# A stochastic mechanism drives fast substrate translocation in the AAA+ machine ClpB

## TL;DR

The study reveals that the ClpB AAA+ machine uses a fast, random motion driven by ATP to move proteins, rather than a step-by-step process.

## Contribution

The paper introduces a novel stochastic mechanism for ATP-driven protein translocation in ClpB AAA+ machines.

## Key findings

- Translocation events in ClpB occur in milliseconds, much faster than ATP hydrolysis.
- Translocation is not strictly unidirectional and is not strongly affected by temperature or ATP concentration.
- ATPγS inhibits rapid translocation and directionality, indicating ATP hydrolysis is essential for the process.

## Abstract

How biological machines harness ATP to drive mechanical work remains a crucial question. Structural studies of protein-translocating AAA+ machines proposed a coupled and sequential translocation process, whereby ATP hydrolysis events lead to short threading steps. Yet, direct real-time observation of these events remains elusive. Here, we employ single-molecule FRET spectroscopy to track substrate translocation through ClpB, a quality control AAA+ machine. We isolate ClpB and its substrate within lipid vesicles and find that translocation events, while dependent on ATP, take milliseconds, much faster than ATP hydrolysis times. Surprisingly, the translocation rate depends weakly on temperature and ATP concentration. Using three-color FRET experiments, we find that translocation events can occur bidirectionally but are not always complete. Replacing ATP with the slowly hydrolysable analog ATPγS abolishes both rapid translocation and directionality. These results indicate a fast, stochastic Brownian-motor-like mechanism, redefining how ATP is coupled with mechanical action in AAA+ machines.

Cells rely on AAA+ machines to move and remodel proteins. Here, authors show that ClpB threads proteins rapidly using ATP to bias diffusive motion, revealing a fast, stochastic Brownian-motor mechanism rather than stepwise power strokes.