Kinetic effects in directional proteasomal degradation of the green fluorescent protein.

The 26S eukaryotic proteasome is an ATP-dependent degradation machine at the center of the ubiquitin-proteasome system that maintains cell viability through unfolding and degradation of ubiquitinated proteins. Its 19S regulatory particle uses a powerful heterohexameric AAA+ ATPase motor that unfolds substrate proteins and threads them through the narrow central pore for degradation within the associated 20S peptidase. In this study, we probe unfolding and translocation mechanisms of the ATPase motor by performing coarse-grained simulations of mechanical pulling of the green fluorescent protein substrate through the pore. To discern factors controlling the N-C or C-N directional processing of the substrate protein, we use three distinct models involving continuous pulling, at constant velocity or constant force, or discontinuous pulling with repetitive forces. Our results reveal asymmetric unfolding requirements in N- and C-terminal pulling upon continuous application of force in accord with the softer mechanical interface near the N-terminal and restraints imposed by the heterogeneous pore surface. By contrast, repetitive force application that mimics variable gripping by the AAA+ motor results in slower unfolding kinetics when the force is applied at the softer N-terminal. This behavior can be attributed to the dynamic competition between, on the one hand, refolding and, on the other, rotational flexibility and translocation of the unfolded N-terminal α-helix. These results highlight the interplay between mechanical, thermodynamic, and kinetic effects in directional degradation by the proteasome.