A single filament biomechanical study of the enteropathogenic Escherichia coli Type III secretion system reveals a high elastic aspect ratio.

Type III secretion systems (T3SSs) are syringe-like protein complexes used by some of the most harmful bacterial pathogens to infect host cells. While the T3SS filament, a long hollow conduit that bridges between bacteria and host cells, has been characterized structurally, very little is known about its physical properties. These filaments should endure shear and normal stresses imposed by the viscous mucosal flow during infection within the intestinal tract. We used atomic force microscopy (AFM) to probe the longitudinal and radial mechanical response of individual T3SS filaments by pulling on filaments extending directly from bacterial surfaces and later pressing into filaments that were detached from the bacteria. The measured longitudinal elastic moduli were higher by about two orders of magnitude than the radial elastic moduli. These proportions are commensurate with the role of the T3SS filament, which requires horizontal flexibility while maintaining its structural integrity to withstand intense stresses during infection.

Nonexponential kinetics captured in sequential unfolding of polyproteins over a range of loads.

While performing under mechanical loads in vivo, polyproteins are vitally involved in cellular mechanisms such as regulation of tissue elasticity and mechano-transduction by unfolding their comprising domains and extending them. It is widely thought that the process of sequential unfolding of polyproteins follows an exponential kinetics as the individual unfolding events exhibit identical and identically distributed (iid) Poisson behavior. However, it was shown that under high loads, the sequential unfolding kinetics displays nonexponential kinetics that alludes to aging by a subdiffusion process. Statistical order analysis of this kinetics indicated that the individual unfolding events are not iid, and cannot be defined as a Poisson (memoryless) process. Based on numerical simulations it was argued that this behavior becomes less pronounced with lowering the load, therefore it is to be expected that polyproteins unfolding under lower forces will follow a Poisson behavior. This expectation serves as the motivation of the current study, in which we investigate the effect of force lowering on the unfolding kinetics of Poly-L8 under varying loads, specifically high (150, 100 â€‹pN) and moderate-low (45, 30, 20 â€‹pN) forces. We found that a hierarchy among the unfolding events still exists even under low loads, again resulting in nonexponential behavior. We observe that analyzing the dwell-time distributions with stretched-exponentials and power laws give rise to different phenomenological trends. Using statistical order analysis, we demonstrated that even under the lowest load, the sequential unfolding cannot be considered as iid, in accord with the power law distribution. Additional free energy analysis revealed the contribution of the unfolded segments elasticity that scales with the force on the overall one-dimensional contour of the energy landscape, but more importantly, it discloses the hierarchy within the activation barriers during sequential unfolding that account for the observed nonexponentiality.

An inside look at a biofilm: Pseudomonas aeruginosa flagella biotracking.

The opportunistic pathogen, Pseudomonas aeruginosa, a flagellated bacterium, is one of the top model organisms for biofilm studies. To elucidate the location of bacterial flagella throughout the biofilm life cycle, we developed a new flagella biotracking tool. Bacterial flagella were site-specifically labeled via genetic code expansion. This enabled us to track bacterial flagella during biofilm maturation. Live flagella imaging revealed the presence and synthesis of flagella throughout the biofilm life cycle. To study the possible role of flagella in a biofilm, we produced a flagella knockout strain and compared its biofilm to that of the wild-type strain. Results showed a one order of magnitude stronger biofilm structure in the wild type in comparison with the flagella knockout strain. This suggests a possible structural role for flagella in a biofilm, conceivably as a scaffold. Our findings suggest a new model for biofilm maturation dynamic which underscores the importance of direct evidence from within the biofilm.

Interplay between Viscoelasticity and Force Rate Affects Sequential Unfolding in Polyproteins Pulled at Constant Velocity.

Polyproteins are unique constructs, comprised of folded protein domains in tandem and polymeric linkers. These macromolecules perform under biological stresses by modulating their response through partial unfolding and extending. Although these unfolding events are considered independent, a history dependence of forced unfolding within polyproteins was reported. Here we measure the unfolding of single poly(I91) octamers, complemented with Brownian dynamics simulations, displaying increasing hierarchy in unfolding-foces, accompanied by a decrease in the effective stiffness. This counters the existing understanding that relates stiffness with variations in domain size and probe stiffness, which is expected to reduce the unfolding forces with every consecutive unfolding event. We utilize a simple mechanistic viscoelastic model to show that two effects are combined within a sequential forced unfolding process: the viscoelastic properties of the growing linker chain lead to a hierarchy of the unfolding events, and force-rate application governs the unfolding kinetics.

