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It is now possible to manipulate individual molecules using a nanopore to read DNA and proteins, or write DNA by inserting mini-genes into cells. Furthermore, development of these methodologies will kick open the door to new biology and chemistry that has been logistically intractable previously. Nanopore technology will place molecular and sub-molecular analysis within the reach of the typical bench-top scientist or clinical lab-no longer limited to genomics or mass spectrometry specialists. Moreover, the prospects for synthetic biology-using nanopores to program or reprogram cells-are promising as well, but have been examined only at the level of a single cell, so far.

Background: In the past two decades, methods have been developed to measure the mechanical properties of single biomolecules. One of these methods, Magnetic tweezers, is amenable to aquisition of data on many single molecules simultaneously, but to take full advantage of this "multiplexing" ability, it is necessary to simultaneously incorprorate many capabilities that ahve been only demonstrated separately. Methods: Our custom built magnetic tweezer combines high multiplexing, precision bead tracking, and bidirectional force control into a flexible and stable platform for examining single molecule behavior. This was accomplished using electromagnets, which provide high temporal control of force while achieving force levels similar to permanent magnets via large paramagnetic beads. Results: Here we describe the instrument and its ability to apply 2-260 pN of force on up to 120 beads simultaneously, with a maximum spatial precision of 12 nm using a variety of bead sizes and experimental techniques. We also demonstrate a novel method for increasing the precision of force estimations on heterogeneous paramagnetic beads using a combination of density separation and bi-directional force correlation which reduces the coefficient of variation of force from 27% to 6%. We then use the instrument to examine the force dependence of uncoiling and recoiling velocity of type 1 fimbriae from Eschericia coli (E. coli) bacteria, and see similar results to previous studies. Conclusion: This platform provides a simple, effective, and flexible method for efficiently gathering single molecule force spectroscopy measurements. [ABSTRACT FROM AUTHOR]

By combining MD simulations and AFS experimental technique, we demonstrated a powerful approach for rational design and single molecule testing of novel inhibitor molecules which can block amyloid-amyloid binding – the first step of toxic amyloid oligomer formation. We designed and tested novel pseudo-peptide amyloid-β (Aβ) inhibitors that bind to the Aβ peptide and effectively prevent amyloid-amyloid binding. First, molecular dynamics (MD) simulations have provided information on the structures and binding characteristics of the designed pseudo-peptides targeting amyloid fragment Aβ (13 − 23). The binding affinities between the inhibitor and Aβ as well as the inhibitor to itself have been estimated using Umbrella Sampling calculations. Atomic Force Spectroscopy (AFS) was used to experimentally test several proposed inhibitors in their ability to block amyloid-amyloid binding – the first step of toxic amyloid oligomer formation. The experimental AFS data are in a good agreement with theoretical MD calculations and demonstrate that three proposed pseudo-peptides bind to amyloid fragment with different affinities and all effectively prevent Aβ-Aβ binding in similar way. We propose that the designed pseudo-peptides can be used as potential drug candidates to prevent Aβ toxicity in Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Mechanical unfolding of mutated apo, disulfide-reduced, monomeric superoxide dismutase 1 protein (SOD1) has been simulated via force spectroscopy techniques, using both an all-atom (AA), explicit solvent model and a coarse-grained heavy-atom Gō (HA-Gō) model. The HA-Gō model was implemented at two different pulling speeds for comparison. The most-common sequence of unfolding in the AA model agrees well with the most-common unfolding sequence of the HA-Gō model, when the same normalized pulling rate was used. Clustering of partially-native structures as the protein unfolds shows that the AA and HA-Gō models both exhibit a dominant pathway for early unfolding, which eventually bifurcates repeatedly to multiple branches after the protein is about half-unfolded. The force-extension curve exhibits multiple force drops, which are concomitant with jumps in the local interaction potential energy between specific β -strands in the protein. These sudden jumps in the potential energy coincide with the dissociation of specific pairs of β -strands, and thus intermediate unfolding events. The most common sequence of β -strand dissociation in the unfolding pathway of the AA model is β -strands 5, 4, 8, 7, 1, 2, then finally β -strands 3 and 6. The observation that β -strand 5 is among the first to unfold here, but the last to unfold in simulations of loop-truncated SOD1, could imply the existence of an evolutionary compensation mechanism, which would stabilize β -strands flanking long loops against their entropic penalty by strengthening intramolecular interactions. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. [ABSTRACT FROM AUTHOR]

Single molecule force spectroscopy (SMFS) was employed to investigate the interaction forces between aliphatic amino, hydroxyl and ether groups and aluminum oxide single crystal surfaces in an aqueous electrolyte at pH = 6. The force studies were based on the variation of the terminal group of polyethylene glycol which was bound via a S Au bond to the gold coated AFM tip. X-ray Photoelectron Spectroscopy (XPS) was performed to characterize the surface chemistry of the substrate. Force distance curves were measured between the PEG-NH 2 , OH and OCH 3 functionalized atomic force microscope (AFM) tip and the non-polar single crystalline Al 2 O 3 (11-20) surface. The experimental results exhibit non-equilibrium desorption events which hint at acid-base interactions of the electron donating hydroxyl and amino groups with Al-ions in the surface of the oxide. The observed desorption forces for the NH 2 , OH/Al 2 O 3 (11-20) were in the range of 100–200 pN. [ABSTRACT FROM AUTHOR]

