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Abstract
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.
Highlights
Polyproteins are biological constructs made of proteins repeats tethered in tandem, often related to physiological activity that involves mechanical stresses, such as muscle contractions,[1,2] mechano-transduction,[3,4] etc
The mechanical response of polyproteins to external forces has been extensively studied using single-molecule force spectroscopy (SMFS) through conformational changes, such as unfolding, folding and collapse, in response to various forms of direct load application protocols.[9−20] In force extension (FX) measurements, direct load is applied to a single polyprotein molecule, which is tethered at both termini, by pulling it at constant velocity
This unfolding history dependency was explained as a biphasic behavior, resulting from two competing effects: (a) increase of domain unfolding probability with each unfolding event along the polyprotein chain (N-effect), and (b) decrease of domain unfolding rates resulting from an increase in the cantileverpolymeric component of the polyprotein after each unfolding event
Summary
Polyproteins are biological constructs made of proteins repeats tethered in tandem, often related to physiological activity that involves mechanical stresses, such as muscle contractions,[1,2] mechano-transduction,[3,4] etc. While previous works focused on the stiffness of the pulling device and the nonlinear elasticity of the polymeric linkers on the unfolding probabilities, this work investigates the relation between the variations in the local stiffness of the polyprotein (linker and folded/unfolded domains) and internal friction of the elongating linker at high extensions and their relation to hierarchical unfolding forces within a polyprotein under tension To this end we performed FX SMFS measurements using atomic force microscopy (AFM) on octameric repeats of.
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