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May 7th, 2025
Version: 2
Weizmann Institute of Science
biophysics
biorxiv

Millisecond bidirectional protein translocation by a AAA+ disaggregation machine

Casier, R.Open in Google Scholar•Levy, D.Open in Google Scholar•Riven, I.Open in Google Scholar•Barak, Y.Open in Google Scholar•Haran, G.Open in Google Scholar

How biological machines utilize ATP to facilitate mechanical work remains heavily contested. ClpB is a quality control machine of the AAA+ family that salvages misfolded and aggregated proteins by forcibly translocating them through its lumen. Structural studies of this and similar machines have suggested a power-stroke translocation mechanism directly coupled to sequential subunit motion. However, functional studies have challenged this deterministic view. Here, we directly track substrate translocation of individual ClpB hexamers trapped within lipid vesicles. Using single-molecule FRET, we find that translocation occurs in milliseconds, far exceeding ATP hydrolysis rate. We further observe partial substrate engagement events and even translocation proceeding in both directions through the lumen of ClpB. Rather than generating discrete power strokes, ATP appears to act by regulating substrate engagement and translocation directionality. These results indicate a fast, stochastic mechanism akin to a Brownian motor, redefining how ATP is coupled with mechanical action in AAA+ machines.

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