As the active anticancer component of Rabdosia Rubescens, oridonin has been proved to show strong anticancer activity in cancer cells, which is also found to be closely related to its specific inhibition effects on the EGFR tyrosine kinase activity. In this study, atomic force microscopy based single molecule force spectroscopy (AFM-SMFS) was used for real-time and in-situ detection of EGF-EGFR interactions in living esophageal cancer KYSE-150 cells to evaluate the anticancer activity of oridonin for the first time. Oridonin was found to induce apoptosis and also reduce EGFR expression in KYSE-150 cells. AFM-SMFS results demonstrated that oridonin could inhibit the binding between EGF and EGFR in KYSE-150 cells by decreasing the unbinding force and binding probability for EGF-EGFR complexes, which was further proved to be closely associated with the intracellular ROS level. More precise mechanism studies based on AFM-SMFS demonstrated that oridonin treatment could decrease the energy barrier width, increase the dissociation off rate constant and decrease the activation energy of EGF-EGFR complexes in ROS dependent way, suggesting oridonin as a strong anticancer agent targeting EGF-EGFR interactions in cancer cells through ROS dependent mechanism. Our results not only suggested oridonin as a strong anticancer agent targeting EGF-EGFR interactions in ROS dependent mechanism, but also highlighted AFM-SMFS as a powerful technique for pharmacodynamic studies by detecting ligand-receptor interactions, which was also expected to be developed into a promising tool for the screening and mechanism studies of drugs. [ABSTRACT FROM AUTHOR]

We studied the interaction of the specific DNA aptamer sgc8c immobilized at the AFM tip with its corresponding receptor, the protein tyrosine kinase-7 (PTK7) embedded in the membrane of acute lymphoblastic leukemia (ALL) cells (Jurkat T-cells). Performing single molecule force spectroscopy (SMFS) experiments, we showed that the aptamer sgc8c bound with high probability (38.3 ± 7.48%) and high specificity to PTK7, as demonstrated by receptor blocking experiments and through comparison with the binding behavior of a nonspecific aptamer. The determined kinetic off-rate (k = 5.16 s) indicates low dissociation of the sgc8c-PTK7 complex. In addition to the pulling force experiments, simultaneous topography and recognition imaging (TREC) experiments using AFM tips functionalized with sgc8c aptamers were realized on the outer regions surface of surface-immobilized Jurkat cells for the first time. This allowed determination of the distribution of PTK7 without any labeling and at near physiological conditions. As a result, we could show a homogeneous distribution of PTK7 molecules on the outer regions of ALL cells with a surface density of 325 ± 12 PTK7 receptors (or small receptor clusters) per μm. [ABSTRACT FROM AUTHOR]

[Ru(bpy)2dppz]2+ and [Ru(phen)2dppz]2+ as the light switches of the deoxyribose nucleic acid (DNA) molecule have attracted much attention and have become a powerful tool for exploring the structure of the DNA helix. Their interactions have been intensively studied because of the excellent photophysical and photochemical properties of ruthenium compounds. In this perspective, this review describes the recent developments in the interactions of these two classic intercalated compounds with a DNA helix. The mechanism of the molecular light switch effect and the selectivity of these two compounds to different forms of a DNA helix has been discussed. In addition, the specific binding modes between them have been discussed in detail, for a better understanding the mechanism of the light switch and the luminescence difference. Finally, recent studies of single molecule force spectroscopy have also been included so as to precisely interpret the kinetics, equilibrium constants, and the energy landscape during the process of the dynamic assembly of ligands into a single DNA helix.

Ligand binding to receptors is one of the most important regulatory elements in biology as it is the initiating step in signaling pathways and cascades. Thus, precisely localizing binding sites and measuring interaction forces between cognate receptor–ligand pairs leads to new insights into the molecular recognition involved in these processes. Here we present a detailed protocol about applying a technique, which combines atomic force microscopy (AFM)-based recognition imaging and force spectroscopy for studying the interaction between (membrane) receptors and ligands on the single molecule level. This method allows for the selection of a single receptor molecule reconstituted into a supported lipid membrane at low density, with the subsequent quantification of the receptor–ligand unbinding force. Based on AFM tapping mode, a cantilever tip carrying a ligand molecule is oscillated across a membrane. Topography and recognition images of reconstituted receptors are recorded simultaneously by analyzing the downward and upward parts of the oscillation, respectively. Functional receptor molecules are selected from the recognition image with nanometer resolution before the AFM is switched to the force spectroscopy mode, using positional feedback control. The combined mode allows for dynamic force probing on different pre-selected molecules. This strategy results in higher throughput when compared with force mapping. Applied to two different receptor–ligand pairs, we validated the presented new mode.

