Weizmann Institute of Science research reveals how AAA+ family machines use an energy-efficient mechanism to thread proteins through a tiny channel, providing inspiration for the development of artificial molecular machines
Key phrase:
Synonyms: Molecular machines, molecular motors, Brownian motor, protein quality control, misfolded proteins, AAA+ family, protein unfolding, ATP, random protein movement, nanomachines
SLUG: molecular-machines-protein-unfolding-brownian-motor-weizmann
Image caption 1: From right: Prof. Gilad Haran, Dr. Inbal Rivan, Dr. Yoav Barak and Dr. Dorit Levy. Credit: Weizmann Institute of Science
Image caption 2: Dr. Rami Cassier, who led the development of the method for real-time monitoring of protein transit in a molecular machine. Credit: Weizmann Institute of Science
Image caption 3: From right: Prof. Gilad Haran, Dr. Yoav Barak, Dr. Inbal Rivan and Dr. Dorit Levy. Credit: Weizmann Institute of Science
SEO Title: This is how molecular machines form tangled proteins
Meta description: Research from the Weizmann Institute reveals an economical mechanism by which molecular machines from the AAA+ family form proteins, helping to understand quality control in cells.
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Trying to untangle a tangle of threads can be frustrating and time-consuming for us, but not for molecular machines – molecules that convert chemical energy into mechanical work and movement. AAA+ family machines are present in the cells of all living things, from bacteria to humans, and are capable, among other things, of recognizing protein chains that have folded into an abnormal structure and rapidly unraveling them. Research conducted in the laboratory of Prof. Gilad Haran At the Weizmann Institute of Science, he deciphered their sophisticated mechanism of action, which combines speed and energy efficiency. The research findings, whichwere recently published in the scientific journal Nature Communications., Revealing how cells perform quality control on their proteins, paving the way to understanding why quality control fails in diseases such as neurodegeneration and cancer, and inspiring the development of efficient and cost-effective man-made molecular machines.
Over the past decade, scientists have managed to photograph the three-dimensional structure of tiny machines from the AAA+ family, by freezing them and viewing them under an electron microscope. It turned out that each machine is made up of six protein subunits connected in a circle and forming a central channel. When a protein chain in a cell becomes tangled, these machines come to its aid and untie its knots by threading it through the channel. But what force pulls on the threads? Until now, it was unclear how the tiny molecular machine converts chemical energy in the cell into an effective physical pulling action. The hypothesis was that this is a "hand chasing hand" mechanism: in each cycle of operation, the machine uses a large amount of energy to throw one "arm" (subunit) forward, grab the protein chain and pull, until it finishes threading the entire thing through the channel. However, this hypothesis was inconsistent with several biophysical observations reported in the literature.
To solve the mystery, scientists led by Dr. Rami Kassier from Prof. Haran's lab developed a method that allows real-time monitoring, rather than through frozen images, of the passage of a protein chain through the molecular machine. They used fluorescent sensors attached to the cheese protein casein and the AAA+ machine operating on it. A green sensor was attached to the casein, an orange one to the machine's inlet, and a red one to the outlet. The sensors were designed so that when they are far apart, only the green one glows, but when the protein passes through the channel, it donates its energy to the orange or red. Based on the intensity of the light in each color, the scientists knew exactly where the protein was at any given moment. To ensure that the protein and the machine would meet again and again, the scientists trapped them in a small fat bubble that does not allow them to escape, but does allow the entry of ATP molecules, which serve as the "fuel" of most molecular machines.
The 2016 Nobel Prize in Chemistry was awarded for the development of man-made molecular machines. The new findings may allow for improved design of such machines.
"The part of the protein that we marked moved through the channel at an enormous speed, within a few milliseconds," describes Prof. Haran, from the Department of Chemical and Biological Physics at the institute. "This is despite the fact that it took the machine more than half a second to break down one ATP molecule and extract energy from it. In this way, the machine turned out to be quite economical and the 'hand chasing hand' mechanism involving bursts of energy and large jumps became less likely - we had to recalculate the trajectory."
Revolving Door – The Molecular Version
The scientists performed two experiments to decipher the role of ATP molecules in the mechanism of the machine. In the first experiment, they replaced them with molecules that have a similar structure but are almost inactive and detected that the movement in the channel became undirected. In the second experiment, the scientists gradually reduced the concentration of ATP without completely eliminating it. They noticed a dramatic decrease in the number of transition events in the channel, but to their surprise, the speed of the transition remained almost unchanged.
"We discovered that the machine uses energy to start the threading process and maintain the direction of movement, but not to forcefully pull the chain and accelerate its movement," explains Prof. Harn. "We propose that the molecular machine is similar to a revolving door - when the protein enters, it can try to move in any direction, but the machine is built so that in the presence of ATP, only movement in a certain direction leads to progress and attempts to move in the opposite direction are blocked. Since proteins move randomly all the time, such a mechanism - known in professional parlance as a 'Brownian engine', after Robert Brown, who was the first to observe the random movement of small particles under a microscope - is very economical."
"Based on these findings and previous studies, we can now speculate exactly what is happening under the hood of the molecular machine," he adds. "Loops on the walls of the channel protrude into its space and, like the wings of a revolving door, determine the preferred direction of movement. The machine uses energy to make sure that the loops (the wings of the door) oscillate in the right direction."
In the final part of the study, the scientists focused on failure events in which the passage through the channel was not completed. "Such events lasted for a long time and we found that during them the protein moved back and forth along the channel until it accidentally exited where it came from," describes Prof. Haran. "This indicates that the channel does not have large energy changes and powerful forces, but rather a mechanism for directing delicate movement, subject to occasional errors."
"In the new study, we were able to peer into the mechanism of an important molecular machine that has been operating in cells for billions of years," says Prof. Haran. "In many diseases, such as neurodegeneration and cancer, the quality control of cell proteins fails and improperly folded proteins accumulate. Understanding the control processes is the basis for discovering in the future why this happens and how to stop it. In addition, AAA+ family machines have many roles beyond quality control; they transport proteins and genetic material and transport them through membranes, and we hypothesize that the 'Brownian motor' we identified also drives these processes."
In 2016, the Nobel Prize in Chemistry was awarded for the development of man-made molecular machines, such as a tiny elevator, an artificial muscle, and a nanocar, and the new findings may allow engineers to improve the design of such machines. "The energy efficiency of the Brownian engine could allow a leap forward in the development of artificial molecular machines," believes Prof. Haran. "Such machines are expected to perform practical work in the future and be integrated into engines and computers."
Dr. Dorit Levy and Dr. Inbal Rivan from the Institute's Department of Chemical and Biological Physics, and Dr. Yoav Barak from the Institute's Department of Chemical Research Infrastructures, also participated in the study.
