Single-molecule Atomic Force Microscopy Research Articles

Strengthening E-cadherin adhesion via antibody-mediated binding stabilization

E-cadherins (Ecads) are a crucial cell-cell adhesion protein with tumor suppression properties. Ecad adhesion can be enhanced by the monoclonal antibody 66E8, which has potential applications in inhibiting cancer metastasis. However, the biophysical mechanisms underlying 66E8-mediated adhesion strengthening are unknown. Here, we use molecular dynamics simulations, site-directed mutagenesis, and single-molecule atomic force microscopy experiments to demonstrate that 66E8 strengthens Ecad binding by stabilizing the primary Ecad adhesive conformation: the strand-swap dimer. By forming electrostatic interactions with Ecad, 66E8 stabilizes the swapped β-strand and its hydrophobic pocket and impedes Ecad conformational changes, which are necessary for rupture of the strand-swap dimer. Our findings identify fundamental mechanistic principles for strengthening of Ecad binding using monoclonal antibodies.

Adsorbing DNA to Mica by Cations: Influence of Valency and Ion Type.

Ion-mediated attraction between DNA and mica plays a crucial role in biotechnological applications and molecular imaging. Here, we combine molecular dynamics simulations and single-molecule atomic force microscopy experiments to characterize the detachment forces of single-stranded DNA at mica surfaces mediated by the metal cations Li+, Na+, K+, Cs+, Mg2+, and Ca2+. Ion-specific adsorption at the mica/water interface compensates (Li+ and Na+) or overcompensates (K+, Cs+, Mg2+, and Ca2+) the bare negative surface charge of mica. In addition, direct and water-mediated contacts are formed between the ions, the phosphate oxygens of DNA, and mica. The different contact types give rise to low- and high-force pathways and a broad distribution of detachment forces. Weakly hydrated ions, such as Cs+ and water-mediated contacts, lead to low detachment forces and high mobility of the DNA on the surface. Direct ion-DNA or ion-surface contacts lead to significantly higher forces. The comprehensive view gained from our combined approach allows us to highlight the most promising cations for imaging in physiological conditions: K+, which overcompensates the negative mica charge and induces long-ranged attractions. Mg2+ and Ca2+, which form a few specific and long-lived contacts to bind DNA with high affinity.

Unfolding and Refolding Proteins Using Single-Molecule AFM.

Single-molecule atomic force microscopy (AFM) allows capturing the conformational dynamics of an individual molecule under force. In this chapter, we describe a protocol for conducting a protein nanomechanical experiment using AFM, covering both the force-extension and force-clamp modes. Combined, these experiments provide an integrated vista of the molecular mechanisms-and their associated kinetics-underpinning the mechanical unfolding and refolding of individual proteins when exposed to mechanical load.

Accurate quantification of the stability of the perylene-tetracarboxylic dianhydride on Au(111) molecule–surface interface

Studying inorganic/organic hybrid systems is a stepping stone towards the design of increasingly complex interfaces. A predictive understanding requires robust experimental and theoretical tools to foster trust in the obtained results. The adsorption energy is particularly challenging in this respect, since experimental methods are scarce and the results have large uncertainties even for the most widely studied systems. Here we combine temperature-programmed desorption (TPD), single-molecule atomic force microscopy (AFM), and nonlocal density-functional theory (DFT) calculations, to accurately characterize the stability of a widely studied interface consisting of perylene-tetracarboxylic dianhydride (PTCDA) molecules on Au(111). This network of methods lets us firmly establish the adsorption energy of PTCDA/Au(111) via TPD (1.74 ± 0.10 eV) and single-molecule AFM (2.00 ± 0.25 eV) experiments which agree within error bars, exemplifying how implicit replicability in a research design can benefit the investigation of complex materials properties.

Crystal structure of Leptospira LSS_01692 reveals a dimeric structure and induces inflammatory responses through Toll-like receptor 2-dependent NF-κB and MAPK signal transduction pathways.