[Display omitted] Classical cadherins are transmembrane proteins whose extracellular domains link neighboring cells, and whose intracellular domains connect to the actin cytoskeleton via β-catenin and α-catenin. The cadherin-catenin complex transmits forces that drive tissue morphogenesis and wound healing. In addition, tension-dependent changes in αE-catenin conformation enables it to recruit the actin-binding protein vinculin to cell–cell junctions, which contributes to junctional strengthening. How and whether multiple cadherin-complexes cooperate to reinforce cell–cell junctions in response to load remains poorly understood. Here, we used single-molecule optical trap measurements to examine how multiple cadherin-catenin complexes interact with F-actin under load, and how this interaction is influenced by the presence of vinculin. We show that force oriented toward the (−) end of the actin filament results in mean lifetimes 3-fold longer than when force was applied towards the barbed (+) end. We also measured force-dependent actin binding by a quaternary complex comprising the cadherin-catenin complex and the vinculin head region, which cannot itself bind actin. Binding lifetimes of this quaternary complex increased as additional complexes bound F-actin, but only when load was oriented toward the (−) end. In contrast, the cadherin-catenin complex alone did not show this form of cooperativity. These findings reveal multi-level, force-dependent regulation that enhances the strength of the association of multiple cadherin/catenin complexes with F-actin, conferring positive feedback that may strengthen the junction and polarize F-actin to facilitate the emergence of higher-order cytoskeletal organization. [ABSTRACT FROM AUTHOR]

BiP (immunoglobulin heavy-chain binding protein) is a Hsp70 monomeric ATPase motor that plays broad and crucial roles in maintaining proteostasis inside the cell. Structurally, BiP is formed by two domains, a nucleotide-binding domain (NBD) with ATPase activity connected by a flexible hydrophobic linker to the substrate-binding domain. While the ATPase and substrate binding activities of BiP are allosterically coupled, the latter is also dependent on nucleotide binding. Recent structural studies have provided new insights into BiP's allostery; however, the influence of temperature on the coupling between substrate and nucleotide binding to BiP remains unexplored. Here, we study BiP's binding to its substrate at the single molecule level using thermo-regulated optical tweezers which allows us to mechanically unfold the client protein and explore the effect of temperature and different nucleotides on BiP binding. Our results confirm that the affinity of BiP for its protein substrate relies on nucleotide binding, by mainly regulating the binding kinetics between BiP and its substrate. Interestingly, our findings also showed that the apparent affinity of BiP for its protein substrate in the presence of nucleotides remains invariable over a wide range of temperatures, suggesting that BiP may interact with its client proteins with similar affinities even when the temperature is not optimal. Thus, BiP could play a role as a "thermal buffer" in proteostasis., (© 2023 The Protein Society.)

The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets. [ABSTRACT FROM AUTHOR]

Lamins are mechanosensitive and elastic components of the nuclear lamina that respond to external mechanical cues by altering gene regulation in a feedback mechanism. Numerous mutations in A-type lamins cause a plethora of diverse diseases collectively termed as laminopathies, the majority of which are characterized by irregularly shaped, fragile, and plastic nuclei. These nuclei are challenged to normal mechanotransduction and lead to disease phenotypes. Here, we review our current understanding of the nucleocytoskeleton coupling in mechanotransduction mediated by lamins. We also present an up-to-date understanding of the methods used to determine laminar elasticity both at the bulk and single molecule level. [ABSTRACT FROM AUTHOR]

The mucus layer covering the gastrointestinal (GI) epithelium is critical in selecting and maintaining homeostatic interactions with our gut bacteria. However, the molecular details of these interactions are not well understood. Here, we provide mechanistic insights into the adhesion properties of the canonical mucus-binding protein (MUB), a large multi-repeat cell-surface adhesin found in Lactobacillus inhabiting the GI tract. We used atomic force microscopy to unravel the mechanism driving MUB-mediated adhesion to mucins. Using single-molecule force spectroscopy we showed that MUB displayed remarkable adhesive properties favouring a nanospring-like adhesion model between MUB and mucin mediated by unfolding of the multiple repeats constituting the adhesin. We obtained direct evidence for MUB self-interaction; MUB-MUB followed a similar binding pattern, confirming that MUB modular structure mediated such mechanism. This was in marked contrast with the mucin adhesion behaviour presented by Galectin-3 (Gal-3), a mammalian lectin characterised by a single carbohydrate binding domain (CRD). The binding mechanisms reported here perfectly match the particular structural organization of MUB, which maximizes interactions with the mucin glycan receptors through its long and linear multi-repeat structure, potentiating the retention of bacteria within the outer mucus layer. [ABSTRACT FROM AUTHOR]

The spore of Bacillus subtilis , a dormant type of cell, is surrounded by a complex multilayered protein structure known as the coat. It is composed of over 70 proteins and essential for the spore to withstand extreme environmental conditions and allow germination under favorable conditions. However, understanding how the properties of the coat arise from the interactions among all these proteins is an important challenge. Moreover, many specific protein–protein interactions among the coat proteins are crucial for coat assembly. In this study, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) was applied to investigate the interaction as a dynamic process between two morphogenetic coat proteins, CotE and CotZ. The unbinding force and kinetic parameters characterizing the interaction between CotE and CotZ were obtained. It is found that there is a strong affinity between CotE and CotZ. Furthermore, the assembly behaviors of CotE and CotZ, individually or in combination, were studied by AFM at solid-liquid interfaces. Our results revealed that CotE–CotZ assembly is dependent on their molar ratios and the interaction between CotE and CotZ involves in the CotE–CotZ assembly. [ABSTRACT FROM AUTHOR]

