venerdì 4 novembre 2016

PROTEINS: The molecular machines

Post n. 29 English

Until the middle of last century, molecular biology was mainly focused on the study of proteins.
At that time, the structure and function of nucleic acids were not yet known, while the large number of functions carried out by proteins were known. There are, for example, proteins in bones and cartilage, they are the fundamental substance of the muscles and the skin. Proteins are hormones and substances that defend us from external agents and others have a transportation function. Inside the cell, nothing moves without the intervention of the proteins. A few examples of their role in the cell will help us understand how every living organism is under the constant control of proteins and how life on our planet without them would have no origin.
With the discovery of the structure of nucleic acids (DNA and RNA) and the genetic code in the fifties of the last century, the molecular biology studies are oriented mainly towards the study of nucleic acids. From these studies, it was found that the DNA is formed by discrete amounts, that is of well-defined portions called Genes and these genes through the genetic code define the protein. The original idea was that each gene was an expression for a protein. This idea through mystical Dawkins Genes Replicator, led to consider the genes as regulators of cell activity. When the sequencing of the DNA was completed, that is counting the genes, it was discovered that it contains about twenty thousand genes, but human cells contain about one hundred thousand different proteins. Therefore, the idea one gene one protein is not valid any more. This conclusion was already clear at the beginning of the new millennium and in fact Carol Ezzel in "Adesso comandano le proteine" Le Scienze 2002 wrote: «Simply applying the data produced by the Human Genome Project - which dethroned the outdated dogma, that one gene codes for a protein - does not solve the problem».  The author also shows us how more vigour the study of proteins and in particular of enzymatic proteins or Proteomics are in those years.
With the continuation of the research a new theoretical framework is proposed by Richard C. Francis in "L’ultimo mistero dell’ereditarietà" in 2011, in reference to the genes states: «The traditional idea is that they are a kind of managerial officials who run the our development. The alternative idea is that the directional function is located at a cellular level and the genes act more as resources available to the cell itself». And Steven Rose in "Geni, cellule e cervello" in 2013 adds: «It is not the DNA that determines the cellular activity, but it is the cell in which the DNA is incorporated that chooses what pieces of DNA to be used to construct certain proteins, when and how: epigenetics».
As we said previously, the cell is under the constant control of proteins and in particular of enzymatic proteins or enzymes. Inside the cell thousands of chemical reactions occur. In the chemical and physical conditions in which the cells of living organisms are, these reactions were very slow or may not occur, and the cells will not survive. Enzymes are catalysts that accelerate and control the speed of the chemical reactions allowing the cells to survive. To have an idea of ​​the action of these substances, Robert M. Stroud in "Una famiglia di proteasi" Le Scienze 1974, writes that without proteolytic enzymes it would take 50 years to digest a meal.
The main feature of enzymes is their specificity, that is, they catalyze only one chemical reaction. A medium-sized cell contains about three thousand different enzymes that control as many reactions. Each of these enzymes is present in multiple copies and for this reason, inside each cell there are about two million enzymes. This raises the problem of understanding how enzymes actually work.
The study of the function of the enzymatic proteins begins with Emil Fischer in 1894. It was already known at that time that enzymes consist of twenty different amino acids that make up all proteins. An average enzyme contains about 300 amino acids that fold to form a globular structure, fundamental to perform its function. The enzymes recognize molecules selectively, each enzyme recognizes only a compound or at most two, referred to as "substrate", and acts on them. The part of the enzyme which recognizes the substrate or the substrates takes the name of "active site." Based on these considerations Fischer formulated the hypothesis of the "key and lock". According to this theory, the active site would have such a form that the substrate fits like a key fits its lock.

For each key, its lock, for each compound, its enzyme.
Without going into too much detail, this model was replaced by the induced adaptation model. As it is known, the enzyme changes the chemical-physical environment of its surroundings. When the substrate enters in this new environment its links slow down, and the substrate takes on a different form called "transition state". The transition state may vary from molecule to molecule and the enzyme adapts to these small structural changes.

Enzymes produced through the evolution process in nearly 4 billion years are really extraordinary substances, true molecular machines. It’s enough to say that, one molecule of catalase alloy and turns around 100 000 molecules of oxygenated water per second and releases the same number of water molecules, and one carbonic anhydrase molecule transforms 600 000 substrates (CO2 and H2O) per second.
Besides the catalytic enzymes have what has been called "social aspect." Mike Williamson writes in "Come funzionano le proteine" 2013: «The social aspect of the enzyme associates it with other components, with a membrane or a protein, or the formation of large complexes through interaction with other enzymes». It was found that only a few proteins act alone, most of them operate in complex and some of them operate even in more complexes. The protein components of a complex may vary from 2 to 100. The protein complexes have an organization similar to highly efficient factories. For example in our cells, the glucose metabolism occurs through tens of chemical reactions. These reactions cannot occur at random, but must be regulated and planned to perfection because the product of a reaction is used for a subsequent reaction.