Leptospirosis is a commonly overlooked zoonotic disease that occurs in tropical and subtropical regions. Recent studies have divided the Leptospira spp. into three groups based on virulence, including pathogenic, intermediate, and saprophytic species. Pathogenic species express a protein family with leucine-rich repeat (LRR) domains, which are less expressed or absent in nonpathogenic species, highlighting the importance of this protein family in leptospirosis. However, the role of LRR domain proteins in the pathogenesis of leptospirosis is still unknown and requires further investigation. In this study, the 3D structure of LSS_01692 (rLRR38) was obtained using X-ray crystallography at a resolution of 3.2 Å. The results showed that rLRR38 forms a typical horseshoe structure with 11 α-helices and 11 β-sheets and an antiparallel dimeric structure. The interactions of rLRR38 with extracellular matrix and cell surface receptors were evaluated using ELISA and single-molecule atomic force microscopy. The results showed that rLRR38 interacted with fibronectin, collagen IV, and Toll-like receptor 2 (TLR2). Incubating HK2 cells with rLRR38 induced two downstream inflammation responses (IL-6 and MCP-1) in the TLR2 signal transduction pathway. The TLR2-TLR1 complex showed the most significant upregulation effects under rLRR38 treatment. Inhibitors also significantly inhibited nuclear factor κB and mitogen-activated protein kinases signals transduction under rLRR38 stimulation. In conclusion, rLRR38 was determined to be a novel LRR domain protein in 3D structure and demonstrated as a TLR2-binding protein that induces inflammatory responses. These structural and functional studies provide a deeper understanding of the pathogenesis of leptospirosis.

Catch Bond-Mediated Adhesion Drives Staphylococcus aureus Host Cell Invasion.

Various viruses and pathogenic bacteria interact with annexin A2 to invade mammalian cells. Here, we show that Staphylococcus aureus engages in extremely strong catch bonds for host cell invasion. By means of single-molecule atomic force microscopy, we find that bacterial surface-located clumping factors bind annexin A2 with extraordinary strength, indicating that these bonds are extremely resilient to mechanical tension. By determining the lifetimes of the complexes under increasing mechanical stress, we demonstrate that the adhesins form catch bonds with their ligand that are capable to sustain forces of 1500-1700 pN. The force-dependent adhesion mechanism identified here provides a molecular framework to explain how S. aureus pathogens tightly attach to host cells during invasion and shows promise for the design of new therapeutics against intracellular S. aureus.

Nanoscale clustering of mycobacterial ligands and DC-SIGN host receptors are key determinants for pathogen recognition.

The bacterial pathogen Mycobacterium tuberculosis binds to the C-type lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) on dendritic cells to evade the immune system. While DC-SIGN glycoconjugate ligands are ubiquitous among mycobacterial species, the receptor selectively binds pathogenic species from the M. tuberculosis complex (MTBC). Here, we unravel the molecular mechanism behind this intriguing selective recognition by means of a multidisciplinary approach combining single-molecule atomic force microscopy with Förster resonance energy transfer and bioassays. Molecular recognition imaging of mycobacteria demonstrates that the distribution of DC-SIGN ligands markedly differs between Mycobacterium bovis Bacille Calmette-Guérin (BCG) (model MTBC species) and Mycobacterium smegmatis (non-MTBC species), the ligands being concentrated into dense nanodomains on M. bovis BCG. Upon bacteria-host cell adhesion, ligand nanodomains induce the recruitment and clustering of DC-SIGN. Our study highlights the key role of clustering of both ligands on MTBC species and DC-SIGN host receptors in pathogen recognition, a mechanism that might be widespread in host-pathogen interactions.

Live-Cell SOFI Correlation with SMLM and AFM Imaging.