The fungus Candida albicans is the most common cause of mycotic infections in immunocompromised hosts. Little is known about the initial interactions between Candida and immune cell receptors, such as the C-type lectin dendritic cell-specific intracellular cell adhesion molecule-3 (ICAM-3)-grabbing non-integrin (DC-SIGN), because a detailed characterization at the structural level is lacking. DC-SIGN recognizes specific Candida-associated molecular patterns, that is , mannan structures present in the cell wall of Candida. The molecular recognition mechanism is however poorly understood. We postulated that small differences in mannan-branching may result in considerable differences in the binding affinity. Here, we exploit atomic force microscope-based dynamic force spectroscopy with single Candida cells to gain better insight in the carbohydrate recognition capacity of DC-SIGN. We demonstrate that slight differences in the N-mannan structure of Candida, that is, the absence or presence of a phosphomannan side chain, results in differences in the recognition by DC-SIGN as follows: (i) it contributes to the compliance of the outer cell wall of Candida, and (ii) its presence results in a higher binding energy of 1.6 kBT. The single-bond affinity of tetrameric DC-SIGN for wild-type C. albicans is ~10.7 kBT and a dissociation constant kD of 23 μM, which is relatively strong compared with other carbohydrate-protein interactions described in the literature. In conclusion, this study shows that DC-SIGN specifically recognizes mannan patterns on C. albicans with high affinity. Knowledge on the binding pocket of DC-SIGN and its pathogenic ligands will lead to a better understanding of how fungal-associated carbohydrate structures are recognized by receptors of the immune system and can ultimately contribute to the development of new anti-fungal drugs. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Spores formed by Bacillus subtilis are surrounded by a protective and multilayered shell, termed the coat, which grants the spores resistance to various environmental stresses and facilitates spore germination. The spore coat consists of more than seventy different proteins, arranged into at least four distinct structural layers: the undercoat, inner coat, outer coat and crust. However, how these proteins, especially the morphogenetic proteins, interact to establish the organized, functional coat layers remains poorly understood. CotY and CotZ as the components of the crust, play a morphogenetic role in the crust assembly around the spore. In this study, the single molecule force spectroscopy was used to investigate the interaction and dynamics between CotY and CotZ at the single-molecule level. The results show that homotypic interactions of CotY and CotZ and the heterotypic interaction between CotY and CotZ exist. Furthermore, the dissociation kinetics of the complexes were studied by monitoring the relationship between the unbinding forces and the loading rates at different pulling velocities. In this way, a series of kinetic parameters regarding the three different complexes were obtained. It revealed the strong interactions between CotY and CotZ, CotY and CotY, and a relatively weak interaction of CotZ and CotZ. [ABSTRACT FROM AUTHOR]

Highly stereoregular syndiotactic polypropylene (s-PP) with high crystallinity and melting temperature has unique physical properties such as high impact resistance and transparency, which is closely related to the chain conformation and crystal structure of s-PP. However, the molecular mechanism underlying the mechanical properties remains unclear, because the complex amorphous-crystalline network and the diverse crystal structure limit the classical characterizations. Here, we investigate the single-molecule force-induced melting in the melt-prepared and solution-prepared s-PP single crystal by using atomic force microscopy (AFM)-based single molecule force spectroscopy (SMFS). Our results show that the mechanical stability slightly increases with the increase of crystal thickness and the crystal thickness also has a significant impact on the melting pathway. The comparison of the melting pathways of s-PP and isotactic polypropylene (i-PP) suggests that the syndiotactic structure and lower tacticity of s-PP will increase the chain friction during slide and therefore suppresses the α c relaxation in the crystal region. Moreover, the difference of the chain folding trajectory in sectors (100) and (010) shows no effect on the mechanical stability. However, the mechanical stability of form I structure was higher than that of form II. [Display omitted] • Force-induced melting of single PP chain from the single crystals were quantified. • The effect of the crystal thickness and chain configuration on mechanical stability were studied. • The mechanical stability of form I structure was higher than that of form II. • The lower tacticity and bigger chain friction of s-PP suppresses the α c relaxation in the crystal region. [ABSTRACT FROM AUTHOR]

Background: The discharge of the Cnidarian stinging organelle, the nematocyst, is one of the fastest processes in biology and involves volume changes of the highly pressurised (150 bar) capsule of up to 50%. Hitherto, the molecular basis for the unusual biomechanical properties of nematocysts has been elusive, as their structure was mainly defined as a stress-resistant collagenous matrix. Results: Here, we characterise Cnidoin, a novel elastic protein identified as a structural component of Hydra nematocysts. Cnidoin is expressed in nematocytes of all types and immunostainings revealed incorporation into capsule walls and tubules concomitant with minicollagens. Similar to spider silk proteins, to which it is related at sequence level, Cnidoin possesses high elasticity and fast coiling propensity as predicted by molecular dynamics simulations and quantified by force spectroscopy. Recombinant Cnidoin showed a high tendency for spontaneous aggregation to bundles of fibrillar structures. Conclusions: Cnidoin represents the molecular factor involved in kinetic energy storage and release during the ultra-fast nematocyst discharge. Furthermore, it implies an early evolutionary origin of protein elastomers in basal metazoans. [ABSTRACT FROM AUTHOR]

Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell-cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 μm² areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding. [ABSTRACT FROM AUTHOR]

