sabato 2 aprile 2016

THE ORIGINS OF PROTEINS: The synthesis of polypeptides

Post n. 26 English

Imagine you have a full water bottle and you place it upside down in a container, which is also full of water. No one, not even those who have never done this experience as a kid would expect the bottle to get empty. Although it’s a bit obvious, it seems an appropriate metaphor to clarify the problem of the synthesis of the fundamental macromolecules of life.
The reaction of hundreds of peptide bonds for the formation of proteins,

of the constituent nucleotides of nucleic acids,

of hundreds of bonds for the formation of nucleic acids,

of sugars

and lipids

give out as a reaction product water also.
These reactions are actually equilibrium reactions, meaning reactions whereby the products, always remain constant after reaching a certain concentration; further formation of products decompose in order to restore the reactants. Reactions of this type are represented by a double arrow, and, in the case of peptides, we simplify them in the synthesis of a dipeptide, where Ala (alanine amino acid) Gly and (Glycine amino acid):
Ala+Gly Ala - Gly + H2O
In aqueous medium, and then in a prebiotic soup, these synthesis reactions cannot occur because of the abundance of water already present, pushes the reaction to the left. In a simplistic way, as the water cannot come out spontaneously from the bottle, if also immersed in a container filled with water, so, in an aqueous environment, the water cannot get out of the reactions mentioned above, spontaneously. And if the water is not produced, the synthesis of the macromolecules essential for life does not take place.
Now, the question is, all living organisms contain water and within them these reactions take place. How is it possible?
Let us return to the metaphor of the water bottle and reactions.
While keeping the bottle upside down, if we want to get out of the water, we would have to take it out of the container, that is, we have to do work, we have to provide energy. In living organisms, there are molecules which provide this energy; Part of this energy is supplied by catalysts, enzymes, lowering the necessary energy for the synthesis reactions, the rest of molecules that serve as "fuel" which release water during the reaction.
In many cases, the enzymes create conditions that do not require “fuel” molecules. As reported by Pier Luigi Luisi in "Sull’origine della vita e della biodiversità" in 2013, «The large size of the enzymes are necessary because in the proximity of the active site a non-aqueous microenvironment forms. These conditions allow the enzyme an extraordinary reactivity which is different from reactions than in an aqueous environment».
Finally, in living organisms, macromolecules formed soon find themselves immersed in aqueous solutions where they are unstable. To avoid the disruption they take on particular structures. We must assume a mechanism that, in the prebiotic era, stabilizes macromolecules.
In conclusion, in all the living organisms the synthesis of macromolecules reactions require a catalysts, energy, non-aqueous microenvironments and stabilization mechanisms. Vital processes must have needed a necessary continuity with the processes that led to the origin of life. So, the same tools you need today for living organisms, surely must have been necessary in the prebiotic era.
So, in order to understand how the formation of macromolecules took place in the prebiotic era, we have to go looking for:
1) Catalysts, 2) non-aqueous microenvironments, 3) some source of energy, 4) a mechanism that stabilizes the macromolecules.
It is challenging the fact that you come to the same conclusion starting from another perspective, that is, through the study of equilibrium reactions.
The substances in the liquid state, gaseous and in solution move and this movement depends on the temperature. The branch of chemistry that studies the movement of particles and deals with the mechanism of the reactions is called chemical kinetics.
The chemical kinetics states that: necessary and sufficient condition for a reaction to occur is that there is a collision and that the shock is effective. A certain energy is therefore necessary to break the bonds that already exist in the reactants. This statement can be represented graphically by placing on the horizontal axis the energy and the vertical axis the reaction coordinate that indicates the course of the reaction.

