Force Spectroscopy Experiments Research Articles

Accelerating a Mechanically Driven anti-Woodward-Hoffmann Ring Opening with a Polymer Lever Arm Effect.

Mechanical forces have previously been used to drive reactions along pathways that violate the orbital symmetry effects captured in the Woodward-Hoffmann rules. Here, we show that a polymer "lever arm effect" can provide a mechanical advantage in accelerating the symmetry forbidden disrotatory ring opening of benzocyclobutene (BCB). Addition of an α-E-alkene to the BCB mechanophore drops the force required to induce reactions on the ∼0.1 s time scale of single-molecule force spectroscopy experiments from 1370 to 920 pN.

Energy landscape investigation by wavelet transform analysis of atomic force spectroscopy data in a biorecognition experiment.

Force fluctuations recorded in an atomic force spectroscopy experiment, during the approach of a tip functionalized with biotin towards a substrate charged with avidin, have been analyzed by a wavelet transform. The observation of strong transient changes only when a specific biorecognition process between the partners takes place suggests a drastic modulation of the force fluctuations when biomolecules recognize each other. Such an analysis allows to investigate the peculiar features of a biorecognition process. These results are discussed in connection with the possible role of energy minima explored by biomolecules during the biorecognition process.

Ligand-Induced Changes of the Apparent Transition-State Position in Mechanical Protein Unfolding

Force-spectroscopic measurements of ligand-receptor systems and the unfolding/folding of nucleic acids or proteins reveal information on the underlying energy landscape along the pulling coordinate. The slope Δx‡ of the force-dependent unfolding/unbinding rates is interpreted as the distance from the folded/bound state to the transition state for unfolding/unbinding and, hence, often related to the mechanical compliance of the sample molecule. Here we show that in ligand-binding proteins, the experimentally inferred Δx‡ can depend on the ligand concentration, unrelated to changes in mechanical compliance. We describe the effect in single-molecule, force-spectroscopy experiments of the calcium-binding protein calmodulin and explain it in a simple model where mechanical unfolding and ligand binding occur on orthogonal reaction coordinates. This model predicts changes in the experimentally inferred Δx‡, depending on ligand concentration and the associated shift of the dominant barrier between the two reaction coordinates. We demonstrate quantitative agreement between experiments and simulations using a realistic six-state kinetic scheme using literature values for calcium-binding kinetics and affinities. Our results have important consequences for the interpretation of force-spectroscopic data of ligand-binding proteins.

Resistive switching and polarization reversal of hydrothermal-method-grown undoped zinc oxide nanorods by using scanning probe microscopy techniques.

This paper reports the localized electrical, polarization reversal, and piezoelectric properties of the individual hexagonal ZnO nanorods, which are grown via the hydrothermal method and textured with [0001] orientation. The studies are conducted with conductive atomic force microscopy (c-AFM) and piezoresponse force microscopy (PFM) techniques. The correlation between the resistance switching and polarization reversal is discussed. The c-AFM results show that there is less variation on the set or reset voltage in nanorod samples, compared to that of the ZnO thin film. With increasing aspect ratio of the nanorods, both set and reset voltages are decreased. The nanorods with low aspect ratio show unipolar resistance switching, whereas both unipolar and bipolar resistance switching are observed when the aspect ratio is larger than 0.26. The PFM results further show the ferroelectric-like property in the nanorods. Comparing with that of the ZnO thin film, the enhanced piezoresponse in the nanorods can be attributed to the size effect. In addition, the piezoresponse force spectroscopy (PFS) experiments are conducted in ambient air, synthetic air, and argon gas. It shows that the depolarization field in the nanorod may be due to the moisture in the environment; moreover, the increased piezoresponse may relate to the absence of oxygen in the environment. It is also shown that the piezoelectric responses increase nonlinearly with the aspect ratio of the nanorods. By comparing the piezoresponse hysteresis loops obtained from the nanorod samples of as-grown, air-annealed and vacuum-annealed, it is found that the oxygen vacancies are the origin of the polarization reversal in ZnO nanorods. Finally, the tradeoff between the electrical and ferroelectric-like properties is also observed.

C-5 Propynyl Modifications Enhance the Mechanical Stability of DNA.

Increased thermal or mechanical stability of DNA duplexes is desired for many applications in nanotechnology or -medicine where DNA is used as a programmable building block. Modifications of pyrimidine bases are known to enhance thermal stability and have the advantage of standard base-pairing and easy integration during chemical DNA synthesis. Through single-molecule force spectroscopy experiments with atomic force microscopy and the molecular force assay we investigated the effect of pyrimidines harboring C-5 propynyl modifications on the mechanical stability of double-stranded DNA. Utilizing these complementary techniques, we show that propynyl bases significantly increase the mechanical stability if the DNA is annealed at high temperature. In contrast, modified DNA complexes formed at room temperature and short incubation times display the same stability as non-modified DNA duplexes.

