Low Force Regime Research Articles

Pincus blob elasticity in an intrinsically disordered protein.

Understanding the dynamic structure of intrinsically disordered proteins (IDPs) is important to deciphering their biological functions. Here, we exploit precision entropic elasticity measurements to infer the conformational behavior of a model IDP construct formed from the disordered tail of the neurofilament low molecular weight protein. The IDP construct notably displays a low-force power-law elastic regime, consistent with the Pincus blob model, which allows direct extraction of the Flory exponent, [Formula: see text], from the force-extension relationship. We find [Formula: see text] increases with added denaturant, transitioning from a nearly ideal chain to a swollen chain in a manner quantitatively consistent with measurements of IDP dimensions from other experimental techniques. We suggest that measurements of entropic elasticity could be broadly useful in the study of IDP structure.

Controlling Aspergillus niger morphology in a low shear-force environment in a rocking-motion bioreactor

The filamentous fungus Aspergillus niger is an important production host in biotechnology. Shear force regimes are one of the key factors that affect macromorphology and product yield. While morphology changes under intensive agitation have been widely investigated, studies at a low shear force regime independently from oxygen limitation has remained a challenge. Therefore, in this study, a 2-dimensional rocking-motion bioreactor is used as an alternative platform for studying the macromorphology under a low shear force regime, but sufficient supply of dissolved oxygen. Talcum macroparticles were added at different concentrations to control the development of a certain macromorphology. Results showed that 0.25% and 1% (ww−1) of talcum led to a mixture of dispersed mycelia and loose clumps, similar to what is obtained in lab-scale stirred tank reactors. At lower talcum concentrations, distinct pellet formation was observed. Quantitative analysis of pellets showed that with 0.05% of talcum, 95% of the pellets had a diameter smaller than 850 µm after 36 h. In case of 0.1% of talcum, 94% ( ± 5.0%) of the pellets had a diameter below 650 µm.The presented approach makes it possible to achieve a certain morphology as, for example, observed in large scale cultivations to study the consequences for product synthesis.

Molecular mechanisms of biofilm formation in Staphylococci bacteria: How antibiotic resistant bacteria is evolving to become more resilient to mechanical force loads.

Among the arsenal of virulence factors used by Staphylococcus aureus bacteria, adhesins play critical roles participating actively on the formation of biofilms. Combining single molecule force spectroscopy in vitro and in silico, we have discovered the extreme mechanostability of the interaction between pathogenic adhesins and proteins of the human extracellular matrix. Here we describe how Staphylococci adhesins use a catch-bond mechanism to hold tight to their human target peptides under high-force loads, while allowing for thermal dissociation in a low-force regime. Furthermore, we employed bioinformatic tools to retrieve and align nearly 200 proteins of the bacterial adhesin superfamily. Using AI-based protein structure prediction, we modelled, together with their human target, adhesins from methicillin susceptible strains of S. aureus (MSSA) and methicillin resistant strains of S. aureus (MRSA). Using GPU-accelerated NAMD 3, thousands of steered molecular dynamics (SMD) simulations were performed using a wide-sampling paradigm. Our protocol reveled that mechanostability is preserved among all strains with a pattern of higher forces for the MRSA strains. In summary, our work shows that the complex formed by Staphylococci adhesins with proteins of the human extracellular matrix is, by far, the most mechanically stable protein complex know to date, surpassing the mechanostability of the widely employed streptavidin:biotin complex by an order of magnitude. To keep such mechanostable complex, a catch-bond mechanism, where bonds live longer under mechanical load, is used by these adhesins. Additionally, we have recently discovered that the mechanostability of the complex is increasing with the evolution of these bacteria to become resistant to methicillin. With increasing prevalence of multidrug resistant bacterial infections, this new finding could be exploited for the development of antiadhesion strategies as an alternative to antibiotics.

Molecular Origins of Force-Dependent Protein Complex Stabilization during Bacterial Infections.