Molecular imprinting is an effective technique for preparing recognition sites which act as synthetic receptors on polymeric surfaces. Herein, we synthesized MIP surfaces with specific binding sites for streptavidin and characterized them at nanoscale by using two different atomic force microscopy (AFM) techniques. While the single molecule force spectroscopy (SMFS) reveals the unbinding kinetics between streptavidin molecule and binding sites, simultaneous topography and recognition imaging (TREC) was employed, for the first time, to directly map the binding sites on streptavidin imprinted polymers. Streptavidin modified AFM cantilever showed specific unbinding events with an unbinding force around 300 pN and the binding probability was calculated as 35.2% at a given loading rate. In order to prove the specificity of the interaction, free streptavidin molecules were added to AFM liquid cell and the binding probability was significantly decreased to 7.6%. Moreover, the recognition maps show that the smallest recognition site with a diameter of around ~ 21 nm which corresponds to a single streptavidin molecule binding site. We believe that the potential of combining SMFS and TREC opens new possibilities for the characterization of MIP surfaces with single molecule resolution under physiological conditions. [ABSTRACT FROM AUTHOR]

Investigation on the folding mode of a single polymer chain in its crystal is significant to the understanding of the mechanism of the fundamental crystallization as well as the engineering of new polymer crystal-based materials. Herein, we use the combined techniques of atomic force microscopy (AFM) imaging and force spectroscopy to pull a single polyethylene oxide (PEO) chain out of its spiral crystal in amyl acetate. From these data, the folding mode of polymer chains in the spiral crystal has been reconstructed. We find that the stems tilt in the typical flat area, leading to the decrease in the apparent lamellar height. While in the area of screw dislocation, the lamellar height gradually increases in the range of several nanometers. These results indicate that the combined techniques present a novel tool to directly unravel the chain folding mode of spiral crystals at single-molecule level. [ABSTRACT FROM AUTHOR]

ABSTRACT We provide theoretical tests of a novel experimental technique to determine mechanostability of proteins based on stretching a mechanically protected protein by single-molecule force spectroscopy. This technique involves stretching a homogeneous or heterogeneous chain of reference proteins (single-molecule markers) in which one of them acts as host to the guest protein under study. The guest protein is grafted into the host through genetic engineering. It is expected that unraveling of the host precedes the unraveling of the guest removing ambiguities in the reading of the force-extension patterns of the guest protein. We study examples of such systems within a coarse-grained structure-based model. We consider systems with various ratios of mechanostability for the host and guest molecules and compare them to experimental results involving cohesin I as the guest molecule. For a comparison, we also study the force-displacement patterns in proteins that are linked in a serial fashion. We find that the mechanostability of the guest is similar to that of the isolated or serially linked protein. We also demonstrate that the ideal configuration of this strategy would be one in which the host is much more mechanostable than the single-molecule markers. We finally show that it is troublesome to use the highly stable cystine knot proteins as a host to graft a guest in stretching studies because this would involve a cleaving procedure. Proteins 2014; 82:717-726. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: Alzheimer's disease is a neurodegenerative disease with no known cure and few effective treatment options. The principal neurotoxic agent is an oligomeric form of the amyloid-β peptide and one of the treatment options currently being studied is the inhibition of amyloid aggregation. In this work, we test a novel pseudopeptidic aggregation inhibitor designated as SG1. SG1 has been designed to bind at the amyloid-β self-recognition site and prevent amyloid-β from misfolding into β sheet. We used atomic force spectroscopy, a nanoscale measurement technique, to quantify the binding forces between two single amyloid peptide molecules. For the first time, we demonstrate that single molecule atomic force spectroscopy can be used to assess the effectiveness of amyloid aggregation inhibitors by measuring the experimental yield of binding and can potentially be used as a screening technique for quick testing of efficacy of inhibitor drugs for amyloid aggregation. [Copyright &y& Elsevier]

Compatible solutes are small, uncharged, zwitter ionic, osmotically active molecules produced and accumulated by microorganisms inside their cell to counteract different kinds of environmental stress. They enhance protein stability without interfering with the metabolic pathways even at molar concentrations. In this paper, we report the stabilizing effects of compatible solutes, ectoine, betaine and taurine on membrane protein bacteriorhodopsin at different concentrations. Using atomic force microscopy based single molecule force spectroscopy the impact of the osmolytes was quantified by measuring the forces required to pull the protein out of the membrane and the change in the persistence lengths of the unfolded polypeptide chain. Increase in unfolding forces were observed, indicating the strengthening of intramolecular interactions, which are vital for protein stability. The decrease in persistence lengths was recorded and showed increasing tendencies of the polypeptide strand to coil up. Interestingly, it was revealed that these molecules have different stabilizing effects on protein unfolding at different concentrations. The results show that the unfolding of single protein provides insight to the structure-dynamic relationship between the protein and compatible solute molecules at sub-nanometer scale. This also helps to understand the molecular mechanism involved in protein stabilization by organic osmolytes. [ABSTRACT FROM AUTHOR]

Highlights: [•] We show stress induced acceleration of platelets shape changes. [•] Time-lapse AFM imaging visualizes two phases of filopodia extrusion and contraction. [•] SMFS reveals a close to 100 percent activity of the integrin αIIbβ3. [ABSTRACT FROM AUTHOR]