Effect of the C-terminal amino acid of the peptide on the structure and mechanical properties of alginate-peptide hydrogels across length-scales.

Alginate is a natural anionic polysaccharide that exhibits excellent biocompatibility and biodegradability. Alginate hydrogels have many different applications in the field of regenerative medicine especially when peptides are conjugated to the alginate backbone. Here, we systematically investigate the effect of six arginine-glycine-aspartic acid (RGD)-containing peptides, G6KRGDY/S, A6KRGDY/S and V6KRGDY/S, on the macroscopic and microscopic physical properties and spatial organization of alginate-peptides hydrogels. Using rheology, small angle X-ray scattering and nanoindentation measurements we show a strong correlation between the macroscopic-bulk properties and the microscopic-local properties of the alginate-peptide hydrogels. Furthermore, our results indicate that the identity of the amino acid at the C-terminal of the peptide plays a major role in determining the structure and mechanical properties of the hydrogel across length-scales, where the presence of tyrosine (Y) terminated peptides introduce more junction-zones and consequently larger stiffness than those terminated with serine (S).

Correlations within polyprotein forced unfolding dwell-times introduce sequential dependency.

Polyproteins, comprised from proteins arrayed in tandem, respond to mechanical loads through partial unfolding and extension. This response to tension that enables their physiological function is related to the ability to dynamically regulate their elasticity. The unique arrangement of their individual mechanical components (proteins and polymeric linkers), and the interactions between them eventually determines their performance. The sequential unfolding-times within a polyprotein are inherently assumed to be independent and identically distributed (iid), thus expected to follow an exponential distribution. Nevertheless, a large body of literature using single molecule force spectroscopy (SMFS) provides evidence that forced unfolding-times of N proteins within a polyprotein do not follow the exponential distribution. Here we use SMFS with Atomic Force Microscopy to measure the unfolding kinetics of Poly-(I91)8 at 180 pN. The unfolding time-intervals were statistically analysed using three common approaches, all exhibiting an N-effect: hierarchical behavior with non-identical unfolding time distributions. Using continuous time random walk approach indicates that the unfolding times display subdiffusive features. Put together with free-energy reconstruction of the whole unfolding polyprotein, we provide physical explanation for this nontrivial behavior, according to which the elongating polypeptide chain with each unfolding event intervenes with the sequential unfolding probabilities and correlates them.

Segmentation and the Entropic Elasticity of Modular Proteins.

Single-molecule force spectroscopy utilizes polyproteins, which are composed of tandem modular domains, to study their mechanical and structural properties. Under the application of external load, the polyproteins respond by unfolding and refolding domains to acquire the most favored extensibility. However, unlike single-domain proteins, the sequential unfolding of the each domain modifies the free energy landscape (FEL) of the polyprotein nonlinearly. Here we use force-clamp (FC) spectroscopy to measure unfolding and collapse-refolding dynamics of polyubiquitin and poly(I91). Their reconstructed unfolding FEL involves hundreds of kB T in accumulating work performed against conformational entropy, which dwarfs the ∼30 kB T that is typically required to overcome the free energy difference of unfolding. We speculate that the additional entropic energy caused by segmentation of the polyprotein to individual proteins plays a crucial role in defining the "shock absorber" properties of elastic proteins such as the giant muscle protein titin.

Deciphering the Mechanical Properties of Type III Secretion System EspA Protein by Single Molecule Force Spectroscopy.

Bacterial pathogens inject virulence factors into host cells during bacterial infections using type III secretion systems. In enteropathogenic Escherichia coli, this system contains an external filament, formed by a self-oligomerizing protein called E. coli secreted protein A (EspA). The EspA filament penetrates the thick viscous mucus layer to facilitate the attachment of the bacteria to the gut-epithelium. To do that, the EspA filament requires noteworthy mechanical endurance considering the mechanical shear stresses found within the intestinal tract. To date, the mechanical properties of the EspA filament and the structural and biophysical knowledge of monomeric EspA are very limited, mostly due to the strong tendency of the protein to self-oligomerize. To overcome this limitation, we employed a single molecule force spectroscopy (SMFS) technique and studied the mechanical properties of EspA. Force extension dynamic of (I91)4-EspA-(I91)4 chimera revealed two structural unfolding events occurring at low forces during EspA unfolding, thus indicating no unique mechanical stability of the monomeric protein. SMFS examination of purified monomeric EspA protein, treated by a gradually refolding protocol, exhibited similar mechanical properties as the EspA protein within the (I91)4-EspA-(I91)4 chimera. Overall, our results suggest that the mechanical integrity of the EspA filament likely originates from the interactions between EspA monomers and not from the strength of an individual monomer.