The unbinding pathway of a protein complex can vary significantly depending on biochemical and mechanical factors. Under mechanical stress, a complex may dissociate through a mechanism different from that used in simple thermal dissociation, leading to different dissociation rates under shear force and thermal dissociation. This is a well-known phenomenon studied in biomechanics whose molecular and atomic details are still elusive. A particularly interesting case is the complex formed by bacterial adhesins with their human peptide target. These protein interactions have a force resilience equivalent to those of covalent bonds, an order of magnitude stronger than the widely used streptavidin:biotin complex, while having an ordinary affinity, much lower than that of streptavidin:biotin. Here, in an in silico single-molecule force spectroscopy approach, we use molecular dynamics simulations to investigate the dissociation mechanism of adhesin/peptide complexes. We show how the Staphylococcus epidermidis adhesin SdrG uses a catch-bond mechanism to increase complex stability with increasing mechanical stress. While allowing for thermal dissociation in a low-force regime, an entirely different mechanical dissociation path emerges in a high-force regime, revealing an intricate mechanism that does not depend on the peptide's amino acid sequence. Using a dynamic network analysis approach, we identified key amino acid contacts that describe the mechanics of this complex, revealing differences in dynamics that hinder thermal dissociation and establish the mechanical dissociation path. We then validate the information content of the selected amino acid contacts using their dynamics to successfully predict the rupture forces for this complex through a machine learning model.

Driven translocation of a semiflexible polymer through a conical channel in the presence of attractive surface interactions

We study the translocation of a semiflexible polymer through a conical channel with attractive surface interactions and a driving force which varies spatially inside the channel. Using the results of the translocation dynamics of a flexible polymer through an extended channel as control, we first show that the asymmetric shape of the channel gives rise to non-monotonic features in the total translocation time as a function of the apex angle of the channel. The waiting time distributions of individual monomer beads inside the channel show unique features strongly dependent on the driving force and the surface interactions. Polymer stiffness results in longer translocation times for all angles of the channel. Further, non-monotonic features in the translocation time as a function of the channel angle changes substantially as the polymer becomes stiffer, which is reflected in the changing features of the waiting time distributions. We construct a free energy description of the system incorporating entropic and energetic contributions in the low force regime to explain the simulation results.

Quantitative spectral electromechanical characterization of soft piezoelectric nanocomposites

We present a modular system for the quantitative characterization of the piezoelectric coefficient of piezoelectric polymers and soft polymeric nanocomposites in the compression mode. Our approach is based on an apparatus providing spectral information on the electro-mechanical response in aselected range of frequencies of compressive loads (10–1200 Hz), with high sensitivity (down to 0.5 pC/N) and automated data acquisition modalities, enabling repeatability and reproducibility of the electro-mechanical characterization in the low-force regime (0.1 N 1.5 N). The system is modular and can be developed to cover the 2 mHz-1.2 kHz frequency range in charge mode and the 2 μHz-1200 Hz in voltage mode. We calibrated and validated the apparatus functionality using a commercial PVDF piezoelectric polymer. The suitability of the system for the quantitative measurements of the piezoelectricity of soft polymeric nanocomposites was then assessed by performing measurements of a novel piezoelectric nanocomposite material. This consisted of a polydimethylsiloxane (PDMS) matrix with embedded BaTiO3 nanoparticles, engineered with functional surface coatings to favor their homogeneous dispersion into the polymer. The proposed system demonstrated to be an effective solution for the systematic characterization of the electro-mechanical conversion properties of soft piezoelectric materials in view of soft robotics and energy harvesting applications.

