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The protein folding process

Understanding the mechanism that controls the folding of proteins is important not only for basic biological research, but may also help in the study of diseases, due to the fact that several diseases - including Alzheimer's - are characterized by misfolding of proteins. Analyzing the folding mechanism of small proteins is relatively simple, since the transition from the unfolded form to the folded form is done in one step. But what about large and complex proteins?

Sitting, from the right: Rita August, Prof. Gilad Haran, Dr. Inbal Rivan, Menachem Farhi. Standing, from the right: Mila Gomanovsky, Dr. Yoav Barak, Nir Zohar and Sharona Zedekani Cohen. Beads

Proteins are the building blocks of the animal world. In fact, all the activities that occur in living cells depend on proteins: movement, respiration, the activity of the immune system and the hormonal system, enzymatic activity - these are just a few examples of their vast range of activity.

The protein begins its life as a long chain, made of "beads" of amino acids. It is the sequence of the beads that determines the primary structure of the protein. But, in order for it to acquire its activity capacity, the protein must "mature", that is, fold into an active three-dimensional structure. The XNUMXD structure of the protein provides important insights into its activity. For this reason, for decades, scientists have been developing tools and methods to decipher the three-dimensional structure of proteins.

But sometimes the way - or the process - is just as important as the final result: how is the transition from the initial, unfolded state of the protein to the final folded structure? Understanding the mechanism that controls the folding of proteins is important not only for basic biological research, but may also help in the study of diseases, due to the fact that several diseases - including Alzheimer's - are characterized by misfolding of proteins. Analyzing the folding mechanism of small proteins is relatively simple, since the transition from the unfolded form to the folded form is done in one step. But what about large and complex proteins?

Analyzing the folding mechanisms of large proteins is somewhat similar to reading maps," he explains Prof. Gilad HaranFrom the Department of Chemical Physics in the Faculty of Chemistry. "You can identify many roads on the map, which all lead to the same point. Some of these routes are straight and simple, while others have to cross mountains and valleys to reach the same destination. Through the mapping we hope to find out whether the proteins use several ways - or several intermediate structural forms - that lead to the final folded structure. And if this is indeed the case - how many such intermediate forms exist? Do they have to go through all the intermediate forms, or can they take shortcuts and bypass some of them? Do they have a preferred route, a chosen sequence of shapes? Do external conditions, such as temperature, affect their behavior?"

To answer these questions Prof. Hearn developed A unique research method. Together with the members of his team - including research students Menachem Farhi, Sharona Sedgani-Cohen and Nir Zohar, former research student Guy Ziv, post-doctoral researcher Inbal Rivan, and researcher Yoav Barak, from the Department of Chemical Research Infrastructures - he chose a certain protein (the enzyme adenylate kinase), and marked it with fluorescent markers at two different points along the chain of amino acids that make it up. When the two markers are far from each other (that is, when the protein is not folded), green light is emitted. When the protein folds, the markers move closer together, and red light is emitted. Thus, by tracking the emitted light, the scientists hoped to map the location of the labeled amino acids, and in this way determine the number of intermediate states required for the protein in the transition from the unfolded to the folded form.

Before getting to work, the scientists had to face another problem: the protein molecules migrate freely, so it is difficult to follow them for a long enough time to analyze them. To overcome the problem, Prof. Haran created a sort of trap: each protein molecule was placed in a transparent vesicle which was attached to a glass surface. This device prevented the protein molecules from moving, and thus the measurements could be made. Another problem was discovered with the fluorescent color markers, which lost their color within a few seconds - too short a period of time to fully follow the folding process. The scientists solved the problem similar to a psychologist trying to decipher a person's current state of mind by putting together pieces of events that appear to him in random order: they used thousands of protein molecules, and analyzed short segments of folding processes - each of which begins at a different point in time. They then used a statistical analysis they developed to "glue" together the short sequences of the folding events, according to the correct chronological order.

The research findings, recently published in the journal Nature Communications, showed that for the protein they tested, there are six possible intermediate forms leading to the final three-dimensional structure. The way in which the protein is chosen to fold depends on external factors, such as temperature or different chemical concentrations. Thus, for example, the greater the concentration of a certain chemical, the more the protein tends to choose a longer and more "tedious" path - when it visits each of the six intermediate forms, one after the other, before reaching the final structure. At lower concentrations of the substance, the protein chooses a shorter path, in which it can use shortcuts that save some of the intermediate forms, but still lead it

to the correct final structure.

Due to the many folding possibilities of protein molecules, examining thousands of molecules one after the other allowed scientists to identify the exact folding mechanism - as opposed to the average result obtained from measuring all the molecules together. The scientists plan to continue studying other protein molecules, and thus examine whether the laws they discovered are valid for all proteins, and to understand more deeply the forces that direct the folding of proteins.