interface to the degree of cell spreading.

Single molecular recognition force spectroscopy study of a DNA aptamer with the target epithelial cell adhesion molecule.

The epithelial cell adhesion molecule (EpCAM) is a tumor-specific antigen for malignancies of the epithelialis lineage. In this study the interaction between the DNA-based EpCAM aptamer (SYL3C) and EpCAM was explored using single molecular recognition force spectroscopy (SMFS). The capability of aptamer SYL3C to recognize the EpCAM protein and the kinetic parameters were investigated.

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.