Estimation of interaction energy and contact stiffness in atomic-scale sliding on a model sodium chloride surface in ethanol.

Friction force microscopy (FFM) in aqueous environments has recently proven to be a very effective method for lattice-resolution imaging of crystal surfaces. Here we demonstrate the use of ethanol for similar measurements on water-soluble materials. Lattice resolved frictional stick-slip traces of a cleaved NaCl(100) surface submerged in ethanol are compared with previous obtained FFM results in ultrahigh vacuum (UHV). We use the Prandtl-Tomlinson framework to estimate the amplitude of the corrugation potential and the contact stiffness. The surface potential amplitude scales with the applied normal loads are in good agreement with data obtained for NaCl measured under UHV conditions, but demonstrates deviations from the ideal periodic potential given by the Prandtl-Tomlinson model. An additional finding is that the use of ethanol allows us to explore higher load ranges without detectable evidence of surface wear. The contact stiffness does not vary significantly with the normal load up to 38 nN, while above it a sudden increase by almost one order of magnitude was observed. Comparing this to previous results suggests that considerable atom rearrangements may occur in the contact region, although the (100) surface structure is preserved by ethanol-assisted diffusion of Na and Cl ions.

The role of near-wall drag effects in the dynamics of tethered DNA under shear flow.

We utilized single-molecule tethered particle motion (TPM) tracking, optimized for studying the behavior of short (0.922 µm) dsDNA molecules under shear flow conditions, in the proximity of a wall (surface). These experiments track the individual trajectories through a gold nanobead (40 nm in radius), attached to the loose end of the DNA molecules. Under such circumstances, local interactions with the wall become more pronounced, manifested through hydrodynamic interactions. To elucidate the mechanical mechanism that affects the statistics of the molecular trajectories of the tethered molecules, we estimate the resting diffusion coefficient of our system. Using this value and our measured data, we calculate the orthogonal distance of the extended DNA molecules from the surface. This calculation considers the hydrodynamic drag effect that emerges from the proximity of the molecule to the surface, using the Faxén correction factors. Our finding enables the construction of a scenario according to which the tension along the chain builds up with the applied shear force, driving the loose end of the DNA molecule away from the wall. With the extension from the wall, the characteristic times of the system decrease by three orders of magnitude, while the drag coefficients decay to a plateau value that indicates that the molecule still experiences hydrodynamic effects due to its proximity to the wall.

Mechanobiology: protein refolding under force.

The application of direct force to a protein enables to probe wide regions of its energy surface through conformational transitions as unfolding, extending, recoiling, collapsing, and refolding. While unfolding under force typically displayed a two-state behavior, refolding under force, from highly extended unfolded states, displayed a more complex behavior. The first recording of protein refolding at a force quench step displayed an initial rapid elastic recoil, followed by a plateau phase at some extension, concluding with a collapse to a final state, at which refolding occurred. These findings stirred a lively discussion, which led to further experimental and theoretical investigation of this behavior. It was demonstrated that the polymeric chain of the unfolded protein is required to fully collapse to a globular conformation for the maturation of native structure. This behavior was modeled using one-dimensional free energy landscape over the end-to-end length reaction coordinate, the collective measured variable. However, at low forces, conformational space is not well captured by such models, and using two-dimensional energy surfaces provides further insight into the dynamics of this process. This work reviews the main concepts of protein refolding under constant force, which is essential for understanding how mechanotransducing proteins operate in vivo.

Single Molecule Force Spectroscopy and Molecular Dynamics Simulations as a Combined Platform for Probing Protein Face-Specific Binding.

Biomolecular interactions frequently occur in orientation-specific manner. For example, prior nuclear magnetic resonance spectroscopy experiments in our lab have suggested the presence of a group of strongly binding residues on a particular face of the protein ubiquitin for interactions with Capto MMC multimodal ligands ("Capto" ligands) (Srinivasan, K.; et al. Langmuir 2014, 30 (44), 13205-13216). We present a clear confirmation of those studies by performing single-molecule force spectroscopy (SMFS) measurements of unbinding complemented with molecular dynamics (MD) calculations of the adsorption free energy of ubiquitin in two distinct orientations with self-assembled monolayers (SAMs) functionalized with "Capto" ligands. These orientations were maintained in the SMFS experiments by tethering ubiquitin mutants to SAM surfaces through strategically located cysteines, thus exposing the desired faces of the protein. Analogous orientations were maintained in MD simulations using suitable constraining methods. Remarkably, despite differences between the finer details of experimental and simulation methodologies, they confirm a clear preference for the previously hypothesized binding face of ubiquitin. Furthermore, MD simulations provided significant insights into the mechanism of protein binding onto this multimodal surface. Because SMFS and MD simulations both directly probe protein-surface interactions, this work establishes a key link between experiments and simulations at molecular scale through the determination of protein face-specific binding energetics. Our approach may have direct applications in biophysical systems where face- or orientation-specific interactions are important, such as biomaterials, sensors, and biomanufacturing.

