Crowding effects on the mechanical stability and unfolding pathways of Ubiquitin

TL;DR

This study uses a coarse-grained model to investigate how cellular crowding influences the mechanical stability and unfolding pathways of ubiquitin, revealing increased unfolding forces and pathway alterations due to crowding effects.

Contribution

It provides new insights into how macromolecular crowding affects protein unfolding forces and pathways using the SOP model and theoretical analysis.

Findings

Crowding increases the force needed to unfold ubiquitin.

Crowding alters unfolding pathways and promotes structural reassociation.

Unfolding force scales with crowding volume fraction as ^{1/3}.

Abstract

The interior of cells is crowded thus making it important to assess the effects of macromolecules on the folding of proteins. Using the Self-Organized Polymer (SOP) model, which is a coarse-grained representation of polypeptide chains, we probe the mechanical stability of Ubiquitin (Ub) monomers and trimers ((Ub)$_3$) in the presence of monodisperse spherical crowding agents. Crowding increases the volume fraction ($\Phi_c$)-dependent average force ($<f_u(\Phi_c)>$), relative to the value at $\Phi_c = 0$, needed to unfold Ub and the polyprotein. For a given $\Phi_c$, the values of $<f_u(\Phi_c)>$ increase as the diameter ($\sigma_c$) of the crowding particles decreases. The average unfolding force $<f_u(\Phi_c)>$ depends on the ratio $\frac{D}{R_g}$, where $D \approx \sigma_c (\frac{\pi}{6 \Phi_c})^{{1/3}}$ with $R_g$ being the radius of gyration of Ub (or (Ub)$_3$) in the unfolded state. Examination of the unfolding pathways shows that, relative to $\Phi_c = 0$, crowding promotes reassociation of ruptured secondary structural elements. Both the nature of the unfolding pathways and $<f_u(\Phi_c)>$ for (Ub)$_3$ are altered in the presence of crowding particles with the effect being most dramatic for the subunit that unfolds last. We predict, based on SOP simulations and theoretical arguments, that $<f_u(\Phi_c) > \sim \Phi_c^{\frac{1}{3\nu}}$, where $\nu$ is the Flory exponent that describes the unfolded (random coil) state of the protein.