Elasticity of single flexible polymer chains in good and poor solvents

Force versus extension curves measure entropic elasticity of single polymer chain in force spectroscopy experiments. A Worm-like Chain model is used to describe force extension experiments with an intrinsic chain parameter called persistence length, which is a measure of local bending flexibility. For flexible polymers, there is a discrepancy in estimates of persistence length in various force regimes. For instance, Atomic Force Microscopy (AFM) based pulling experiments report anomaly low values which are also inconsistent with magnetic tweezers experiments. To understand this, we investigate the role of coupling between microscopic force probe and intrinsic elasticity of polyethylene glycol chain in AFM-based experiments. We perform experiments using oscillatory rheology by providing an external excitation of fixed frequency to the probe. We show that a proper quantification of elastic response measured directly by oscillatory technique deviates significantly from conventional force-extension curves. The persistence length obtained by fitting WLC to stiffness extension data matches well with equilibrium tweezers experiments. In addition, for polystyrene chain in poor solvent no deviation in elastic response is observed between oscillatory and constant velocity pulling experiments. However, such deviation is seen for polystyrene in good solvent. We attribute this to hydrophobic interaction between monomers of polystyrene in water. Our results suggest that oscillatory rheology on single polymer chains provide quantitative estimate of its elastic response. The consistency in values of persistence length using magnetic tweezers experiments in low force regime and the AFM experiments in high force regime suggests that WLC is successful in describing the polymer elasticity in the force range typically probed in AFM experiments.

Controlling the electronic bands of a 2D semiconductor by force microscopy

In this work, we investigate the transverse transport properties of few-layers MoS2 using a Conductive Atomic Force Microscopy based technique. We find that the system changes between a low-force regime, characterized by a nearly-ideal contact between the MoS2 flake and the substrate, and a high-force regime, for which this contact starts to become highly non-ideal. We propose a 3-diode model that effectively describes the current-voltage characteristics of few-layers MoS2. From this model, we estimate how fast the energy gaps of two-dimensional MoS2 materials change as a function of the applied force. From our analysis, we estimate that MoS2-Au Schottky barrier heights change at the rate of 0.21, 0.23, and 0.78 meV nN−1 for the few-layers, three-layers, and two-layers MoS2, respectively. Our work opens up new possibilities of investigating and controlling the electronic properties of 2D semiconducting materials.

Dodecyltrimethylammonium bromide surfactant effects on DNA: Unraveling the competition between electrostatic and hydrophobic interactions.

We present a new study on the interaction of the DNA molecule with the surfactant dodecyltrimethylammonium bromide (DTAB), performed mainly with optical tweezers. Single-molecule force spectroscopy experiments performed in the low-force entropic regime allowed a robust characterization of the DNA-DTAB interaction, unveiling how the surfactant changes the mechanical properties of the biopolymer, the binding parameters, and the competition of the two mechanisms involved in the interaction: electrostatic attraction between the cationic surfactant heads and the negative phosphate backbone of the DNA and hydrophobic interactions between the tails of the bound DTAB molecules, which can result in DNA compaction in solution depending on the quantity of bound surfactant. Finally, force clamp experiments with magnetic tweezers and gel electrophoresis assays confirm that DTAB compacts DNA depending not only on the surfactant concentration but also on the conformation of the biopolymer in solution. The present study provides new insights on general aspects of the DNA-surfactant complexes formation, contributing to the fundamental knowledge of the physics of such interactions.

Force Response of Polypeptide Chains from Water-Explicit MD Simulations

Using molecular dynamics simulations in explicit water, the force–extension relations for the five homopeptides polyglycine, polyalanine, polyasparagine, poly­(glutamic acid), and polylysine are investigated. From simulations in the low-force regime the Kuhn length is determined, from simulations in the high-force regime the equilibrium contour length and the linear and nonlinear stretching moduli, which agree well with quantum-chemical density-functional theory calculations, are determined. All these parameters vary considerably between the different polypeptides. The augmented inhomogeneous partially freely rotating chain (iPFRC) model, which accounts for side-chain interactions and restricted dihedral rotation, is demonstrated to describe the simulated force–extension relations very well. We present a quantitative comparison between published experimental single-molecule force–extension curves for different polypeptides with simulation and model predictions. The thermodynamic stretching properties of polypeptides are investigated by decomposition of the stretching free energy into energetic and entropic contributions.