Standard optical imaging is diffraction-limited and lacks the resolving power to visualize many of the organelles and proteins found within the cell. The advent of super-resolution techniques overcame this barrier, enabling observation of subcellular structures down to tens of nanometers in size; however these techniques require or are typically applied to fixed samples. This raises the question of how well a fixed-cell image represents the system prior to fixation. Here we present the addition of live-cell Super-Resolution Optical Fluctuation Imaging (SOFI) to a previously reported correlative process using Single Molecule Localization Microscopy (SMLM) and Atomic Force Microscopy (AFM). SOFI was used with fluorescent proteins and low laser power to observe cellular ultrastructure in live COS-7 cells. SOFI-SMLM-AFM of microtubules showed minimal changes to the microtubule network in the 20 min between live-cell SOFI and fixation. Microtubule diameters were also analyzed through all microscopies; SOFI found diameters of 249 ± 68 nm and SMLM was 71 ± 33 nm. AFM height measurements found microtubules to protrude 26 ± 13 nm above the surrounding cellular material. The correlation of SMLM and AFM was extended to two-color SMLM to image both microtubules and actin. Two target SOFI was performed with various fluorescent protein combinations. rsGreen1-rsKAME, rsGreen1-Dronpa, and ffDronpaF-rsKAME fluorescent protein combinations were determined to be suitable for two target SOFI imaging. This correlative application of super-resolution live-cell and fixed-cell imaging revealed minimal artifacts created for the imaged target structures through the sample preparation procedure and emphasizes the power of correlative microscopy.

Compliant mechanical response of the ultrafast folding protein EnHD under force

Ultrafast folding proteins have become an important paradigm in the study of protein folding dynamics. Due to their low energetic barriers and fast kinetics, they are amenable for study by both experiment and simulation. However, single molecule force spectroscopy experiments on these systems are challenging as these proteins do not provide the mechanical fingerprints characteristic of more mechanically stable proteins, which makes it difficult to extract information about the folding dynamics of the molecule. Here, we investigate the unfolding of the ultrafast protein Engrailed Homeodomain (EnHD) by single-molecule atomic force microscopy experiments. Constant speed experiments on EnHD result in featureless transitions typical of compliant proteins. However, in the force-ramp mode we recover sigmoidal curves that we interpret as a very compliant protein that folds and unfolds many times over a marginal barrier. This is supported by a simple theoretical model and coarse-grained molecular simulations. Our results show the ability of force to modulate the unfolding dynamics of ultrafast folding proteins.

Characterizing the Biophysical Properties of Adhesive Proteins in Live Cells Using Single-Molecule Atomic Force Microscopy.

Cell adhesion proteins play essential roles in the formation, regeneration, and maintenance of tissue. However, the molecular mechanisms by which cells regulate the conformation and binding properties of adhesion proteins are poorly understood. These biophysical properties can be resolved, with single-molecule resolution, using atomic force microscopy (AFM). Here, we outline how AFM force measurements can be used to study the conformation, cytoskeletal linkage, binding strength, and force-dependent bond lifetimes of adhesion proteins in live cells.

Single-molecule AFM study of hyaluronic acid softening in electrolyte solutions

Investigation of hyaluronic acid (HA) morphology and mechanical properties at a single-molecule level is important for the development of HA based biomaterials. We have developed the atomic force microscopy (AFM) based approach for quantitative characterization of conformation of HA molecules. HA molecules adsorbed on a modified graphitic surface form oriented linear segments. Conformation of HA molecules can be considered as two-dimensional quasi-projection of a three-dimensional conformation locally straightened by a substrate. The persistence length and Young's modulus of biomolecules estimated using wormlike chain model decrease from 15.7 to 9.9 nm, and from ∼21 to ∼13 GPa, respectively, when KCl concentration increases from 0 to 100 mM. The dependence of the persistence length on ionic strength supports the Odijk-Skolnick-Fixman model of polyelectrolyte stiffening in electrolyte solution. The obtained results represent a new insight into the conformation and mechanical characteristics of HA molecules and complement the characterization of this biopolymer by bulk methods.

Following the Aggregation of Human Prion Protein on Heparin Functionalized Gold Surface in Real Time.

The aggregation of the prion protein (PrP) plays a key role in the development of prion diseases and is believed to be an autocatalytic process with a very high kinetic barrier. Intensive studies have focused on overcoming the kinetic barriers under extremely nonphysiological in vitro conditions by altering the pH of PrP solution on solid surfaces, such as gold, mica, and a lipid bilayer. Importantly, sulfated glycosaminoglycans (GAGs), including heparin, were found to be associated with PrP misfolding and aggregation, suggesting GAGs have catalytic roles in PrP aggregation processes. However, the exact role and details of GAGs in the PrP aggregation are not clear and need a thorough perusal. Here, we investigate the PrP aggregation process on a heparin functionalized gold surface by in situ, real-time monitoring of the atomic scale details of the whole aggregation process by single molecule atomic force microscopy (AFM), combining simultaneous topographic and recognition (TREC) imaging and single molecule force spectroscopy (SMFS). We observed the whole aggregation process for full-length human recombinant PrP (23-231) aggregation on the heparin modified gold surface, from the formation of oligomers, to the assembly of protofibrils and short fibers, and the formation of elongated mature fibers. Heparin is found to promote the PrP aggregation by facilitating the formation of oligomers during the early nucleation stage.