Abstract: Flexible polymer linkers play an important role in various imaging and probing techniques that require surface immobilization, including atomic force microscopy (AFM). In AFM force spectroscopy, polymer linkers are necessary for the covalent attachment of molecules of interest to the AFM tip and the surface. The polymer linkers tether the molecules and provide their proper orientation in probing experiments. Additionally, the linkers separate specific interactions from nonspecific short-range adhesion and serve as a reference point for the quantitative analysis of single molecule probing events. In this report, we present our results on the synthesis and testing of a novel polymer linker and the identification of a number of potential applications for its use in AFM force spectroscopy experiments. The synthesis of the linker is based on the well-developed phosphoramidate (PA) chemistry that allows the routine synthesis of linkers with predetermined lengths and PA composition. These linkers are homogeneous in length and can be terminated with various functional groups. PA linkers with different functional groups were synthesized and tested in experimental systems utilizing different immobilization chemistries. We probed interactions between complementary DNA oligonucleotides; DNA and protein complexes formed by the site-specific binding protein SfiI; and interactions between amyloid peptide (Aβ42). The results of the AFM force spectroscopy experiments validated the feasibility of the proposed approach for the linker design and synthesis. Furthermore, the properties of the tether (length, functional groups) can be adjusted to meet the specific requirements for different force spectroscopy experiments and system characteristics, suggesting that it could be used for a large number of various applications. [Copyright &y& Elsevier]

Abstract: Single molecule force spectroscopy involves loading a chemical bond using an atomic force microscope and measuring the rupture forces required to break that bond. In 20years since its inception this technique developed into a robust way to extract a nearly complete set of the information about the bond that includes the bond energy, the kinetic parameters of the bond, and the geometry of the transition state. In this article we review the basic physics of the measurements, the model that is used for data interpretation, and go over the ways to extract the bond information from the experimental data. We also discuss several practical aspects of the measurements that are helpful to the planning and analysis of single molecule force spectroscopy experiments. [Copyright &y& Elsevier]

Abstract: Atomic force microscopy (AFM) is widely used in the biological sciences. Despite 25years of technical developments, two popular modes of bioAFM, imaging and single molecule force spectroscopy, remain hindered by relatively poor force precision and stability. Recently, we achieved both sub-pN force precision and stability under biologically useful conditions (in liquid at room temperature). Importantly, this sub-pN level of performance is routinely accessible using a commercial cantilever on a commercial instrument. The two critical results are that (i) force precision and stability were limited by the gold coating on the cantilevers, and (ii) smaller yet stiffer cantilevers did not lead to better force precision on time scales longer than 25ms. These new findings complement our previous work that addressed tip-sample stability. In this review, we detail the methods needed to achieve this sub-pN force stability and demonstrate improvements in force spectroscopy and imaging when using uncoated cantilevers. With this improved cantilever performance, the widespread use of nonspecific biomolecular attachments becomes a limiting factor in high-precision studies. Thus, we conclude by briefly reviewing site-specific covalent-immobilization protocols for linking a biomolecule to the substrate and to the AFM tip. [Copyright &y& Elsevier]

The role of the collagen-platelet interaction is of crucial importance to the haemostatic response during both injury and pathogenesis of the blood vessel wall. Of particular interest is the high affinity interaction of the platelet transmembrane receptor, alpha 2 beta 1, responsible for firm attachment of platelets to collagen at and around injury sites. We employ single molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) to study the interaction of the I-domain from integrin alpha 2 beta 1 with a synthetic collagen related triple-helical peptide containing the high-affinity integrin-binding GFOGER motif, and a control peptide lacking this sequence, referred to as GPP. By utilising synthetic peptides in this manner we are able to study at the molecular level subtleties that would otherwise be lost when considering cell-to-collagen matrix interactions using ensemble techniques. We demonstrate for the first time the complexity of this interaction as illustrated by the complex multi-peaked force spectra and confirm specificity using control blocking experiments. In addition we observe specific interaction of the GPP peptide sequence with the I-domain. We propose a model to explain these observations. [ABSTRACT FROM AUTHOR]

Single molecule force spectroscopy is a technique that can be used to probe the interaction force between individual biomolecular species. We focus our attention on the tip and sample coupling chemistry, which is crucial to these experiments. We utilised a novel approach of mixed self-assembled monolayers of alkanethiols in conjunction with a heterobifunctional crosslinker. The effectiveness of the protocol is demonstrated by probing the biotin-avidin interaction. We measured unbinding forces comparable to previously reported values measured at similar loading rates. Specificity tests also demonstrated a significant decrease in recognition after blocking with free avidin. [ABSTRACT FROM AUTHOR]

Abstract: Atomic force microscopy (AFM) and AFM-based force spectroscopy was used to study the desorption of individual chitosan polymer chains from substrates with varying chemical composition. AFM images of chitosan adsorbed onto a flat mica substrate show elongated single strands or aggregated bundles. The aggregated state of the polymer is consistent with the high level of flexibility and mobility expected for a highly positively charged polymer strand. Conversely, the visualization of elongated strands indicated the presence of stabilizing interactions with the substrate. Surfaces with varying chemical composition (glass, self-assembled monolayer of mercaptoundecanoic acid/decanethiol and polytetrafluoroethylene (PTFE)) were probed with chitosan modified AFM tips and the corresponding desorption energies, calculated from plateau-like features, were attributed to the desorption of individual polymer strands. Desorption energies of 2.0±0.3×10−20 J, 1.8±0.3×10−20 J and 3.5±0.3×10−20 J were obtained for glass, SAM of mercaptoundecanoic/dodecanethiol and PTFE, respectively. These single molecule level results can be used as a basis for investigating chitosan and chitosan-based materials for biomaterial applications. [ABSTRACT FROM AUTHOR]

facile approach to assembled virus film with tunable structure is presented. Rod-like tobacco mosaic virus (TMV) was selected as the prototype in this study for its anisotropic structural feature. TMV can either 'lie down' or 'stand up' on gold substrate by tuning the solution pH. A quartz crystal microbalance with dissipation monitoring was used to monitor the pH-dependent self-assembly behavior of TMV nanoparticles, and atomic force microscopy and single molecule force spectroscopy further confirmed the different assembly structures. [ABSTRACT FROM AUTHOR]