Non-contact microfluidic mechanical property measurements of single apoptotic bodies

BackgroundCells exchange information by secreting micro­ and nanosized extracellular vesicles (EVs), ranging from exosomes (30–100 nm) to apoptotic bodies (ABs, 1–5 μm). There is still much to understand about fundamental EV biological, physical, and chemical properties before clinical applications can be developed. EV mechanical properties have only been measured with atomic force microscopy (AFM) with its problematic adhesion and hard substrate effects. To understand EV mechanical behavior in less extreme mechanical conditions relevant to blood flow and many soft tissue environments, a non-contact measurement technique is needed. MethodsWe measured the mechanical properties of single microscale ABs derived from human blood plasma using non-contact microfluidics. EVs were gently stretched in extensional flow, similar to a traditional tensile test, and a linear mechanical model was applied to estimate mechanical stiffnesses from the observed stretching. ResultsThe effective shear elastic modulus of ABs in non-contact flow conditions is approximately 5.6 ± 0.5 Pa, 7 orders of magnitude lower than previously reported AFM-measured biological exosome stiffnesses and 200 times smaller than suspended cells. ConclusionsApoptotic bodies are very soft in fluid environments and exhibit lower effective stiffnesses than suspended cells. By measuring ABs in a natural fluid environment and low-force regime without hard probes and surfaces, we achieved closer agreement with linear mechanical theory and therefore more accurate stiffness measurements. General significanceAFM manufacturers and users should consider implementing new mechanical models to interpret AFM force indentation curves so that accurate extracellular vesicle mechanical properties can be extracted.

Molecular interactions and forces of adhesion between single human neural stem cells and gelatin methacrylate hydrogels of varying stiffness

Single Cell Force Spectroscopy was applied to measure the single cell de-adhesion between human neural stem cells (hNSC) and gelatin methacrylate (GelMA) hydrogel with varying modulus in the range equivalent to brain tissue. The cell de-adhesion force and energy were predominately generated via unbinding of complexes formed between RGD groups of the GelMA and cell surface integrin receptors and the de-adhesion force/energy were found to increase with decreasing modulus of the GelMA hydrogel. For the softer GelMA hydrogels (160 Pa and 450 Pa) it was proposed that a lower degree of cross-linking enables a greater number of polymer chains to bind and freely extend to increase the force and energy of the hNSC-GelMA de-adhesion. In this case, the multiple polymer chains are believed to act together in parallel like ‘molecular tensors’ to generate tensile forces on the bound receptors until the cell detaches. Counterintuitively for softer substrates, this type of interaction gave rise to higher force loading rates, including the appearance of high and low dynamic force regimes in de-adhesion rupture force versus loading rate analysis. For the stiffer GelMA hydrogel (900 Pa) it was observed that the extension and elastic restoring forces of the polymer chains contributed less to the cell de-adhesion. Due to the apparent lower extent of freely interacting chains on the stiffer GelMA hydrogel the intrinsic RGD groups are presumed to be “more fixed” to the substrate. Hence, the cell de-adhesion is suggested to be mainly governed by the discrete unbinding of integrin-RGD complexes as opposed to elastic restoring forces of polymer chains, leading to smaller piconewton rupture forces and only a single lower dynamic force regime. Intriguingly, when integrin antibodies were introduced for binding integrin α5β1, β1- and αv-subunits it was revealed that the cell modifies the de-adhesion force depending on the substrate stiffness. The antibody binding supressed the de-adhesion on the softer GelMA hydrogel while on the stiffer GelMA hydrogel caused an opposing reinforcement in the de-adhesion. Statement of SignificanceConceptual models on cell mechanosensing have provided molecular-level insight to rationalize the effects of substrate stiffness. However most experimental studies evaluate the cell adhesion by analysing the bulk material properties. As such there is a discrepancy in the scale between the bulk properties versus the nano- and micro-scale cell interactions. Furthermore there is a paucity of experimental studies on directly measuring the molecular-level forces of cell-material interactions.Here we apply Single Cell Force Spectroscopy to directly measure the adhesion forces between human neural stem cells and gelatin-methacrylate hydrogel. We elucidate the mechanisms by which single cells bind and physically interact with hydrogels of varying stiffness. The study highlights the use of single cell analysis tools to probe molecular-level interactions at the cell-material interface which is of importance in designing material cues for regulating cell function.