Direct hOGG1-Myc interactions inhibit hOGG1 catalytic activity and recruit Myc to its promoters under oxidative stress

The base excision repair (BER) glycosylase hOGG1 (human oxoguanine glycosylase 1) is responsible for repairing oxidative lesions in the genome, in particular oxidised guanine bases (oxoG). In addition, a role of hOGG1 in transcription regulation by recruitment of various transcription factors has been reported. Here, we demonstrate direct interactions between hOGG1 and the medically important oncogene transcription factor Myc that is involved in transcription initiation of a large number of genes including inflammatory genes. Using single molecule atomic force microscopy (AFM), we reveal recruitment of Myc to its E-box promoter recognition sequence by hOGG1 specifically under oxidative stress conditions, and conformational changes in hOGG1-Myc complexes at oxoG lesions that suggest loading of Myc at oxoG lesions by hOGG1. Importantly, our data show suppression of hOGG1 catalytic activity in oxoG repair by Myc. Furthermore, mutational analyses implicate the C28 residue in hOGG1 in oxidation induced protein dimerisation and suggest a role of hOGG1 dimerisation under oxidising conditions in hOGG1-Myc interactions. From our data we develop a mechanistic model for Myc recruitment by hOGG1 under oxidising, inflammatory conditions, which may be responsible for the observed enhanced gene expression of Myc target genes.

Molecular mechanism for strengthening E-cadherin adhesion using a monoclonal antibody

E-cadherin (Ecad) is an essential cell-cell adhesion protein with tumor suppression properties. The adhesive state of Ecad can be modified by the monoclonal antibody 19A11, which has potential applications in reducing cancer metastasis. Using X-ray crystallography, we determine the structure of 19A11 Fab bound to Ecad and show that the antibody binds to the first extracellular domain of Ecad near its primary adhesive motif: the strand-swap dimer interface. Molecular dynamics simulations and single-molecule atomic force microscopy demonstrate that 19A11 interacts with Ecad in two distinct modes: one that strengthens the strand-swap dimer and one that does not alter adhesion. We show that adhesion is strengthened by the formation of a salt bridge between 19A11 and Ecad, which in turn stabilizes the swapped β-strand and its complementary binding pocket. Our results identify mechanistic principles for engineering antibodies to enhance Ecad adhesion.

Force-induced hydrogen bonding between single polyformaldehyde chain and water

To explore the nature of water insolubility of polyformaldehyde (POM), we utilize single-molecule atomic force microscopy (AFM) and molecular dynamics (MD) simulations to study the possible interactions between POM and water at the single-chain level. Single-molecule force-extension curves of POM obtained in nonane and deionized water showed a marked deviation in the middle force region, which is similar to that observed in polyethylene glycol (PEG). The results indicate that force-induced hydrogen bonding between POM and water occurs upon initial stretching, followed by a dehydration process upon high force stretching. MD simulations show that POM chain itself is able to form H-bonds with water, but this potential is heavily hindered by the native conformation in the crystal. This finding not only provides new insight into the understanding of water insolubility of POM, but also helps the rational design of advanced polymer materials in the future.

Cadherins can dimerize via asymmetric interactions.

Cadherins are essential cell-cell adhesion proteins that interact in two distinct conformations: X-dimers and strand-swap dimers. Both X-dimers and strand-swap dimers are thought to exclusively rely on symmetric sets of interactions between key amino acids on both cadherin binding partners. Here, we use single-molecule atomic force microscopy and computer simulations to show that symmetry in cadherin binding is dispensable and that cadherins can also interact in a novel conformation that asymmetrically incorporates key elements of both strand-swap dimers and X-dimers. Our results clarify the biophysical rules for cadherin binding and demonstrate that cadherins interact in a more diverse range of conformations than previously understood.