Understanding how monomeric proteins fold under in vitro conditions is crucial to describing their functions in the cellular context. Significant advances in theory and experiments have resulted in a conceptual framework for describing the folding mechanisms of globular proteins. The sizes of proteins in the denatured and folded states, cooperativity of the folding transition, dispersions in the melting temperatures at the residue level, and timescales of folding are, to a large extent, determined by N, the number of residues. The intricate details of folding as a function of denaturant concentration can be predicted by using a novel coarse-grained molecular transfer model. By watching one molecule fold at a time, using single-molecule methods, investigators have established the validity of the theoretically anticipated heterogeneity in the folding routes and the N-dependent timescales for the three stages in the approach to the native state. Despite the successes of theory, of which only a few examples are documented here, we conclude that much remains to be done to solve the protein folding problem in the broadest sense. [ABSTRACT FROM AUTHOR]

Abstract: Crystalline bacterial cell surface layers (S-layers) show the ability to recrystallize into highly regular pattern on solid supports. In this study, the genetically modified S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177, carrying a hexa-histidine tag (His6-tag) at the C-terminus, was used to generate functionalized two-dimensional nanoarrays on a silicon surface. Atomic force microscopy (AFM) was applied to explore the topography and the functionality of the fused His6-tags. The accessibility of the His6-tags was demonstrated by in-situ anti-His-tag antibody binding to the functional S-layer array. The metal binding properties of the His6-tag was investigated by single molecule force microscopy. For this purpose, newly developed tris–NTA was tethered to the AFM tips via a flexible polyethylene glycol (PEG) linker. The functionalized tips showed specific interactions with S-layer containing His6-tags in the presence of nickel ions. Thus the His6-tag is located at the outer surface of the S-layer and can be used for stable but reversible attachment of functional tris–NTA derivatives. [Copyright &y& Elsevier]

Polymerized barnacle glue was studied by atomic force microscopy (AFM), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and chemical staining. Nanoscale structures exhibiting rod-shaped, globular and irregularly-shaped morphologies were observed in the bulk cement of the barnacle Amphibalanus amphitrite (=Balanus amphitrite) by AFM. SEM coupled with energy dispersive X-ray (EDX) provided chemical composition information, making evident the organic nature of the rod-shaped nanoscale structures. FTIR spectroscopy gave signatures of β-sheet and random coil conformations. The mechanical properties of these nanoscale structures were also probed using force spectroscopy and indentation with AFM. Indentation data yielded higher elastic moduli for the rod-shaped structures when compared with the other structures in the bulk cement. Single molecule AFM force-extension curves on the matrix of the bulk cement often exhibited a periodic sawtooth-like profile, observed in both the extend and retract portions of the force curve. Rod-shaped structures stained with amyloid protein-selective dyes (Congo red and thioflavin-T) revealed that about 5% of the bulk cement were amyloids. A dominant 100 kDa cement protein was found to be mechanically agile, using repeating hydrophobic structures that apparently associate within the same protein or with neighbors, creating toughness on the 1–100 nm length scale. [ABSTRACT FROM PUBLISHER]

Abstract: By combining single-molecule atomic force microscopy (AFM), proline mutagenesis and steered molecular dynamics (SMD) simulations, we investigated the mechanical unfolding dynamics and mechanical design of the third fibronectin type III domain of tenascin-C (TNfn3) in detail. We found that the mechanical stability of TNfn3 is similar to that of other constituting FnIII domains of tenascin-C, and the unfolding process of TNfn3 is an apparent two-state process. By employing proline mutagenesis to block the formation of backbone hydrogen bonds and introduce structural disruption in β sheet, we revealed that in addition to the important roles played by hydrophobic core packing, backbone hydrogen bonds in β hairpins are also responsible for the overall mechanical stability of TNfn3. Furthermore, proline mutagenesis revealed that the mechanical design of TNfn3 is robust and the mechanical stability of TNfn3 is very resistant to structural disruptions caused by proline substitutions in β sheets. Proline mutant F88P is one exception, as the proline mutation at position 88 reduced the mechanical stability of TNfn3 significantly and led to unfolding forces of <20 pN. This result suggests that Phe88 is a weak point of the mechanical resistance for TNfn3. We used SMD simulations to understand the molecular details underlying the mechanical unfolding of TNfn3. The comparison between the AFM results and SMD simulations revealed similarities and discrepancies between the two. We compared the mechanical unfolding and design of TNfn3 and its structural homologue, the tenth FnIII domain from fibronectin. These results revealed the complexity underlying the mechanical design of FnIII domains and will serve as a starting point for systematically analyzing the mechanical architecture of other FnIII domains in tenascins-C, and will help to gain a better understanding of some of the complex features observed for the stretching of native tenascin-C. [Copyright &y& Elsevier]

