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Updated: Oct 20, 2025

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution
Published on: August 16, 2016
Ultrafast pore-loop dynamics in a AAA+ machine point to a Brownian-ratchet mechanism for protein translocation
Hisham Mazal1, Marija Iljina1, Inbal Riven1
1Department of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot 761001, Israel.
Abstract:
AAA+ ring–shaped machines, such as the disaggregation machines ClpB and Hsp104, mediate ATP-driven substrate translocation through their central channel by a set of pore loops. Recent structural studies have suggested a universal hand-over-hand translocation mechanism with slow and rigid subunit motions. However, functional and biophysical studies are in discord with this model. Here, we directly measure the real-time dynamics of the pore loops of ClpB during substrate threading, using single-molecule FRET spectroscopy. All pore loops undergo large-amplitude fluctuations on the microsecond time scale and change their conformation upon interaction with substrate proteins in an ATP-dependent manner. Conformational dynamics of two of the pore loops strongly correlate with disaggregation activity, suggesting that they are the main contributors to substrate pulling. This set of findings is rationalized in terms of an ultrafast Brownian-ratchet translocation mechanism, which likely acts in parallel to the much slower hand-over-hand process in ClpB and other AAA+ machines.
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