Single-molecule Force Spectroscopy Research Articles

Two-Molecule Force Spectroscopy on Proteins.

Many elastomeric proteins, which play important roles in a wide range of biological processes, exist as parallel/antiparallelly arranged dimers or multimers to perform their mechanobiological functions. For example, in striated muscle sarcomeres, the giant muscle protein titin exists as hexameric bundles to mediate the passive elasticity of muscles. However, it has not been possible to directly probe the mechanical properties of such parallelly arranged elastomeric proteins. And it remains unknown if the knowledge obtained from single-molecule force spectroscopy studies can be directly extrapolated to such parallelly/antiparallelly arranged systems. Here, we report the development of atomic force microscopy (AFM)-based two-molecule force spectroscopy to directly probe the mechanical properties of two elastomeric proteins that are arranged in parallel. We developed a twin-molecule approach to allow two parallelly arranged elastomeric proteins to be picked up and stretched simultaneously in an AFM experiment. Our results clearly revealed the mechanical features of such parallelly arranged elastomeric proteins during force-extension measurements and allowed for the determination of mechanical unfolding forces of proteins in such an experimental setting. Our study provides a general and robust experimental strategy to closely mimic the physiological condition of such parallel elastomeric protein multimers.

Oligo[2]catenane That Is Robust at Both the Microscopic and Macroscopic Scales

Polycatenanes are extremely attractive topological architectures on account of their high degrees of conformational freedom and multiple motion patterns of the mechanically interlocked macrocycles. However, exploitation of these peculiar structural and dynamic characteristics to develop robust catenane materials is still a challenging goal. Herein, we synthesize an oligo[2]catenane that showcases mechanically robust properties at both the microscopic and macroscopic scales. The key feature of the structural design is controlling the force-bearing points on the metal-coordinated core of the [2]catenane moiety that is able to maximize the energy dissipation of the oligo[2]catenane via dissociation of metal-coordination bonds and then activation of sequential intramolecular motions of circumrotation, translation, and elongation under an external force. As such, at the microscopic level, the single-molecule force spectroscopy measurement exhibits that the force to rupture dynamic bonds in the oligo[2]catenane reaches a record high of 588 ± 233 pN. At the macroscopic level, our oligo[2]catenane manifests itself as the toughest catenane material ever reported (15.2 vs 2.43 MJ/m3). These fundamental findings not only deepen the understanding of the structure-property relationship of poly[2]catenanes with a full set of dynamic features but also provide a guiding principle to fabricate high-performance mechanically interlocked catenane materials.

DNA at conductive interfaces: What can atomic force microscopy offer?

This review is devoted to the analysis of the capabilities of atomic force microscopy (AFM) in the study of the behavior of DNA at conducting solid–liquid and solid-air interfaces under electrochemical control and after the influence of the potential differences. The first part of the review presents the main aspects of AFM, the physical and chemical properties of DNA, and the behavior of DNA at conductive interfaces under the action of an electric field. The second part highlights the main categories of AFM applicability for DNA analysis at conducting interfaces from the point of view of “electrical stimulus-structure”, “process-structure”, and “process-structure-electrochemical properties” correlation. The applicability of AFM's main capabilities is shown: (i) visualization (of both monolayers and single molecules and hybridization complexes of DNA on a conductive surface), (ii) single-molecule force spectroscopy under electrochemical control, (iii) manipulation of DNA position at the nanoscale level. This review can be a starting point for further multidisciplinary DNA studies at electrochemical interfaces.

Single-Chain Mechanics at Low Temperatures: A Study of Three Kinds of Polymers from 153 to 300 K

The single-chain mechanics of three kinds of polymers (i.e., polydimethylsiloxane, polyoxymethylene, and poly(methyl methacrylate)) has been investigated from room temperature to 153 K by atomic force microscopy-based single-molecule force spectroscopy in high vacuum and a theoretical model. The stretched part of the individual polymer chain can be regarded as an isolated chain. In the broad range of temperature, the isolated chain shows a gradual change in single-chain mechanics, which can be depicted nicely by an elastic model. Further analysis shows that with thermal fluctuation, the single bonds (e.g., Si–O or C–O) in the backbone of an isolated chain can rotate freely in the temperature range in the timescale of single-molecule manipulation. To our best knowledge, this is the first experimental study on the single-chain mechanics at cryogenic temperatures, which validates the theoretical prediction that the transition temperature of an isolated chain is much lower than that of the corresponding bulk polymer material.