Measuring biological materials mechanics with atomic force microscopy - Mechanical unfolding of biopolymers.

Biopolymers, such as polynucleotides, polypeptides and polysaccharides, are macromolecules that direct most of the functions in living beings. Studying the mechanical unfolding of biopolymers provides important information about their molecular elasticity and mechanical stability, as well as their energy landscape, which is especially important in proteins, since their three‐dimensional structure is essential for their correct activity. In this primer, we present how to study the mechanical properties of proteins with atomic force microscopy and how to obtain information about their stability and energetic landscape. In particular, we discuss the preparation of polyprotein constructs suitable for AFM single molecule force spectroscopy (SMFS), describe the parameters used in our force‐extension SMFS experiments and the models and equations employed in the analysis of the data. As a practical example, we show the effect of the temperature on the unfolding force, the distance to the transition state, the unfolding rate at zero force, the height of the transition state barrier, and the spring constant of the protein for a construct containing nine repeats of the I27 domain from the muscle protein titin.Highlights1. Atomic force microscopy (AFM) can be used to study the mechanical unfolding of polymers.2. AFM provides a direct measurement of unfolding (unbinding) forces.3. Force measurements for different rates provide information about the distance to the transition state and the unfolding rate at zero force.

Single-molecule visualization of conformation and dynamics of P-glycoprotein

P-glycoprotein (Pgp) is an ATP-binding cassette transporter which effluxes foreign bodies out of a cell. It non-selectively binds a variety of oncology drugs and is overexpressed in cancer cells, making it a leading cause of multi-drug resistance. Understanding Pgp’s mechanism of action is a critical research effort. Single-molecule atomic force microscopy (AFM) is a technique that directly visualizes membrane-external protrusions of proteins embedded in native lipid environments at physiologically relevant temperatures and ionic strength.

Understanding the Extraordinary Flexibility of Polydimethylsiloxane through Single-Molecule Mechanics

Linear (un-cross-linked) polydimethylsiloxane (PDMS) exists as a viscous fluid at room temperature even though its molecular weight reaches a high level (105 Da). Accordingly, one can infer the extraordinary flexibility of PDMS. However, the mechanism of the extraordinary flexibility is still unclear at present. Herein the single-chain mechanics of PDMS is measured by single-molecule atomic force microscopy, which is consistent with the results from quantum mechanical calculations. Then, the effects of several factors of microscopic structures of PDMS on the macroscopic mechanical properties are qualitatively analyzed. With the extremely low elasticity of the individual chain, the large effective cross-sectional area, and the very weak intra/interchain noncovalent interactions and their synergistic effect, the relatively small modulus of PDMS is reasonable. We further propose a method to estimate the modulus of an extended chain crystal of a polymer from the single-chain force–extension curve. For the polymer crystals in which only van der Waals forces are involved in the intra/interchain noncovalent interactions, the estimated moduli are very close to the measured values, which indicates that this method should be valid for PDMS. It is found that the estimated modulus of PDMS is smaller than those of most polymers, which directly shows its extraordinary flexibility at the macroscopic level. This work may be helpful for bridging the gap between the single-molecule and macroscopic properties of polymer materials.

The Impact of Fast-Rise-Time Electromagnetic Field and Pressure on the Aggregation of Peroxidase upon Its Adsorption onto Mica

Our present study concerns the influence of the picosecond rise-time-pulsed electromagnetic field, and the impact of nanosecond pulsed pressure on the aggregation state of horseradish peroxidase (HRP) as a model enzyme. The influence of a 640 kV/m pulsed electromagnetic field with a pulse rise-time of ~200 ps on the activity and aggregation state of an enzyme is studied by the single-molecule atomic force microscopy (AFM) method. The influence of such a field is shown to lead to aggregation of the protein and to a decrease in its enzymatic activity. Moreover, the effect of a shock wave with a pressure front rise-time of 80 ns on the increase in the HRP aggregation is demonstrated. The results obtained herein can be of use in modeling the impact of electromagnetic and pressure pulses on enzymes and on whole living organisms. Our results are also important for taking into account the effect of pulsed fields on the body in the development of drugs, therapeutic procedures, and novel highly sensitive medical diagnosticums.