Search

Protein-based biomaterials are in high demand due to their high biocompatibility, non-toxicity, and biodegradability. In this study, we explore the bacterial E. coli secreted protein A (EspA), which self-assembles into long extracellular filaments, as a potential building block for new protein-based biomaterials. We investigated the morphological and mechanical properties of EspA filaments and how protein engineering can modify them. Our study include three types of filaments: natural EspA filaments, full-length recombinant EspA filaments, and truncated recombinant EspA filaments lacking a third of the original codon region. The recombinant EspA proteins formed curly, thin filaments with higher longitudinal elasticity (shorter persistence length) compared to the natural, linear filaments. Additionally, the recombinant filaments had a radial elastic modulus about an order of magnitude lower than the natural filaments. The truncated recombinant filaments had a higher radial modulus than the full-length ones, and unlike the purely elastic natural filaments, recombinant filaments were less compliant with the applied force that penetrated them. These differences underscore the potential to modulate EspA filament properties through protein sequence mutations. Our findings suggest EspA as a fundamental element for developing a new biomaterial with a hierarchical structure, enabling the fabrication of macroscopic substances from self-assembled EspA-modulated filaments.

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 ​pN) and moderate-low (45, 30, 20 ​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.