Synthesis and Ring-Opening Metathesis Polymerization of a Strained trans-Silacycloheptene and Single-Molecule Mechanics of Its Polymer.

The cis- and trans-isomers of a silacycloheptene were selectively synthesized by the alkylation of a silyl dianion, a novel approach to strained cycloalkenes. The trans-silacycloheptene (trans-SiCH) was significantly more strained than the cis isomer, as predicted by quantum chemical calculations and confirmed by crystallographic signatures of a twisted alkene. Each isomer exhibited distinct reactivity toward ring-opening metathesis polymerization (ROMP), where only trans-SiCH afforded high-molar-mass polymer under enthalpy-driven ROMP. Hypothesizing that the introduction of silicon might result in increased molecular compliance at large extensions, we compared poly(trans-SiCH) to organic polymers by single-molecule force spectroscopy (SMFS). Force-extension curves from SMFS showed that poly(trans-SiCH) is more easily overstretched than two carbon-based analogues, polycyclooctene and polybutadiene, with stretching constants that agree well with the results of computational simulations.

Discrimination between cyclic nucleotides in a cyclic nucleotide-gated ion channel.

Cyclic nucleotide-gated ion channels are crucial in many physiological processes such as vision and pacemaking in the heart. SthK is a prokaryotic homolog with high sequence and structure similarities to hyperpolarization-activated and cyclic nucleotide-modulated and cyclic nucleotide-gated channels, especially at the level of the cyclic nucleotide binding domains (CNBDs). Functional measurements showed that cyclic adenosine monophosphate (cAMP) is a channel activator while cyclic guanosine monophosphate (cGMP) barely leads to pore opening. Here, using atomic force microscopy single-molecule force spectroscopy and force probe molecular dynamics simulations, we unravel quantitatively and at the atomic level how CNBDs discriminate between cyclic nucleotides. We find that cAMP binds to the SthK CNBD slightly stronger than cGMP and accesses a deep-bound state that a cGMP-bound CNBD cannot reach. We propose that the deep binding of cAMP is the discriminatory state that is essential for cAMP-dependent channel activation.

PH-Dependent PdS3 Site in α3DIV Revealed by Single-Molecule Force Spectroscopy.

Lead is a toxic metal harmful to human beings because of its long history and wide use in human society. The interaction of lead with proteins is one of the most common ways it exerts toxicity. Due to its thiophilic property, lead targets thiol-rich proteins with high affinity and forms stable Pb-S bonds, which may replace the originally bound metal ion and incapacitate the protein/enzyme. Thus, the knowledge of the Pb-S bonds in proteins is important. To study Pb-S bonds, we chose a de novo designed protein α3DIV, able to bind Pb(II) via its three cysteines, as the model protein. Using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), we proved the formation of a triangular pyramidal PbS3 site in α3DIV by detecting specific Pb-S bond rupture signals, including the previously undetected Pb-S(Cys67) bond. Moreover, the pH-dependent weakening of Pb-S bond strength was revealed and quantified, leading to the dissociation of the PbS3 site at pH 4.5.

Anisotropic Friction in a Ligand-Protein Complex.

The effect of an externally applied directional force on molecular friction is so far poorly understood. Here, we study the force-driven dissociation of the ligand-protein complex biotin-streptavidin and identify anisotropic friction as a not yet described type of molecular friction. Using AFM-based stereographic single molecule force spectroscopy and targeted molecular dynamics simulations, we find that the rupture force and friction for biotin-streptavidin vary with the pulling angle. This observation holds true for friction extracted from Kramers' rate expression and by dissipation-corrected targeted molecular dynamics simulations based on Jarzynski's identity. We rule out ligand solvation and protein-internal friction as sources of the angle-dependent friction. Instead, we observe a heterogeneity in free energy barriers along an experimentally uncontrolled orientation parameter, which increases the rupture force variance and therefore the overall friction. We anticipate that anisotropic friction needs to be accounted for in a complete understanding of friction in biomolecular dynamics and anisotropic mechanical environments.

Revealing the Flexibility of Inorganic Sub-Nanowires by Single-Molecule Force Spectroscopy

Revealing the Flexibility of Inorganic Sub-Nanowires by Single-Molecule Force Spectroscopy

Single-Molecule Force Spectroscopy Reveals Cation-π Interactions in Aqueous Media Are Highly Affected by Cation Dehydration.

