Amazing stuff!
"... Machines from the AAA+ family, which exist in the cells of all living organisms from bacteria to humans, can, among their many functions, recognize misfolded protein chains and swiftly unravel them.
Researchers ... have deciphered this sophisticated mechanism, which is both fast and remarkably efficient. Their findings ... reveal how cells perform quality control on their proteins, and may help explain why this control fails in diseases such as neurodegeneration and cancer. They may also provide inspiration for the development of highly efficient artificial molecular machines. ...
When a protein chain in the cell becomes entangled or misfolded, these machines come to the rescue, unraveling the chain by threading it through the channel. ...
Until now, it was unclear how a tiny molecular machine converts chemical energy within the cell into an effective mechanical pulling action. ...
To address this question, the researchers ... developed a method that allowed them to monitor, in real time rather than through frozen snapshots, the passage of a protein chain through the molecular machine. They used fluorescent sensors attached to the milk protein casein and to the AAA+ machine that processes it. A green sensor was attached to the casein, an orange sensor to the machine’s entrance and a red sensor to its exit. ...
“Loops in the channel wall protrude into its interior and, like the wings of a revolving door, determine the preferred direction of movement. The machine uses energy to ensure that these loops oscillate in the correct direction.” ...
In the final stage of the study, the researchers focused on failure events, in which threading through the channel was not completed. “These events lasted a relatively long time,” ... “We found that in their course, the protein moved back and forth within the channel until it mistakenly exited from the same end where it had entered. This indicates that there are no large energy fluctuations or powerful forces inside the channel, but rather a subtle motion-guiding mechanism that is occasionally prone to error.” ..."
From the 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."
Fig. 1: Observation of multiple fast events of translocation through the lumen of ClpB facilitated by porous vesicles.
Fig. 4: Translocation of casein in both the forward and backward directions is observed in three-color experiments.
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