Direct Equilibrium Protein Folding-Unfolding of Mechanically Labile Alpha Helical Protein by Atomic Force Microscopy

The direct observation of protein folding and unfolding at/near equilibrium by atomic force microscopy (AFM) has been challenging due to the technological burden of limited force precision and resolution of AFM. Using chemically etched low-drift cantilevers and protein handles that are easy to pick up, here we demonstrate on a commercial AFM, the direct observation of the at/near equilibrium folding-unfolding dynamics of protein G-related albumin-binding domain (GA), an all-α protein consisting of a three-helix bundle. Unfolding and refolding of GA occurred under a low-force regime (<10 pN) previously difficult to observe using AFM. GA unfolded/refolded at/near equilibrium as evidenced by rapid hopping between folded and unfolded states. The high stability of our experimental platform allowed for constant separation experiments to probe at (near)-equilibrium kinetics of GA in real-time. Our results indicate GA unfolding occurs by two-state fashion at low force (∼10 pN) and displays high malleability, a large distance to the unfolded transition state. Finally, binding of human serum albumin (HSA) brought GA unfolding/folding out of equilibrium, where a near two-fold increase in unfolding force was observed relative to unbound GA. Taken together, these techniques further extend the capability of commercial AFMs towards studying protein unfolding/refolding at equilibrium as well as increasingly complex biomolecular systems in the context of biological function.

Nitrosative stress affects the interaction of integrin alphaIIbbeta3 with its ligands

Binding of integrin alphaIIbbeta3 (αiibβ3) to its ligands is a highly restricted and regulated mechanism. Any modification of the protein structure yields a dysfunctional role, especially in a redox environment. Here, we examine the effect of nitrosative stress on the αiibβ3 reconstituted into nanodiscs. Using single molecule force spectroscopy, we measured the interaction between αiibβ3 and its ligand RGD and found that in the presence of exogenous nitric oxide (NO) two force regimes are generated: a low force regime of ~100pN indicating the presence of integrin in a normal status, and a broad spectrum of high force regime (~210–450pN) suggesting the protein modification/aggregation. By high resolution atomic force microscopy imaging, we demonstrate that both NO and nitrite (a stable product formed from NO) are involved in destabilizing the transmembrane protein complex leading to release of αiibβ3 from the lipid bilayer and protein aggregation. Our experimental setup opens new ways for testing in a membrane environment the effect of radical species on integrins under clinically relevant conditions.

Nanotribology of hydrogels with similar stiffness but different polymer and crosslinker concentrations

HypothesisThe stiffness has been found to regulate hydrogel performances and applications. However, the key interfacial properties of hydrogels, like friction and adhesion are not controlled by the stiffness, but are altered by the structure and composition of hydrogels, like polymer volume fraction and crosslinking degree. ExperimentsColloidal probe atomic force microscopy has been use to investigate the relationship between tribological properties (friction and adhesion) and composition of hydrogels with similar stiffness, but different polymer volume fractions and crosslinking degrees. FindingsThe interfacial normal and lateral (friction) forces of hydrogels are not directly correlated to the stiffness, but altered by the hydrogel structure and composition. For normal force measurements, the adhesion increases with polymer volume fraction but decreases with crosslinking degree. For lateral force measurements, friction increases with polymer volume fraction, but decreases with crosslinking degree. In the low normal force regime, friction is mainly adhesion-controlled and increases significantly with the adhesion and polymer volume fraction. In the high normal force regime, friction is predominantly load-controlled and shows slow increase with normal force.

Single-Chain Polymer Models Incorporating the Effects of Side Groups: An Approach to General Polymer Models

High elasticity is one of the most important characteristics of polymers. The entropy-driven elastic behavior dominates the low force regime. The entropic elasticity of single polymer chains can be...

Temperature dependent mechanical unfolding of calixarene nanocapsules studied by molecular dynamics simulations.