Cation-π interactions underlie many important processes in biology and materials science. However, experimental investigations of cation-π interactions in aqueous media remain challenging. Here, we studied the cation-π binding strength and mechanism by pulling two hydrophobic polymers with distinct cation binding properties, i.e., poly-pentafluorostyrene and polystyrene, in aqueous media using single-molecule force spectroscopy and nuclear magnetic resonance measurement. We found that the interaction strengths linearly depend on the cation concentrations, following the order of Li^{+}<NH_{4}^{+}<Na^{+}<K^{+}. The binding energies are 0.03-0.23 kJ mol^{-1} M^{-1}. This order is distinct from the strength of cation-π interactions in gas phase and may be caused by the different dehydration ability of the cations. Taken together, our method provides a unique perspective to investigate cation-π interactions under physiologically relevant conditions.

Chain Sliding versus β-Sheet Formation upon Shearing Single α-Helical Coiled Coils.

Coiled coils (CCs) are key building blocks of biogenic materials and determine their mechanical response to large deformations. Of particular interest is the observation that CC-based materials display a force-induced transition from α-helices to mechanically stronger β-sheets (αβT). Steered molecular dynamics simulations predict that this αβT requires a minimum, pulling speed-dependent CC length. Here, de novo designed CCs with a length between four to seven heptads are utilized to probe if the transition found in natural CCs can be mimicked with synthetic sequences. Using single-molecule force spectroscopy and molecular dynamics simulations, these CCs are mechanically loaded in shear geometry and their rupture forces and structural responses to the applied load are determined. Simulations at the highest pulling speed (0.01nm ns-1 ) show the appearance of β-sheet structures for the five- and six-heptad CCs and a concomitant increase in mechanical strength. The αβT is less probable at a lower pulling speed of 0.001nm ns-1 and is not observed in force spectroscopy experiments. For CCs loaded in shear geometry, the formation of β-sheets competes with interchain sliding. β-sheet formation is only possible in higher-order CC assemblies or in tensile-loading geometries where chain sliding and dissociation are prohibited.

Increasing the Mechanical Stability of Polymer-Gold Interfacial Connection: A Parallel Covalent Strategy.

Thiol-gold (S-Au) chemistry has been widely used in coating and functionalizing gold surfaces because it is robust and highly efficient. However, recent studies have shown that the S-Au-based self-assembled monolayers can lead to significant instability under external mechanical loading (e.g., in a swelled polymer film). Such instability limits further applications of S-Au chemistry-based functional materials. Here, we report a surface-modifying procedure based on a parallel covalent strategy. By employing dendritic macromolecules as a "middle layer" between the gold surface and polymer, the interfacial connecting strength increased by at least 350% as revealed by atomic force microscopy-based single molecule force spectroscopy (AFM-SMFS). The ultimate cleavage structure is confirmed to be an amide bond by control SMFS experiments, fluorescent microscopy, and dynamic force spectroscopy. This study/concept paves the way to prepare stable stimuli-responsive polymer brushes on solid surfaces and study mechanophores with high force stability.

Simultaneous Force and Darkfield Measurements Reveal Solvent-Dependent Axial Control of Optically Trapped Gold Nanoparticles

Single molecule force spectroscopy using optical tweezers (OT) has enabled nanoresolved measurements of dynamic biological processes but not of synthetic molecular mechanisms. Standard OT probes made from silica or polystyrene are incompatible with trapping in organic solvents for solution phase chemistry or with force-detected absorption spectroscopies. Here, we demonstrate optical trapping of gold nanoparticles in both aqueous and organic conditions using a custom OT and darkfield instrument which can uniquely measure force and scattering spectra of single gold nanoparticles (Au NPs) simultaneously. Our work reveals that standard models of trapping developed for aqueous conditions cannot account for the trends observed in different media here. We determine that higher pushing forces mitigate the increase in trapping force in higher index organic solvents and lead to axial displacement of the particle which can be controlled through trap intensity. This work develops a new model framework incorporating axial forces for understanding nanoparticle dynamics in an optical trap. These results establish the combined darkfield OT with Au NPs as an effective OT probe for single molecule and single particle spectroscopy experiments, with three-dimensional nanoscale control over NP location.