A Flexible Nanoarray Approach for the Assembly and Probing of Molecular Complexes

Immobilization is a key step involved in probing molecular interactions using single-molecule force spectroscopy methods, including atomic force microscopy (AFM). To our knowledge, we describe a novel approach termed flexible nanoarray (FNA) in which the interaction between the two internally immobilized amyloid β peptides is measured by pulling of the tether. The FNA tether was synthesized with nonnucleotide phosphoramidite monomers using the DNA synthesis chemistry. The two anchoring points for immobilization of the peptides inside the tether were incorporated at defined distances between them and from the ends of the polymer. Decamers of amyloid β peptide capable of dimer formation were selected as a test system. The formation of the peptide dimers was verified by AFM force spectroscopy by pulling the tether at the ends. In these experiments, the thiolated end of the FNA tether was covalently immobilized on the AFM substrate functionalized with maleimide. The other end of the FNA tether was functionalized with biotin to form a noncovalent link with the streptavidin functionalized AFM tip during the approach stage. The dimers’ rupture fingerprint was unambiguously identified on the force curves by its position and the force value. The FNA design allowed reversible experiments in which the monomers were allowed to associate after the rupture of the dimers by performing the approach stage before the rupture of the biotin-streptavidin link. This suggests that the FNA technique is capable of analyzing multiple intermolecular interactions in the same molecular complex. The computational analysis showed that the tethered peptides assemble into the same dimer structure as that formed by nontethered peptides, suggesting that the FNA tether has the necessary flexibility to enable assembly of the dimer even during the course of the force spectroscopy experiment.

Adhesion energy of single wall carbon nanotube loops on various substrates

The physics of adhesion of one-dimensional nano structures such as nanotubes, nano wires, and biopolymers on different material substrates is of great interest for the study of biological adhesion and the development of nano electronics and nano mechanics. In this paper, we present force spectroscopy experiments of a single wall carbon nanotube loop using our home-made interferometric atomic force microscope. Characteristic force plateaux during the peeling process allows us to access to quantitative values of the adhesion energy per unit length on various substrates: graphite, mica, platinum, gold and silicon. By combining a time-frequency analysis of the deflexion of the cantilever, we access to the dynamic stiffness of the contact, providing more information on the nanotube configurations and its intrinsic mechanical properties.

Spectrin–ankyrin interaction mechanics: A key force balance factor in the red blood cell membrane skeleton

As major components of red blood cell (RBC) cytoskeleton, spectrin and F-actin form a network that covers the entire cytoplasmic surface of the plasma membrane. The cross-linked two layered structure, called the membrane skeleton, keeps the structural integrity of RBC under drastically changing mechanical environment during circulation. We performed force spectroscopy experiments on the atomic force microscope (AFM) as a means to clarify the mechanical characteristics of spectrin–ankyrin interaction, a key factor in the force balance of the RBC cytoskeletal structure. An AFM tip was functionalized with ANK1-62k and used to probe spectrin crosslinked to mica surface. A force spectroscopy study gave a mean unbinding force of ~30pN under our experimental conditions. Two energy barriers were identified in the unbinding process. The result was related to the well-known flexibility of spectrin tetramer and participation of ankyrin 1–spectrin interaction in the overall balance of membrane skeleton dynamics.

Correlation of breaking forces, conductances and geometries of molecular junctions.

Electrical and mechanical properties of elongated gold-molecule-gold junctions formed by tolane-type molecules with different anchoring groups (pyridyl, thiol, amine, nitrile and dihydrobenzothiophene) were studied in current-sensing force spectroscopy experiments and density functional simulations. Correlations between forces, conductances and junction geometries demonstrate that aromatic tolanes bind between electrodes as single molecules or as weakly-conductive dimers held by mechanically-weak π − π stacking. In contrast with the other anchors that form only S-Au or N-Au bonds, the pyridyl ring also forms a highly-conductive cofacial link to the gold surface. Binding of multiple molecules creates junctions with higher conductances and mechanical strengths than the single-molecule ones.