For the reaction of formation of the proteins that we schematized in the equilibrium reaction of the dipeptide, Gly + Ala Ala - Gly + H2O, X represents the reactants (amino acids) and Y the compounds (peptides).
This chart takes us step by step to the understand of how the formation reaction of the peptides in the prebiotic era, could occur in particular:
1) going in search of minerals in the prebiotic era, which functioned as a catalysts, black arrow on the left, that lowered the energy needed for the reaction to take place.
2) Looking for conditions that have further reduced the energy required for the reaction, that is, the red arrow to the left, in the final non-aqueous microenvironments.
3) finding a source of energy to overcome the remaining energy barrier and getting the products.
4) finding a mechanism to stabilize the reaction products, lowering their energy to Y*. This is because the products are unstable. As indicated by the small red arrow to the right, the energy required to go from the products of the reaction to the reactants is low and may also take place at room temperature.
We have come to exactly the same conclusions we got analyzing the reactions of formation of macromolecules, which occur in living organisms.
So let’s try to give an answer to these 4 points. In order not to lose the continuity completeness, I didn’t divide this article in two separate ones, that’s why it’s è particularly long. However, in order to our thoughts, at the end of each of these points the conclusions are highlighted.
1) The search for the catalysts.
1) The search for inorganic catalysts that could have assisted in the prebiotic era, the formation of proteins has gained a considerable number of scientists’ attention. Since silicon is the most common element of the Earth's crust and silicates cover more than 90% of the crust, the research is directed towards these minerals and, in particular, to the components of their disintegration: the clays.
Many researchers have shown that it is possible to obtain polypeptides using the amino acids in the presence of clay.
As reported by Graham Cairns-Smith in "Clays and origin of life: Frontiers of Life" in 1998:
M. Paecht-Horowitz and other colleagues (1970) were able to polymerize activated amino acids in the presence of montmorillonite. N. Lahav, D. White and S. Chang (1978) were able to assemble molecules of glycine in the presence of montmorillonite, through a cycle in which alternated absence and presence of water. Even Bujdak et al. 1995 obtained similar results. Cairns-Smith adds, presumably, polymerization is favoured by the absence of water. In fact, in the experiment of Lahav and Chang the temperature bounced between 25 to 95 degrees.
We remind that the clays contain colloidal silica and amorphous silica (silica gel) and as reported by Antonio Cadeddu in "Genesi di una teoria scientifica" in 1998: “In the midst of other chemical-physical factors acting on the enzymatic processes, we should give special attention to the structure of colloidal formations. [...] According Smuk, also inorganic gels with their very elementary structure can influence the direction of the catalytic reactions. Thus, for example, the addition of silica to an aqueous solution of acetic acid and methyl alcohol gel facilitates the catalytic synthesis of the ester”.  Note that in this reaction one of the products is just water, such as in protein synthesis. On the other hand, the use of the silica gel in the industry is well known both, as catalyst and as a dehydrating agent.
And so, if it is possible that the clays have catalysed the protein synthesis reaction, in the prebiotic era, why isn’t the matter not considered closed? The problem is that the clays:
A) they are not selective in the sense that they do not recognize amino Right acids from Levo and therefore they catalyse the two forms.
B) Do not recognize the 20 biological amino acids among the approximately 60 present in the prebiotic era and catalyse any type of amino acid, giving rise to any polymer. But the chemical evolution requires particular polymers to perform specific functions within the proto organisms. Ultimately, the presence of clay and / or amorphous silica catalyses generically the bond between amino acids, i.e. the peptide bond:
R'COOH + R "NH2 R'C=O-NH-R''+ H2O
The only selective mineral in relation to amino acids, as we have seen elsewhere, is the Sol silica, the colloidal silica. The explanation for this diversity has been deeply illustrated in previous articles:
solutions in contact with surfaces give rise to double electrical layers. In the presence of clays, the electric field inside the double layer has parallel and equidistant lines of force, which means, the field is uniform. Since the amino acid molecules have helical dipoles, they cannot enter the interior of the electrical double layer of the clay. Instead, the electrical double layer that is generated in contact with the colloidal silica has an internal electric field with helical lines of force where the amino acids may accumulate.
The catalytic action of the clay acts from a distance on all the amino acids, while that of the colloidal silica takes place inside of the electrical double layer and is selective.
1) Conclusion: the clays are good catalysts, but the colloidal silica is the only mineral that can function as selective catalyst.
2) The search for non-aqueous microenvironments.
2) Let’s go back to the reaction Ala +Gly Ala - Gly + H2O and imagine that you have 100 molecules of Ala and 100 Gly molecules that after an hour have reached equilibrium resulting in 10 molecules of Ala-Gly and 10 water molecules (quantity and time are chosen randomly). These quantities, in time, do not vary anymore, they remain constant. If the reaction is carried out in the presence of a catalyst, instead of waiting for an hour equilibrium is reached in a few minutes. So, they will always form 10 molecules of Ala-Gly and 10 of water that remain constant over time. Therefore, the catalyst accelerates the reaction but does not move the chemical equilibrium.