Investigation on protein dimerization and evaluation of medicine effects by single molecule force spectroscopy

Monitoring the dimerization state of the mesenchymal-epithelial transition factor (Met) was essential for in-depth understanding of the tumor signal transduction network. At present, the dimerization activation pathway of Met protein was mainly studied at the macro level, while the research at the single molecule level was far from comprehensive. Herein, the dimerization activation of Met protein's extracellular domain induced by ligand hepatocyte growth factor (HGF) was dynamically studied by single-molecule force spectroscopy. Met protein was immobilized on a biomimetic lipid membrane for ensuring its physiological environment, and then the Met dimers were recognized by bivalent probe which was formed by two Met-binding aptamers. Then the dimeric state of Met protein could be distinguished from monomeric state of Met protein through some parameters, (such as unimodal ratio, bimodal ratio and separation work). The unimodal indicates the occurrence of single molecule binding event, and the bimodal represents the occurrence of double binding event (also represents the presence of Met dimer). Before HGF treatment, most of the Met protein on the lipid membrane was still in the form of monomer, so the unimodal ratio in the force curve was larger (78.8 ± 5.2%), and the bimodal ratio was smaller (17.0 ± 4.1%). After HGF treatment, the unimodal ratio decreased to 54.0 ± 7.4%, and the bimodal ratio increased to 43.2 ± 7.3%. It was due to the formation of dimers after the binding of Met protein on the fluidity lipid membrane with HGF. In addition, the average separation work increased to about 2 times after HGF treatment. Given that studies of Met protein dimerization inhibitors have contributed to the development of more potent and safe inhibitors to significantly inhibit tumor metastasis, the effects of different medicines (including anticoagulant medicines, different antibiotics and anti-cancer medicines) on the dimerization activation of Met protein were then explored by the platform described above. The results showed that anticoagulant medicines heparin and its analogs can significantly inhibit HGF-mediated Met protein activation, while different antibiotics and anticancer medicines had no significant effect on the dimerization of Met protein. This work provided a platform for studying protein dimerization as well as for screening Met protein dimerization inhibitors at the single-molecule level.

Revealing the Flexibility of Inorganic Sub-nanowires by Single-Molecule Force Spectroscopy

Revealing the Flexibility of Inorganic Sub-nanowires by Single-Molecule Force Spectroscopy

Quantification of carboxylate-bridged di-zinc site stability in protein due ferri by single-molecule force spectroscopy.

Carboxylate-bridged diiron proteins belong to a protein family involved in different physiological processes. These proteins share the conservative EXXH motif, which provides the carboxylate bridge and is critical for metal binding. Here, we choose de novo-designed single-chain due ferri protein (DFsc), a four-helical protein with two EXXH motifs as a model protein, to study the stability of the carboxylate-bridged di-metal binding site. The mechanical and kinetic properties of the di-Zn site in DFsc were obtained by atomic force microscopy-based single-molecule force spectroscopy. Zn-DFsc showed a considerable rupture force of ~200 pN, while the apo-protein is mechanically labile. In addition, multiple rupture pathways were observed with different probabilities, indicating the importance of the EXXH-based carboxylate-bridged metal site. These results demonstrate carboxylate-bridged di-metal site is mechanically stable and improve our understanding of this important type of metalloprotein.

Modulation of non-covalent wet adhesion and cohesion via proximally immobilized non-lysine pair structure

Marine mussels have evolved the secretion of diversified mussel foot proteins to firmly attach to wet, salt-crusty, corroded, and fouled surfaces. Biomimicry of this robust wet adhesion via translating sticky biological molecules is still at an impasse mainly due to the lack of available materials and methodology to fully validate the underline mechanisms of adhesion and cohesion. Increasing sources of evidence have shown that the pair structure of protonated 3,4-dihydroxyphenylalanine (DOPA) and lysine contributes to the adhesion on substrates. However, it is still an uncultivated field to study the roles of those commonly distributed lysine and non-lysine pair structures in the adhesion to different surfaces, while remaining the intermolecular cohesion strength of the pair structures. Herein, the adhesive and cohesive properties of precisely designed single-molecule domains were systematically analyzed using single-molecule force spectroscopy after surface modification of probe tips. It was revealed a record-high adhesion force of ∼ 1000 pN for DOPA-lysine on cation-rich substrates (mica) and tyrosine-Lys on cation-deficient substrates (hydrophilic modified silicon nitride, Si3N4(OH)) due to an identified hydrophobic force. DOPA-phenylalanine and DOPA-tryptophan pairs were found to show versatile strong adhesion to both cation-rich and -deficient substrates, spanning from 750 pN to 2250 pN. Furthermore, our results revealed that Phe-Lys had an extraordinarily robust cohesion strength, up to 12,000 pN due to cation-π interactions. This study broadens our view of peptides adhesion, provides single molecule proofs toward understanding the mechanism of pair structures in natural adhesive proteins, as well as the design principles of the artificial adhesive layer.