Reconciling bond strength of a slip bond at low loading rates with rebinding

Conventional theory predicts that molecular bond strength becomes trivial at very low loading rates, which, however, failed in some experiments probably due to hidden rebinding. Here we propose average bond strength as an alternative physical quantity to be measured in dynamic force spectroscopy experiments. Our analysis indicates that there exists an asymptotic value of average bond strength at low loading rates, which, nevertheless, increases with the spring constant of the loading system. This finding is subsequently confirmed with the Monte Carlo simulation. The proposed average bond strength can be informative in the understanding of the intriguing behaviors of molecular bonds at low loading rates.

Extraction of accurate biomolecular parameters from single-molecule force spectroscopy experiments.

The atomic force microscope (AFM) is able to manipulate biomolecules and their complexes with exquisite force sensitivity and distance resolution. This capability, complemented by theoretical models, has greatly improved our understanding of the determinants of mechanical strength in proteins and revealed the diverse effects of directional forces on the energy landscape of biomolecules. In unbinding experiments, the interacting partners are usually immobilized on their respective substrates via extensible linkers. These linkers affect both the force and contour length (Lc) of the complex at rupture. Surprisingly, while the former effect is well understood, the latter is largely neglected, leading to incorrect estimations of Lc, a parameter that is often used as evidence for the detection of specific interactions and remodeling events and for the inference of interaction regions. To address this problem, a model that predicts contour length measurements from single-molecule forced-dissociation experiments is presented that considers attachment position on the AFM tip, geometric effects, and polymer dynamics of the linkers. Modeled data are compared with measured contour length distributions from several different experimental systems, revealing that current methods underestimate contour lengths. The model enables nonspecific interactions to be identified unequivocally, allows accurate determination of Lc, and, by comparing experimental and modeled distributions, enables partial unfolding events before rupture to be identified unequivocally.

Using Atomic Force Microscopy to Evaluate the Nanostructure and Nanomechanics of Warm Mix Asphalt

AbstractIn this paper, various Atomic Force Microscopy (AFM) techniques are utilized to study the effects of different warm mix asphalt (WMA) additives on the nanostructure and microstructure as well as the adhesive and cohesive properties of an asphalt binder. To achieve this objective, a control asphalt binder was modified with three different types of WMA additives, which included: Advera (a synthetic zeolite), Evotherm, and Sasobit (a commercial wax). The AFM tapping-mode imaging technique was used to examine the nanostructure and microstructure of the different asphalt material considered. In addition, AFM force spectroscopy experiments were conducted using chemically functionalized AFM tips to measure the nanoscale level adhesive and cohesive forces for the control and WMA asphalt binders before and after moisture conditioning. The results of the AFM imaging showed that while the Sasobit additive has reduced the width of the so-called “bee-like” structures within the asphalt binder, the other WMA ad...

Bringing Force Probe Molecular Dynamics Simulations Closer to Experiments

The interaction between Streptavidin and Biotin is one of the strongest known non covalent bonds in nature. For this reason, many experimental and computational studies have been done on this complex, which revealed many details on binding and unbinding of biotin, which turned out to be an astonishingly complex process.However, taken all these studies together and despite considerable technical advances over the past years, there is still a time scale gap remaining, i.e., the loading rates possible in force spectroscopy experiments and those achievable in molecular dynamics (MD) simulations hardly overlap.Recent high speed force spectroscopy (HS-FS) experiments by Scheuring offer the possibility to reach pulling velocities up to 5 mm/s.Here we present extensive MD simulations covering pulling velocities from 0.5 mm/s to 50 m/s, thus providing an overlap of one order of magnitude with experimental time scales. Further, to resemble the AFM experiment as closely as possible in the simulations, we modeled the PEG-linker between Biotin and the cantilever as a wormlike chain and chose a spring constant of the pulling potential to match the stiffness of the cantilever in HS-FS experiments done by Scheuring.Over the whole interval, agreement of the probability distributions of unbinding forces is seen. Taken together, these data cover unbinding time scales over 10 orders of magnitude, which offers a unique opportunity to compare against several proposed transition state treatments of this process.

Quantitating membrane bleb stiffness using AFM force spectroscopy and an optical sideview setup.