Abstract: Tenascin-X is an extracellular matrix protein and binds a variety of molecules in extracellular matrix and on cell membrane. Tenascin-X plays important roles in regulating the structure and mechanical properties of connective tissues. Using single-molecule atomic force microscopy, we have investigated the mechanical properties of bovine tenascin-X in detail. Our results indicated that tenascin-X is an elastic protein and the fibronectin type III (FnIII) domains can unfold under a stretching force and refold to regain their mechanical stability upon the removal of the stretching force. All the 30 FnIII domains of tenascin-X show similar mechanical stability, mechanical unfolding kinetics, and contour length increment upon domain unfolding, despite their large sequence diversity. In contrast to the homogeneity in their mechanical unfolding behaviors, FnIII domains fold at different rates. Using the 10th FnIII domain of tenascin-X (TNXfn10) as a model system, we constructed a polyprotein chimera composed of alternating TNXfn10 and GB1 domains and used atomic force microscopy to confirm that the mechanical properties of TNXfn10 are consistent with those of the FnIII domains of tenascin-X. These results lay the foundation to further study the mechanical properties of individual FnIII domains and establish the relationship between point mutations and mechanical phenotypic effect on tenascin-X. Moreover, our results provided the opportunity to compare the mechanical properties and design of different forms of tenascins. The comparison between tenascin-X and tenascin-C revealed interesting common as well as distinguishing features for mechanical unfolding and folding of tenascin-C and tenascin-X and will open up new avenues to investigate the mechanical functions and architectural design of different forms of tenascins. [Copyright &y& Elsevier]

Abstract: Multidimensional energy landscapes are an intrinsic property of proteins and define their dynamic behavior as well as their response to external stimuli. In order to explore the energy landscape and its implications on the dynamic function of proteins dynamic force spectroscopy and steered molecular dynamics (SMD) simulations have proved to be important tools. In this study, these techniques have been employed to analyze the influence of the direction of the probing forces on the complex of an antibody fragment with its peptide antigen. Using an atomic force microscope, experiments were performed where the attachment points of the 12 amino acid long peptide antigen were varied. These measurements yielded clearly distinguishable basal dissociation rates and potential widths, proving that the direction of the applied force determines the unbinding pathway. Complementary atomistic SMD simulations were performed, which also show that the unbinding pathways of the system are dependent on the pulling direction. However, the main barrier to be crossed was independent of the pulling direction and is represented by a backbone hydrogen bond between GlyH-H40 of the antibody fragment and GluOε-6peptide of the peptide. For each pulling direction, the observed barriers can be correlated with the rupture of specific interactions, which stabilize the bound complex. Furthermore, although the SMD simulations were performed at loading rates exceeding the experimental rates by orders of magnitude due to computational limitations, a detailed comparison of the barriers that were overcome in the SMD simulations with the data obtained from the atomic force microscope unbinding experiments show excellent agreement. [Copyright &y& Elsevier]

Abstract: Single molecule force spectroscopy (SMFS) is a powerful means in studying the intramolecular and intermolecular interactions in polymer and supramolecular systems, thus opening new horizons for nanomechanic chemistry. This feature article concerns mainly how to employ AFM-based SMFS to obtain some of the important information which is not available by conventional characterizing methods, including single chain mechanics, the interfacial conformation and adhesive energy of polymers, interaction between macromolecules and small molecules, and the direct measurement of intermolecular forces. In addition, some of the recent advances in using the SMFS to bottom–up assembly of functional nanostructure are also discussed. [Copyright &y& Elsevier]

Abstract: Group B Streptococci (GBS) is a leading cause of sepsis and meningitis in neonates and immunocompromised adults in western countries. GBS do not bind to fibronectin (Fn) in solution, but will bind to Fn adsorbed onto a solid surface. The reason for the specificity of this binding is unknown. Single molecule force spectroscopy was used to test the hypothesis that GBS, through streptococcal C5a peptidase (ScpB) molecules present on the surface of the bacteria, binds to a motif created by the juxtaposition of multiple adjacent Fn molecules. Atomic force microscopy (AFM) topographical images of adsorbed Fn deposited from various Fn coating concentrations were used to determine the Fn surface concentration. ScpB was tethered to an AFM tip with all surface modifications characterized by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. At the lowest Fn coverages the probability of observing a ScpB–Fn binding event increased linearly with Fn surface coverage. As an Fn monolayer was reached the probability of a ScpB–Fn binding event occurring increased markedly (∼50 fold), with a concomitant increase in the rupture force from 17pN to 33pN. These results are consistent with the hypothesis that ScpB binds to a motif created by the juxtaposition of multiple Fn molecules. [Copyright &y& Elsevier]

Abstract: We have recently developed a new method for directly measuring the spring constant of single molecules and molecular complexes on a real-time basis [L.A. Chtcheglova, G.T. Shubeita, S.K. Sekatskii, G. Dietler, Biophys. J. 86 (2004) 1177]. The technique combines standard force spectroscopy with a small dithering of tip. Changes in the amplitude of the oscillations are measured as a function of the pulling-off force to yield the spring constant of the complex. In this report, we present the first results of combination of this approach with the force-clamp spectroscopy. The standard atomic-force microscope has been supplemented with an electronic unit, which is capable of realizing an arbitrary force function, and permits the force-loading regime to be interrupted at any time. Using this method, the time needed to rupture a single bond can be measured as a function of the force that is required to maintain the complex in a stretched condition. The energy landscape of the avidin–biotin complex is explored and discussed. [Copyright &y& Elsevier]