Simulated Force Quench Dynamics Shows GB1 Protein Is Not a Two State Folder.

Single molecule force spectroscopy is a useful technique for investigating mechanically induced protein unfolding and refolding under reduced forces by monitoring the end-to-end distance of the protein. The data is often interpreted via a "two-state" model based on the assumption that the end-to-end distance alone is a good reaction coordinate and the thermodynamic behavior is then ascribed to the free energy as a function of this one reaction coordinate. In this paper, we determined the free energy surface (PMF) of GB1 protein from atomistic simulations in explicit solvent under different applied forces as a function of two collective variables (the end-to-end-distance, and the fraction of native contacts ρ). The calculated 2-d free energy surfaces exhibited several distinct states, or basins, mostly visible along the ρ coordinate. Brownian dynamics (BD) simulations on the smoothed free energy surface show that the protein visits a metastable molten globule state and is thus a three state folder, not the two state folder inferred using the end-to-end distance as the sole reaction coordinate. This study lends support to recent experiments that suggest that GB1 is not a two-state folder.

The elastic free energy of a tandem modular protein under force.

Recent studies have provided a theoretical framework for including entropic elasticity in the free energy landscape of proteins under mechanical force. Accounting for entropic elasticity using polymer physics models has helped explain the hopping behavior seen in single molecule experiments in the low force regime. Here, we expand on the construction of the free energy of a single protein domain under force proposed by Berkovich et al. to provide a free energy landscape for N tandem domains along a continuous polypeptide. Calculation of the free energy of individual domains followed by their concatenation provides a continuous free energy landscape whose curvature is dominated by the worm-like chain at forces below 20 pN. We have validated our free energy model using Brownian dynamics and reproduce key features of protein folding. This free energy model can predict the effects of changes in the elastic properties of a multidomain protein as a consequence of biological modifications such as phosphorylation or the formation of disulfide bonds. This work lays the foundations for the modeling of tissue elasticity, which is largely determined by the properties of tandem polyproteins.

Probing the effect of force on HIV-1 receptor CD4.

Cell-surface proteins are central for the interaction of cells with their surroundings and are also associated with numerous diseases. These molecules are exposed to mechanical forces, but the exact relation between force and the functions and pathologies associated with cell-surface proteins is unclear. An important cell-surface protein is CD4, the primary receptor of HIV-1. Here we show that mechanical force activates conformational and chemical changes on CD4 that may be important during viral attachment. We have used single-molecule force spectroscopy and analysis on HIV-1 infectivity to demonstrate that the mechanical extension of CD4 occurs in a time-dependent manner and correlates with HIV-1 infectivity. We show that Ibalizumab, a monoclonal antibody that blocks HIV-1, prevents the mechanical extension of CD4 domains 1 and 2. Furthermore, we demonstrate that thiol/disulfide exchange in CD4 requires force for exposure of cryptic disulfide bonds. This mechanical perspective provides unprecedented information that can change our understanding on how viruses interact with their hosts.

Nanomechanics of HaloTag tethers.

The active site of the Haloalkane Dehydrogenase (HaloTag) enzyme can be covalently attached to a chloroalkane ligand providing a mechanically strong tether, resistant to large pulling forces. Here we demonstrate the covalent tethering of protein L and I27 polyproteins between an atomic force microscopy (AFM) cantilever and a glass surface using HaloTag anchoring at one end and thiol chemistry at the other end. Covalent tethering is unambiguously confirmed by the observation of full length polyprotein unfolding, combined with high detachment forces that range up to ∼2000 pN. We use these covalently anchored polyproteins to study the remarkable mechanical properties of HaloTag proteins. We show that the force that triggers unfolding of the HaloTag protein exhibits a 4-fold increase, from 131 to 491 pN, when the direction of the applied force is changed from the C-terminus to the N-terminus. Force-clamp experiments reveal that unfolding of the HaloTag protein is twice as sensitive to pulling force compared to protein L and refolds at a slower rate. We show how these properties allow for the long-term observation of protein folding-unfolding cycles at high forces, without interference from the HaloTag tether.

