Sprecher
Beschreibung
We have recently constructed a modular chimera that mimics the functional architecture of the 26S proteasome, coupling protein unfolding and proteolysis.[1] The nanomachine consists of two sequentially aligned compartments, A(p97) and B(aCt), made by scaffolded DNA origami cages. The first compartment contains p97, an ATP-dependent unfoldase, that mechanically unfolds the substrate. This then translocates into the second compartment, where it is proteolytically degraded by alpha-chymotrypsin. We observed that the substrate of p97, the protein I3, is unfolded by A(p97) three times faster than by non-compartmentalized p97, and proteolysis by B(aCt) is five times faster than by non-compartmentalized alpha-chymotrypsin. These findings confirm the widely observed enhanced performance of DNA-scaffolded enzymes relative to their freely diffusing counterparts.[2, 3] A key finding from our previous study, although not yet fully understood, is the uniform spatial orientation of p97, whereby its N-terminal domains consistently align toward one of the cage’s apertures. In this work, we propose a strategy for studying the dynamics of p97 in different settings while monitoring motor’s operation and relative orientation at the single-molecule level. For this purpose, we will engineer an encaged p97 that functions like a molecular pulley, actively drawing the substrate protein toward itself for unfolding. We envision that the obtained results will contribute to a deeper understanding of p97’s operation and will help to shed light onto the mechanisms underlying the enhanced reactivity and preferred orientation of natural and artificial compartmentalized biological nanomachines.
- Huang J, et al. 2024. Nat Nanotechnol 19: 1521-31
- Huang J, et al. 2023. Small 19: e2202253
- Lin P, et al. 2025. Chempluschem 90: e202400483