How do you shift the equation to the right?
There exists a chemical principle that in our case one could synthesize like this: remove the water and the equilibrium moves to the right, towards the formation of the dipeptide. Which is like saying: empty the container and the water will come out spontaneously from the bottle.
The problem of how to eliminate or reduce the water and therefore its action on the synthesis reactions has always been associated to heat. In 1963, Sidney Fox warmed an amino acid solution to a temperature of 130 °C to obtain a mixture of polymers that has called "proteinoid". The heat has in this case two functions: remove the water favouring the reaction, increase the thermal agitation of the amino acid molecules in order to have an effective impact. As we have seen above, even in the presence of clays as catalysts, the heat that is supplied retains the same purpose.
Can the heat really be used to remove water and provide energy for the synthesis reactions?
In 1978, Richard E. Dickerson in "L’evoluzione chimica e l’origine della vita" Le Scienze, made a proposal: The evaporation caused by the sun, in a freshwater pond; but then he adds: "One objection to this proposal is that several of the important precursors of biological molecules, such as hydrogen cyanide, cyanogen, formaldehyde, acetaldehyde and ammonia are themselves volatile". Note that these substances are precursors of the ribose and the nitrogenous bases that constitute nucleic acids. Therefore, if you remove the water through heating you also eliminate the precursors of the nucleic acids and thus blocks the formation of the basic substances required for the origin of life, which is like cutting the branch on which you are sitting on. In conclusion, we can’t use heat to remove the water but we must create dry compartments in aqueous solutions.
These compartments exist and are found in aqueous solutions in contact with the various clay components; are double electrical layers similar to micro capacitors, which we have extensively discussed. As we remember Giuseppe Bianchi in "Electrochemistry" 1963 "[...], have a tendency to be expelled from the electrical double layer species with lower dielectric constant to be replaced from species to higher dielectric constant." Amino acids have a high dielectric constant and helical dipoles. They can then enter the electrical double layer of the colloidal silica expelling the water molecules. In the absence of water, the equilibrium is shifted to the right, towards the formation of peptides.
2) Conclusion: inside of aqueous solutions in contact with the colloidal silica contained in clays, are formed microenvironments non-aqueous, as occurs in living organisms near the enzymes. The balance Ala + Gly Ala-Gly + H2O, the water removed, is moved to the right, towards the formation of peptides.
3) The search for a source of energy.
3) Not being able to use the heat as an energy source for the synthesis of polypeptides, many scientists went looking for energy-rich molecules such as HCN or inorganic pyrophosphate that, in the prebiotic soup, functioned as "fuel".
As reported by R. F. Doolittle and P. Bork in “La modularità delle proteine" Le Scienze Quaderni 1996, around 1970 M. G. Rossmann proposed that proteins were made up of modules (domains), which appeared early in the history of life and assembled in different combinations. This hypothesis was confirmed. Many proteins are made up of modules (or domains) with a number of amino acid residues between 45 and 70. As we have seen elsewhere, the Ferredoxin all derive from a primordial protein of 27 amino acids, considered one of the first proteins to have appeared during the process that gave rise to life. Today it is sure that when a polypeptide chain contains 30-40 amino acid residues (some authors will push to 20 amino acids), begin to have cohesive forces sufficient to assume a predominant form. So, we will use a polypeptide of 30 amino acids as the reference and see how to synthesize them in prebiotic soup.
The HCN (hydrogen cyanide) is an energy-rich molecule and was present in the prebiotic era. The reaction of the formation of a polypeptide with 30 amino acids should follow this path. A molecule of amino acid would have to initially react with HCN and subsequently with a second amino acid molecule obtaining a dipeptide. The dipeptide obtained would have to again react with HCN, and then with another amino acid molecule obtaining a tripeptide, and so on for 30 molecules. Reactions of this type are thermodynamically possible have a 100% efficiency. For the formation of a single peptide bond, however, it takes at least five intermediate reactions. The synthesis, in this way, of a 30 amino acid polypeptide, required that in the prebiotic soup eventually there had to be about 150 consecutive chemical reactions for a single molecule of polypeptide.
The primordial soup brings prebiotic chemistry to evanescence.
To clarify the question of the source of energy it is perhaps appropriate to review some thermodynamic criteria and abandon the idea of ​​prebiotic soup.
Imagine we have a beaker containing 100 grams of water and add a bit of alcohol. The thermodynamics provides us with the analytical tools to demonstrate that the ethyl alcohol is miscible with water.
Now imagine a glass surface where we deposit a drop of water. Close to the water, we deposit a drop of ethyl alcohol. The drop of expanding alcohol is approaches the drop of water. Since the two miscible substances we expect that as soon as the alcohol reaches the drop of water, they mix.

