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

[Image: see text] 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.