Rate limit of protein elastic response is tether dependent.

The elastic restoring force of tissues must be able to operate over the very wide range of loading rates experienced by living organisms. It is surprising that even the fastest events involving animal muscle tissues do not surpass a few hundred hertz. We propose that this limit is set in part by the elastic dynamics of tethered proteins extending and relaxing under a changing load. Here we study the elastic dynamics of tethered proteins using a fast force spectrometer with sub-millisecond time resolution, combined with Brownian and Molecular Dynamics simulations. We show that the act of tethering a polypeptide to an object, an inseparable part of protein elasticity in vivo and in experimental setups, greatly reduces the attempt frequency with which the protein samples its free energy. Indeed, our data shows that a tethered polypeptide can traverse its free-energy landscape with a surprisingly low effective diffusion coefficient D(eff) ~ 1,200 nm(2)/s. By contrast, our Molecular Dynamics simulations show that diffusion of an isolated protein under force occurs at D(eff) ~ 10(8) nm(2)/s. This discrepancy is attributed to the drag force caused by the tethering object. From the physiological time scales of tissue elasticity, we calculate that tethered elastic proteins equilibrate in vivo with D(eff) ~ 10(4)-10(6) nm(2)/s which is two to four orders magnitude smaller than the values measured for untethered proteins in bulk.

Accurate quantification of diffusion and binding kinetics of non-integral membrane proteins by FRAP.

Non-integral membrane proteins frequently act as transduction hubs in vital signaling pathways initiated at the plasma membrane (PM). Their biological activity depends on dynamic interactions with the PM, which are governed by their lateral and cytoplasmic diffusion and membrane binding/unbinding kinetics. Accurate quantification of the multiple kinetic parameters characterizing their membrane interaction dynamics has been challenging. Despite a fair number of approximate fitting functions for analyzing fluorescence recovery after photobleaching (FRAP) data, no approach was able to cope with the full diffusion-exchange problem. Here, we present an exact solution and matlab fitting programs for FRAP with a stationary Gaussian laser beam, allowing simultaneous determination of the membrane (un)binding rates and the diffusion coefficients. To reduce the number of fitting parameters, the cytoplasmic diffusion coefficient is determined separately. Notably, our equations include the dependence of the exchange kinetics on the distribution of the measured protein between the PM and the cytoplasm, enabling the derivation of both k(on) and k(off) without prior assumptions. After validating the fitting function by computer simulations, we confirm the applicability of our approach to live-cell data by monitoring the dynamics of GFP-N-Ras mutants under conditions with different contributions of lateral diffusion and exchange to the FRAP kinetics.

Hopping around an entropic barrier created by force.

We use Langevin dynamics to investigate the role played by the recently discovered force-induced entropic energy barrier on the two-state hopping phenomena that has been observed in single RNA, DNA and protein molecules placed under a stretching force. Simple considerations about the free energy of a molecule readily show that the application of force introduces an entropic barrier separating the collapsed state of the molecule, from a force-driven extended conformation. A notable characteristic of the force induced barrier is its long distances to transition state, up to tens of nanometers, which renders the kinetics of crossing this barrier highly sensitive to an applied force. Langevin dynamics across such force induced barriers readily demonstrates the hopping behavior observed for a variety of single molecules placed under force. Such hopping is frequently interpreted as a manifestation of two-state folding/unfolding reactions observed in bulk experiments. However, given that such barriers do not exist at zero force these reactions do not take place at all in bulk.

Collapse dynamics of single proteins extended by force.

Single-molecule force spectroscopy has opened up new approaches to the study of protein dynamics. For example, an extended protein folding after an abrupt quench in the pulling force was shown to follow variable collapse trajectories marked by well-defined stages that departed from the expected two-state folding behavior that is commonly observed in bulk. Here, we explain these observations by developing a simple approach that models the free energy of a mechanically extended protein as a combination of an entropic elasticity term and a short-range potential representing enthalpic hydrophobic interactions. The resulting free energy of the molecule shows a force-dependent energy barrier of magnitude, DeltaE =epsilon(F - F(c))(3/2), separating the enthalpic and entropic minima that vanishes at a critical force F(c). By solving the Langevin equation under conditions of a force quench, we generate folding trajectories corresponding to the diffusional collapse of an extended polypeptide. The predicted trajectories reproduce the different stages of collapse, as well as the magnitude and time course of the collapse trajectories observed experimentally in ubiquitin and I27 protein monomers. Our observations validate the force-clamp technique as a powerful approach to determining the free-energy landscape of proteins collapsing and folding from extended states.