However, as the drop of expanding alcohol approaches the water, this moves away continuously; water and alcohol do not mix; It is exactly the opposite of what we expected according to thermodynamics.
Take another example. In a beaker containing 100 grams of water we add a bit of H2SO4 (sulfuric acid). The thermodynamics states: the H2SO4 can be mixed with water and in addition heat is produced.
Now we place four water drops on the sides of an imaginary square on a glass surface.

At the centre of the four drops, we place micro drops of sulfuric acid. Since the two substances can be mixed, we expect that as soon as the sulfuric acid reaches the drops of water get mixed. Well, also in this case the sulfuric acid does not mix with water but tries to find an escape route too. Do not try to replace ethyl alcohol with concentrated nitric acid (HNO3), you might be reminded of the existence of a "purpose".
Why do these experiments run on the contrary to the thermodynamics?
The reason is the following: when a reaction is made to take place in a beaker, both walls of the beaker and that of the air above (i.e., the set of the whole) have an influence on the process. Moreover, we took a quantity of water such that the effect of the outline of the process is practically negligible. These are the processes that the thermodynamics studies, processes that regard large numbers, large-scale processes, whereby the boundary is negligible. The drops are small masses where the boundary is predominant, in which the glass-alcohol interaction (or sulfuric acid), and air-alcohol (or sulfuric acid), and the glass-water and air-water interaction predominate. This type of thermodynamics, we can define at a small scale, has never been studied and we do not know how to study it. As these experiments show, thermodynamics at small scales can move spontaneously in the opposite direction compared to the same thermodynamics at large scales.
3a) Conclusion, a dynamic at small scales, cannot be explained by classical thermodynamics.
There are researches, which states that at small scales (nanoscales) the second law of thermodynamics, for a short time, can be neglected, or that the laws we have studied in school no longer apply at a nanoscale. I do not have the competence to enter into the merits of this research. Here we want to state that the second law of thermodynamics is always valid. It, however, at small scales, does not provide appropriate analytical tools. As we lack the appropriate analytical tools, what we can do is to follow a logical thought that is thermodynamically legitimate.
How can thermodynamics at small scales help us understand the origin of the polypeptide? And then, is there really a need for a source of energy for the synthesis of polypeptides?
Imagine, in the prebiotic era, a heterogeneous solution of clay where the colloidal silica is present and where the concentration of amino acids is 0.1g / L. In an aqueous solution pH around 7, the amino acids are in the form of dipolar ion +NH3-CHR-COO-. If two molecules of amino acids are located next to each other they are oriented with the positive charge towards the negative and give rise to ammonium salts R'-COO- NH3+ -R. This type of orientation, energetically more stable, is one necessary for the formation of peptide bonds. As we have suggested elsewhere, the colloidal silica retains on its surface the Levo amino acids, leaving out the Destro amino acids in the waters. The levo amino acids accumulate inside the electric double layer of colloidal silica and expel H2O. They then, as already suggested, are located one next to the other, adsorbed as a function of their specific electrokinetic potential and definitely oriented towards the formation of peptide bonds.
The double electrical layers are in equilibrium with the solution, but at the same time represent separate compartments within the solution.
As the number of different amino acids molecules adsorbed on the surface is difficult to imagine, tens, maybe hundreds. We take as plausible the 30 molecules.
We are therefore to in presence of amino acids in solution and groups of 30 amino acids inside the double electrical layer of the colloidal silica. We find within these micro-capacitors the molecules of amino acids:
a) Next to each other in a chain of ammonium salts.
b) The bonds of the functional groups are weaker because they are subject to interactions with polar covalent bonds of the colloidal silica. The silica acts as a catalyst, by lowering the activation energy.
c) Are subjected to the electrostatic pressure of the double layer.
d) The water, within these compartments, is reduced if not absent, and the equilibrium is shifted towards the formation of the products.
Ultimately, the energy required for the synthesis, is minimized.
Within these compartments classical thermodynamics, is of no help, and we must turn to a thermodynamics at small scales. As we have seen with the experiments described above, thermodynamics at small scales proceeds in the opposite direction to the thermodynamics at large scales. It is possible, that within the double electrical layers, a thermodynamics at small scales comes in, whereby the formation of the polypeptide becomes a spontaneous process. With the formation of a polypeptide of 30 amino acids 29 water molecules are formed. These molecules, freed in a poor water environment if not dry, find space where to move by increasing the entropy of the system and promoting the synthesis of the polypeptide.
The addition of energy is not necessary, as the thermal agitation is already sufficient. The spontaneity of the process is provided by the increase of entropy.
3b) Conclusion: within the double electrical layers, it is possible that thermodynamics at small scales has worked, whereby the formation of the polypeptide becomes a spontaneous process. No energy input is required, the thermal agitation is enough and the spontaneity of the process is provided by the increase of entropy. The synthesis of polypeptides is therefore a spontaneous process with an increase of entropy: Chaos from the order
4) The search for a polypeptide stabilization mechanism.
4) The colloidal silica particles have a very short life. If a colloidal silica particle,
which has synthesized a polypeptide on its surface, meets other of colloidal silica particles, it will form amorphous silica. The electrical interactions between colloidal silica particles are so strong they deform each other; so much, so that the amorphous silica does not change the plane of polarized light. The polypeptide, not finding the electrical interactions, detaches from the surface and goes into solution. Since the colloidal silica
was an helical coil structure the same will be for the polypeptides. As we have seen elsewhere, this structure is unstable in solution but contains within it both positive and negative charges. It before it’s decomposed by water in single amino acids, spontaneously it establishes links between the positive and negative charges that stabilize the helical structure: the α-helix is more ordered and more stable with Y * potential energy.
The total chaos is greater because of the energy that is released at the time of the formation of strong hydrogen bonds. The energy is released as heat, which increases the agitation of the water molecules, and so the overall disorder, which is, the entropy.