Using atomistic molecular dynamics simulations, we study the temperature dependence of the mechanical unfolding of a model supramolecular complex, a dimer of interlocked calixarene capsules. This system shows reversible transitions between two conformations that are stabilized by different networks of hydrogen bonds. We study the forced dissociation and formation of these networks as a function of temperature and find a strong impact of the nonequilibrium conditions imposed by pulling the system mechanically. The kinetics of the transition between the two conformations is ideally suited to investigate the range of validity of the stochastic models employed in the analysis of force dependent kinetic rates obtained from experimental or simulation data. These models usually assume activated dynamics for the relevant transitions, and therefore, the analytical expressions for the kinetic rates are of an Arrhenius form. A study of the temperature- and force-dependent kinetics by simulation allows an analysis of the transition rates without any model assumption. We find that the temperature dependence of the rates is well described by an Arrhenius law for each value of the force. This enables us to determine the activation free energy and the bare kinetic rate as a function of force independent of each other. In accord with the common model assumptions, we find that the activation free energy decreases with increasing force. The force dependence of the bare rates is compatible with the results of model calculations in the low force regime, and deviations are observed at high forces.

Effect of cooling rate on the structure and nanotribology of Ag–Cu nano-eutectic alloys

Wear mechanisms of three Ag–Cu eutectic alloy samples cooled at different rates from the melt have been investigated by friction force microscopy. The eutectic phase exhibits a lamellar structure where the interlamellar thickness decreases with increasing cooling rate. The hardness of the samples decreases with decreasing thickness of the lamellae. In the low normal force regime (Fn ≤ 1000 nN) friction is governed by shearing and the relevant contact area can be well described by the Johnson–Kendal–Roberts model. At higher normal force values, the surface is worn, and friction can be described by the ploughing friction coefficient. A ploughing friction coefficient is determined, which is positively correlated with the hardness of the Ag–Cu eutectic alloy samples cooled at different rates, while the wear volume negatively correlates with the hardness.

Bioinspired passive variable recruitment of fluidic artificial muscles

This article presents a novel, passive approach to creating variable actuator recruitment in bundles of fluidic artificial muscles. The passive recruitment control approach is inspired by the functionality of mammalian muscle tissues, in which a single activation signal from the nervous system sequentially triggers contraction of progressively larger actuation elements until the required force is generated. Biologically, this behavior is encoded by differences in electrical resistance properties between smaller and larger muscle-fiber groups. The approach presented here produces analogous behavior using a uniform applied pressure to all fluidic artificial muscles while creating differential pressure responses and threshold pressures among the fluidic artificial muscles via tailored bladder elasticity parameters. A model for using elastic bladder stiffness to control an artificial muscle bundle with a single valve is explored and used to compare a bundle of fluidic artificial muscles with both low and high threshold pressure units to a single fluidic artificial muscle of equivalent displacement and force capability. The results of this analysis indicate the efficacy of using this control method; it is advantageous in cases where a wide range of displacements and forces are necessary and can increase efficiency when the system primarily operates in a low-force regime but requires occasional bursts of high-force capability.

Surface-induced effects in fluctuation-based measurements of single-polymer elasticity: A direct probe of the radius of gyration.

Single-molecule measurements of polymer elasticity are powerful, direct probes of both biomolecular structure and principles of polymer physics. Recent work has revealed low-force regimes in which biopolymer elasticity is understood through blob-based scaling models. However, the small tensions required to observe these regimes have the potential to create measurement biases, particularly due to the increased interactions of the polymer chain with tethering surfaces. Here, we examine one experimentally observed bias, in which fluctuation-based estimates of elasticity report an unexpectedly low chain compliance. We show that the effect is in good agreement with predictions based on quantifying the exclusion effect of the surface through an image-method calculation of available polymer configurations. The analysis indicates that the effect occurs at an external tension inversely proportional to the polymer's zero-tension radius of gyration. We exploit this to demonstrate a self-consistent scheme for estimating the radius of gyration of the tethered polymer. This is shown in measurements of both hyaluronic acid and poly(ethylene glycol) chains.