AFM-based force spectroscopy in combination with optical microscopy is a powerful tool for investigating cell mechanics and adhesion on the single cell level. However, standard setups featuring an AFM mounted on an inverted light microscope only provide a bottom view of cell and AFM cantilever but cannot visualize vertical cell shape changes, for instance occurring during motile membrane blebbing. Here, we have integrated a mirror-based sideview system to monitor cell shape changes resulting from motile bleb behavior of Xenopus cranial neural crest (CNC) cells during AFM elasticity and adhesion measurements. Using the sideview setup, we quantitatively investigate mechanical changes associated with bleb formation and compared cell elasticity values recorded during membrane bleb and non-bleb events. Bleb protrusions displayed significantly lower stiffness compared to the non-blebbing membrane in the same cell. Bleb stiffness values were comparable to values obtained from blebbistatin-treated cells, consistent with the absence of a functional actomyosin network in bleb protrusions. Furthermore, we show that membrane blebs forming within the cell-cell contact zone have a detrimental effect on cell-cell adhesion forces, suggesting that mechanical changes associated with bleb protrusions promote cell-cell detachment or prevent adhesion reinforcement. Incorporating a sideview setup into an AFM platform therefore provides a new tool to correlate changes in cell morphology with results from force spectroscopy experiments.

Estimation of the free energy of adsorption of a polypeptide on amorphous SiO2 from molecular dynamics simulations and force spectroscopy experiments.

Estimating the free energy of adsorption of materials-binding peptides is fundamental to quantify their interactions across bio/inorganic interfaces, but is difficult to achieve both experimentally and theoretically. We employ a combination of molecular dynamics (MD) simulations and dynamical force-spectroscopy experiments based on atomic force microscopy (AFM) to estimate the free energy of adsorption ΔGads of a (GCRL) tetrapeptide on amorphous SiO2 in pure water. The results of both equilibrium, advanced sampling MD and non-equilibrium, steered MD are compared with those of two different approaches used to extract ΔGads from the dependence of experimentally measured adhesion forces on the applied AFM loading rates. In order to obtain unambiguous peak forces and bond loading rates from steered MD trajectories, we have developed a novel numerical protocol based on a piecewise-harmonic fit of the adhesion work profile along each trajectory. The interpretation of the experiments has required a thorough quantitative characterization of the elastic properties of polyethylene glycol linker molecules used to tether (GCRL)15 polypeptides to AFM cantilevers, and of the polypeptide itself. All obtained ΔGads values fall within a relatively narrow window between -5 and -9 kcal mol(-1), but can be associated with large relative error bars of more than 50%. Among the different approaches compared, Replica Exchange with Solute Tempering simulations augmented with MetaDynamics (RESTMetaD) and fitting of dynamic force spectroscopy experiments with the model of Friddle and De Yoreo lead to the most reliable ΔGads estimates.

Small Peptide Binding Stiffens the Ubiquitin-like Protein SUMO1

Posttranslational modification by small ubiquitin-like modifiers (SUMOs), known as SUMOylation, is a key regulatory event in many eukaryotic cellular processes in which SUMOs interact with a large number of target proteins. SUMO binding motifs (SBMs) are small peptides derived from these target proteins that interact noncovalently with SUMOs and induce conformational changes. To determine the effect of SBMs on the mechanical properties of SUMO1 (the first member of the human SUMO family), we performed single-molecule force spectroscopy experiments on SUMO1/SBM complexes. The unfolding force of SUMO1 (at a pulling speed of 400 nm/s) increased from ∼130 pN to ∼170 pN upon binding to SBMs, indicating mechanical stabilization upon complexation. Pulling-speed-dependent experiments and Monte Carlo simulations measured a large decrease in distance to the unfolding transition state for SUMO1 upon SBM binding, which is by far the largest change measured for any ligand binding protein. The stiffness of SUMO1 (measured as a spring constant for the deformation response along the line joining the N- and C-termini) increased upon SBM binding from ∼1 N/m to ∼3.5 N/m. The relatively higher flexibility of ligand-free SUMO1 might play a role in accessing various conformations before binding to a target.

Single-Molecule Force Spectroscopy on Unfolded and Intrinsically Disordered Proteins

Transcription factors and cell-signaling proteins contain a hyper abundance of domains or segments that are intrinsically disordered (ID) under native conditions. In many cases, intrinsically disordered proteins (IDPs) or ID segments undergo coupled folding and binding and this transition is associated to its biological role. Many techniques for dissecting folding are not readily amenable to IDPs because they are aggregation prone and insoluble. Single-molecule methods provide an avenue to mitigate these issues. We have performed single-molecule force spectroscopy experiments on a Trimethylamine N-oxide (TMAO) refolded IDP and found that this IDP unfolds cooperatively under mechanical force. The data are rich in information and reveal that what appears to be a two-state transition in bulk is actually multi-state at the single-molecule level. We found that the analysis methods typically employed in such experiments may not be applicable because of the high concentrations of TMAO. To address these issues, we have designed unfolded mutants of T4 lysozyme using a random mutagenesis phenotypic screen. We are actively performing both bulk and single-molecule experiments to characterize unfolded mutants of T4 Lysozyme and TMAO refolded IDPs.