The energy needed to decompose the α-helix, as shows us the big red arrow on the right, it is now greater and is not available at room temperature.
4) Conclusion: The formation of the more ordered and stable structure of the α-helix stabilizes even more the polypeptide because it increases the universal entropy: Chaos from order.
Finally, following the chart, we represent the four points and their answers.
1) Go in search of minerals in the prebiotic era, which may have functioned as catalysts and lowered (down arrows on the left) the energy needed for the reaction to take place.
1) The clays catalyse the synthesis of amino acids but are not selective. The colloidal silica is the only mineral that can function as selective catalyst.
2) Look for conditions that have further reduced the activation energy, that is, the red arrow to the left, and ultimately dehydrated environments or where the action of water on the reaction is much reduced.
2) Inside of aqueous solutions in contact with the colloidal silica contained in clays, they are formed microenvironments non-aqueous, as occurs in living organisms in the vicinity of the enzymes. The balance Ala + Gly Ala-Gly + H2O, the water removed, is moved to the right, towards the formation of peptides.
3) Find a source of energy to overcome the remaining energy barrier and get the products.
3a) A dynamic at small scales, cannot be explained by classical thermodynamics.
3b) Within the double electrical layers, it is possible for thermodynamics to work at small scales where the formation of the polypeptide becomes a spontaneous process. You do not need any application of energy, thermal agitation is already enough and the spontaneity of the process that is provided by the increase of entropy. The polypeptide synthesis is therefore a spontaneous process due to increasing entropy: Chaos from order.
4) Find a mechanism to stabilize the reaction products, lowering their energy to Y*. This is because as indicated by the small red arrow to the right, the energy required to go from the products of reaction to the reactants is low and could take place at room temperature.
4) The formation of the more ordered and stable structure dell'α-helix more stabilizes the polypeptide because the universal entropy increases and then, again: Chaos from order.
The "Arrow of time" carries with it the synthesis of polypeptides.

                                                                                                Giovanni Occhipinti

Translated by: Sydney Isae Lukee