Enzymes regulate each of these reactions. They, one after the other, in a coordinated manner and with extraordinary efficiency, as in an assembly line, transform the substrate providing the product for the next enzyme. Every product of a reaction immediately finds another enzyme, to be further transformed and everything has to work perfectly up to the final products.
We have said that an average enzyme contains about 300 amino acid residues that fold into a globular structure.
But how did it get to such a large molecule? And how is the globular structure formed?
Around 1970 it was suggested that proteins were formed by domains, which are amino acid sequences that have not changed throughout evolutionary process. Rossman in 1974 identified a domain of about 70 amino acids present in many enzymes and proposed that this domain was of prebiotic origin. Today many believe that the domains, in the prebiotic era, were smaller and mainly consisted of ꭤ-helices of about 20 amino acids. The large protein molecules could have originated from the aggregation and evolution of small domains. The different arrangement of these domains, or the combination of a new domain to an existing enzyme, generates a new function. This leads to the conclusion, as Russell F. Doolittle wrote already in 1985 in "The Proteins" Scientific American: «[...], so the vast majority of proteins must be derived from a small number of archetypes».
The globular structure of an enzymatic protein is called tertiary structure. The primary structure is a long chain that indicates the position of each amino acid in the protein molecule. If the enzyme stayed the same it would have no function, and it would quickly degrade. Before the degradation happens, the secondary structure is formed where several amino acids are organized in helices and sheets. Finally, it forms the globular structure, that is, the tertiary structure. The globular structure of a protein (fold) is a consequence of the primary structure, but from the knowledge of the primary structure, we are not able to go back to its tertiary structure. In the second half of the last century, X-ray analysis have given a big contribution to the knowledge of the tertiary structure. It was thought that knowing the structure one could retrieve the function. In fact, in 1985 Russell F. Doolittle (cited work) wrote: «One of the major objectives of the protein has been in-depth knowledge of their structure in order to be able to understand their function». Today there are thousands of protein structures but going back to their function is a difficult task.
Nevertheless, as described by Peter M. Hoffmann in “Gli ingranaggi di Dio” 2014, modern equipment with sensors that operate at nano scales, sophisticated optical microscopes that can detect the light emitted by a single molecule and so-called Laser pliers, have enabled us to understand how the enzymes reach the globular structure and how they work.
The process that the primary structure leads to the tertiary structure is under thermodynamic control, that is, the molecules tend to place themselves in a position, which corresponds to a minimum of energy, a more stable state. In short, like a stone rolling down a hill until it reaches the valley. But if the stone along its descent meets a terrace it is likely to stop halfway. The enzymes are in a similar situation when the primary structure must reach the globular structure. In fact, a globular protein which comprises of about 150 amino acids could curl up in countless ways. According to Anfinsen 1045 would be the number of possible randomly generated conformations. Even though most of these conformations are impossible, in a single enzymatic protein there would still be a huge number of low energy conformations, where the difference in energy between the different conformations is small. As seen from the diagram (called diagram of the energy landscape) the folding process should lead the enzyme to have a minimum of energy.

elaborazione da: Treccani

A slight change of the surrounding environment could block the enzyme in an intermediate minimum, like the stone blocked halfway by a terrace.
What did evolution invent to avoid this risk? The guides: Chaperone and Chaperonin.
To understand how Chaperones work Mike Williamson (cited work) used the following metaphor: «In the nineteenth century, a single woman was often accompanied by a chaperone in public, an older or married woman who prevented her friend from engaging inappropriately contacts with the opposite sex. By analogy, a chaperone protein acts by preventing unstructured proteins to engage 

in unwanted interaction [...]». The chaperonin lead instead is the primary structure to the right conformation. They have the shape of a barrel with a cavity inside with which the protein assimilates the right conformation and then it is released into the environment.
However, despite everything it is possible that a protein may still not be structured properly, damaged, or no longer useful to the cell. What did evolution invent? Quality control and Recycling: ubiquitin and proteasome.
Ubiquitin is a small protein molecule, stable and very abundant in cells. The ubiquitin recognizes and binds to proteins to degrade; proteins are so labelled. From this moment the fate of the protein to be degraded is marked. The complex protein ubiquitin is recognized by another protein complex: the proteasome. The Proteasome is a real molecular machine: a digester. Also in the shape of a barrel, as soon as it comes into contact with the protein-ubiquitin complex, the proteasome dismantles it, puts ubiquitin into the circulation of the cell and swallows the protein to be degraded. The degradation leads to peptides of about seven residues that are released into the cell to be reused.


