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.

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.