Single Cell Force Spectroscopy Analysis for Acinetobacter Baylyi Mutation Aggregation

Acinetobacter baylyi ADP1 is an ideal bacterial strain for synthetic biology and metabolic engineering due to its high natural transformability and capability for processing diverse chemical compounds. For the production of compounds within industrial bioreactors, it is optimal to have a uniform dispersal of bacterial cells within solution, but mutations in bacterial cells may generate surface properties leading to cell aggregation. During long-term culture in the laboratory, mutants of the bacterium A. baylyi ADP1 originated a distinctive phenotype of cell aggregation. Genome sequencing and the analysis of gene knockouts showed this aggregation to be due to mutations in the per and pgi genes, and a reduction in bioemulsifier production. Qualitative analysis of Atomic Force Microscopy (AFM) visualizations identified altered appearances of cell surfaces correlating with the difference in cell aggregation phenotype. AFM force spectroscopy experiments were then conducted to compare the adhesive and viscoelastic properties of aggregating cells to non-aggregating cells. The most distinctive difference found for force spectroscopy measurements was for a four-fold difference in nN in adhesion that was attributable to pgi. Overall, this experiment has resulted in a multilevel approach for the evaluation and detection of a cell aggregation phenotype in mutant strains of A. baylyi ADP1.

Mechanical Aspects of Protein Thermostability

Because thermophilic proteins are stable and functional at high temperature but generally lack activity under ambient conditions, it was suggested that thermal stability can correspond to enhanced mechanical rigidity. While this concept has initially found wide experimentally support, its generality has been later questioned. It is then natural to inquire the correlation between protein thermal and mechanical stabilities using single-molecule force-spectroscopy experiments. Here, we will present results of a combination of computational techniques, mainly based on Molecular Dynamics simulations, to tackle these problems for several mesophilic/thermophilic homologues of different molecular complexity.By applying innovative network analysis to map the protein conformational landscape, we first question the universality of the rigidity paradigm. In fact we show that for several paris of homologues, thermophiles can be as flexible as their mesophilic variant with important effects coming from the local partition of rigid/flexible regions, protein oligomerization state, or the time and length scales considered.We will then focus on the correlation among thermostability and mechanical resistance. The object under investigation is a pair of mesophilic/thermophilic cold-shock proteins for which experimental data are available. We have first implemented an enhanced-sampling algorithm which allows us to explore the proteins’ free-energy landscape over a wide range of temperatures, and we have used constant-force steered MD to investigate the effect of mechanical force. Our results suggest that thermal stability does not guarantee a mechanical one and that the associated unfolding mechanisms, of which our simulations provide extensive details, are very different. We also aim at identifying the weak-points of the protein mechanical stability and how more stable mutants can be designed. If such mutants also exhibit increased thermal stability, our approach could prove useful in numerous applications.

Role of monomer arrangement in the amyloid self-assembly

Assembly of amyloid proteins into aggregates requires the ordering of the monomers in oligomers and especially in such highly organized structures as fibrils. This ordering is accompanied by structural transitions leading to the formation of ordered β-structural motifs in proteins and peptides lacking secondary structures. To characterize the effect of the monomer arrangements on the aggregation process at various stages, we performed comparative studies of the yeast prion protein Sup35 heptapeptide (GNNQQNY) along with its dimeric form CGNNQQNY-(d-Pro)-G-GNNQQNY. The (d-Pro)-G linker in this construct is capable of adopting a β-turn, facilitating the assembly of the dimer into the dimeric antiparallel hairpin structure (AP-hairpin). We applied Atomic Force Microscopy (AFM) techniques to follow peptide–peptide interactions at the single molecule level, to visualize the morphology of aggregates formed by both constructs, thioflavin T (ThT) fluorescence to follow the aggregation kinetics, and circular dichroism (CD) spectroscopy to characterize the secondary structure of the constructs. The ThT fluorescence data showed that the AP-hairpin aggregation kinetics is insensitive to the external environment such as ionic strength and pH contrary to the monomers the kinetics of which depends dramatically on the ionic strength and pH. The AFM topographic imaging revealed that AP-hairpins primarily assemble into globular aggregates, whereas linear fibrils are primary assemblies of the monomers suggesting that both constructs follow different aggregation pathways during the self-assembly. These morphological differences are in line with the AFM force spectroscopy experiments and CD spectroscopy measurements, suggesting that the AP-hairpin is structurally rigid regardless of changes of environmental factors.