Towards a new modular polyprotein system compatible with single-molecule force spectroscopy by atomic force microscopy and magnetic tweezers.

Single-molecule force spectroscopy (SMFS) has provided multiple advancements in the understanding of the free energy landscape of proteins under force. Specific covalent tethering of proteins to surfaces has improved both the yield and the quality of data acquisition in Atomic Force Spectroscopy (AFS) and Magnetic Tweezers (MT). Given the complementary information obtained by AFS and MT, we propose a modular polyprotein system with HaloTag specific covalent tethering to surfaces, in order to enable testing of the same protein preparation by both techniques. The modular design allows to easily include the proteins of interest in the system via SnoopTag/SnoopCatcher and SpyTag/SpyCatcher orthogonal spontaneous isopeptide bonding and can exploit cohesin-dockerin reversible interaction. The system also includes fingerprinting protein markers for improved accuracy by concurrent experiments and elastin-like polypeptides to reduce non-specific interactions with the surface. In summary, the proposed modular polyprotein strategy is intended to provide an accessible and versatile setup, easily adaptable to different proteins of interest, and includes recent improvements in sample preparation to increase the yield, quality and accuracy of SMFS experiments.

Nucleosome chaperone activity of LEDGF and HDGF2 characterized with single molecule force spectroscopy.

The nucleosome, two turns of DNA wrapped around a histone octamer, is the fundamental structure of DNA organization in eukaryotic cells. Lens epithelium-derived growth factor (LEDGF) and hepatoma-derived growth factor 2 (HDGF2) are recently discovered proteins that facilitate transcription without ATP hydrolysis, suggesting roles as nucleosome chaperones during the integration of lentiviral DNA, including HIV-1 DNA, into infected cell genomes. Both proteins have methyl-lysine reading PWWP domains that can recognize H3K36me2/3 marks. To better understand these activities, we used optical tweezers to stretch reconstituted DNA nucleosome arrays containing twelve Widom 601 positioning sequences with unmodified (WT) and methylated histones (mimicking the H3K36me2/3 modifications), in the presence of LEDGF and HDGF2. To test the function of these nucleosome chaperone proteins during HIV-1 DNA integration, we exposed the modified nucleosomes to HIV-1 integrase (IN) together with equal concentrations of LEDGF and HDGF2. Analysis of force-extension data showed that while some destabilization of the inner wrapping of nucleosomes occurs due to histone methylation, exposure to chaperones results in further destabilization. Notably, the outer half turn unwrapping of nucleosomes that typically occurs at lower forces disappears with chaperone binding. Kinetic analysis of histone-DNA interactions in the presence of these chaperones via survival probability and confocal fluorescence measurements indicates that LEDGF not only facilitates nucleosome unwrapping but also moderately promotes nucleosome reassembly. These results offer insight into the functions of LEDGF and HDGF2 as nucleosome chaperone proteins and their effect on IN binding to the nucleosome array.

Simulation-based inference of single-molecule force spectroscopy.

Single-molecule force spectroscopy (smFS) is a powerful approach to studying the microscopic mechanisms of molecular self-organization in biological cells and complex materials. However, extracting quantitative reduced models from these experiments is still challenging because coupling the molecule with the ever-present experimental measuring device introduces artifacts that blur the measurements. Performing statistical inference within the Bayesian framework is an attractive way to extract quantitative models from smFS experiments. But even minimalistic models lead to mathematically intractable likelihoods---ubiquitous in the computational sciences---that hinder standard approaches based on likelihood optimization. We develop a computational framework that performs Bayesian inference to extract reduced models based on diffusive dynamics on a free energy landscape. We build on a new generation of likelihood-free inference methods called simulation-based inference (SBI). By encoding a parametric mechanistic model into a simulator in combination with probabilistic deep learning, SBI enables us to directly estimate the Bayesian posterior, avoiding any likelihood calculation. Using synthetic data, we show how we can systematically disentangle the measurement of hidden molecular properties from experimental artifacts. By integrating physics-based parametric models with machine learning density estimation, SBI enables accurate Bayesian inference for models with an intractable likelihood. It paves the way for more complex and realistic models that would otherwise be ruled out due to their mathematical intractability. SBI is general, conceptually transparent, easy to use, and broadly applicable to other types of biophysical experiments.