We do not know when, within humans, began to transport and exchange goods. The cell had already invented 3.5 billion years ago: microtubules, kinesin, dynein, myosin and actin.
Within the cell there was heavy traffic, as documented by us Robert Day Allen in "The microtubule as intracellular molecular motor" The Sciences in 1987, it was already known since the nineteenth century by Joseph Leidy. By the mid-sixties of the last century microtubules were discovered, channels with a diameter of about 25 nanometers wide and about 100 000 nanometers long (the dimensions understandable to us, is as if we have 1 cm diameter tubes and 4000 cm long , 40 meters). Microtubules consist of long intertwined protein chains: tubulin. It was immediately understood that microtubules inside the cell had structural functions. It was Allen and his collaborators, around 1985, to discover that in addition to the structural features microtubules were in fact the routes used by cells to transport materials with the help of two other proteins: actin and myosin. Later it was discovered that other proteins, dynein and kinesin were involved in the transport of materials within the cell.
The movements of kinesin and dynein in microtubules and myosin that moves on protein actin filaments, real molecular machines, seem human like as the image taken from Hoffmann’s mentioned essay depicts.

The upper part is a vesicle that contains the nutrients, the body rests on two feet moving on the microtubule. It mimics the way we walk and always keeps one foot on the ground that is the microtubule. To raise a foot energy is needed, ATP. A kind of firecracker exploding under foot, it detaches from the microtubule and swings advancing it. For a clearer idea of his movement, you can watch a great video on YouTube, for example:
These videos are not imaginary animation or computer generated. The videos were for sure processed but real.
As confirmed by Hoffmann in his essay, they were obtained with special tools which can film the movement of a single molecule.
Hoffmann, after seeing the video of the myosin moving along actin filaments as a little creature with two legs, wrote: «Is it possible that the molecule is alive? No, not in the true sense of the term. Watching it parading before us, we can see how all of these machines, interacting in a controlled manner, can create a living being. It is here, no doubt, where life begins».
Finally, in a very concise way we will describe three types of machines, the molecular copiers: Helicase, Polymerase, and RNApolymerase.
The DNA is a double stranded molecule which contains the information necessary for the synthesis of proteins. The two helices are held together by weak bonds (hydrogen bonds). Because these bonds are millions, the DNA molecule is very stable. Furthermore, to protect the entire molecule from possible degradation it is enveloped by numerous proteins. During cell division, the DNA must be copied, and a copy transferred to the new cell. To copy the DNA we must first separate the two helices. The copying of DNA involves a large number of proteins, but the main task is done by helicase enzyme. The helicase is a ring-like protein similar to a six-pointed star. It makes its way through the protein molecules that wrap around the DNA and once it reaches the DNA it opens up, wraps it in its interior and closes again.


This molecular machine opens, first of all, the double helix and therefore as a pair of scissors moving along the DNA separating the two helices. Two other proteins, polymerases and topoisomerases, are in charge of the double helices reconstruction from the constituent elements found in the cell. Like perfectly coordinated engines, the helicase cuts DNA, polymerase reconstructs the double helix and another polymerase (topoisomerase) follows and reconstructs another double helix in the opposite direction. At the end we will have two identical copies, one for each cell.

The RNA polymerase is the protein that transcribes a piece of DNA into a messenger RNA for protein synthesis. It attaches to the DNA and then moves along the double helix until it finds the correct sequence from which to start: the promoter. RNA polymerase is a powerful molecular machine which does everything by itself. As soon as it finds the promoter the polymerase detaches the two helices, moving along the filaments, transcribes a piece of DNA (gene) into messenger RNA, corrects errors and once the copy process is concluded it sews the DNA and walks away.


As we have explained, in the cells they operate as assembly lines, guides, quality control and recycling, transportation of materials and we might even add protein pumps and molecular motors. These molecular machines definitely have always existed. They were certainly much more rudimentary, but they had to be there from the beginning. The origin of life without these molecular machines is unlikely.
We too have invented the assembly lines, quality control and recycling, pumps, electric motors and the so on, but behind our inventions there was always an idea, a mind.
So, who is behind the cellular inventions? Which is: what is life?

                                                                                      Giovanni Occhipinti

Translated by: Sydney Isae Lukee

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