mercoledì 9 dicembre 2015

THE ORIGINS OF PROTEINS: The choice of the amino acids

Post n. 25 English

As seen and mentioned by Miller and other researchers and even in meteorites about 60 amino acids have been identified, why is it that only 20 have become part of the proteins? And then: were all twenty amino acids really present in the prebiotic era? And if they weren’t all present, how many and which were the amino acids constituent of the first proteins? And how were they chosen?
The 20 amino acids in question are:
Gly (glycine),    Ala (alanine),    Val (Valine),    Leu (leucine),       Ileu (isoleucine),       Pro (Proline),      Asp (aspartic acid),       Glu (Glutamic acid),       Ser (serine),             Thr (threonine ),     Gln (glutamine),       Asn (asparagine),         Met (Methionine),  Cys (cysteine),     Phe (phenylalanine),     Tyr (Tyrosine),           Trp (tryptophan),         Hys (histidine),  Arg (arginine),  Lys (Lysine).
The greatest part of scientists, which are studied the origin of life, are of the opinion that the number of amino acids, when life began, was certainly less than today. Few, however, are researchers who declare the numbers and names. Among them was Mario Ageno, student of Enrico Fermi and the father of biophysics in Italy, who tackled in a comprehensive way the issue. He began from a Miller's work of 1974, which reviews the results of all the experiments conducted up to that time and proves, beyond any doubt, the prebiotic synthesis of 12 amino acids synthesis:
Gly, Ala, Val, Leu, Ileu, Pro, Asp, Glu, Ser, Thr, Gln, Asn
Miller shows how, adding H2S (hydrogen sulphide) to the primitive-atmosphere model (CH4, NH3, H2O, H2),
is easily obtained.
While the
was obtained from Khane and Sagan by exposing under ultraviolet radiation a mixture of: CH4, C2H6, NH3, H2O, H2S.
Also through pyrolysis processes and reactions in solution
Phe, Tyr, Trp
are obtained.
Finally, the synthesis of Pro and pipecolic acid are obtained
Arg, Lys.
The Histidine is the only amino acid that has never been synthesized via prebiotic procedure.
Of course, it is not excluded, in fact it is very likely, that the processes of synthesis of the amino acids in the prebiotic era, in many cases, have followed other steps than those described above.
We focus for the moment on the 12 amino acids for sure prebiotic origin and see later what other processes of abiotic synthesis of amino acids can be considered plausible.
Ageno suggests starting from a formal study of the genetic code, ie the law of correspondence between l`RNA and amino acids.
The RNA is the nucleic acid that carries the information for the synthesis of proteins.
The constituents of the RNA are:
The phosphate group: (H2PO4)-.
A sugar, ribose

Four nitrogenous bases: A (adenine), G (guanine), U (uracil), C (cytosine).

A phosphate group, a ribose molecule, and an any of the four molecule bases give rise to four different aggregates, which are called nucleotides. As an example, we show the Adenosine-5-phosphate.

By tying together on average 300 different nucleotides, you get a macromolecule: RNA

(Figures from: "Lezioni di Biofisica)" by Mario Ageno

In the RNA, the three adjacent nucleotide bases are given the name of triplets. For example, in the figure the three UAC bases make up a triplet (codon). If in the figure, there were GUA, we would be in the presence of another triplet, and so on. Starting from this RNA macromolecule, through a process now quite complex, proteins are assembled. Each triplet corresponds to a specific amino acid, one and only one, and that law of correspondence, represented by a 3:1 is called: genetic code. All living organisms on our planet use the same genetic code, it is therefore universal.
 Having available four nucleotides, the ways in which we can dispose them 3 by 3 are 43 = 64. Three of these triplets are used as end signal (t.), then, in theory, RNA contains the information for 61 amino acids. Since the amino acids in all living organisms are only 20, the genetic code is degenerate meaning that more triplets code for the same amino acid.

For example, the triplets in 1st, 2nd, and 3rd  position, GUU, GUC, GUA and GUG, all encode the same amino acid: the valine (Val).
In reference to the law of correspondence, Mario Ageno wondered if the genetic code had been from the beginning 3:1; is possible that in primitive times it was different for example 2:1? In "Lezioni di Biofisica" 1984 Ageno excludes such a possibility, because, in that case, all metabolic processes made with a code 2: 1 would be lost in the transition to a code 3: 1 and evolution would have begun again, then he adds: «However, it is possible that at the beginning not all three positions were “active”: maybe the first and second and the third had the spacing function».
But he does not elaborate on the consequences of such an eventuality on the number and choice of the amino acids in the prebiotic era, which we will face in the continuation of the article.
In relation to the amino acids present in the prebiotic era, analyzing the genetic code, Mario Ageno writes: «The same formal structure of the genetic code suggests that the two amino acid methionine (Met) and tryptophan (Trp) appeared for sure recently».
He then takes into account the studies on the amino acid sequence of proteins, in particular hemoglobin and cytochrome, contained in different species. He analyzes a particular type of protein considered of ancient origins: the Ferrodoxine. As known, one of the evolutionary processes that leads to new proteins is the doubling of the gene. This gene will express a double-length protein but consists of two identical amino acid sequences that with time, due to the effect of the mutations will make them differ. However, it is often possible to go back to the original protein, and it is what has been done with Ferrodoxine. It can be shown that these proteins derive all from an original protein of 27 amino acids, considered one of the first proteins appeared during the process that gave rise to life.
This ancient protein is made up of nine different amino acids, which are:

Gly, Ala, Val, Glu, Asp, Pro, Cys, Ser, Ileu

Following a different reasoning, Jukes also adds Leu. As you see all, except Cys, are part of the 12 amino acids, of sure prebiotic origin, by Miller's review. Ageno then suggests, that the number of amino acids in the prebiotic period could be about half of the current ones.
This conclusion, however, creates problems.
If in the prebiotic era only 10 amino acids were present, many triplets would be meaningless. For example, four triplets with C and G in the first and second position and two triplets with A and G in the 1st and 2nd position all encode Arg. Since Arg is not among the ten listed above, and not even among the 12 of Miller's review, these triplets would be meaningless triplets. But Ageno categorically rules out the existence of meaningless triplets in the prebiotic era, because isolated mutations would have disrupted protein synthesis with lethal results. To solve the problem, he introduces the "wobbling" phenomenon. He imagine that the central letter -U- indicate a hydrophobic amino acid (which does not bind with H2O) while -A- indicate a hydrophilic amino acid (which binds with H2O), leaving the precise specification of the evolution results.
This explanation, however, creates other problems. Why is it that Arg and Ser despite being hydrophilic not have A in second position but C? And why doesn’t the Pro have no U in second place despite being a hydrophobic? Yet in both first and second case there should be triplets available because, as seen, more triplets encode the same amino acid, for example the Pro could have replaced one of the Leu triplets.
It should also be added that the appearance of a new amino acid also results in the opening of a new metabolic pathway. It's pretty hard to imagine that life in early appearance and evolution, could be subject to the stress of constantly creating such a large number of new metabolic pathways. Ultimately, the presence of the ten amino acids listed above is credible, in the prebiotic era, however, since meaningless triplets it could not exist, there cannot be the final number. We can conclude that the problem of how many amino acids there were in the prebiotic era, which ones and how they were chosen is still unresolved until here.
Let’s consider Miller's work. As seen, both among the 12 amino acids of the experiment, among the components of the ancient protein, Pro is present. But as Ageno suggests the synthesis of Pro involves the synthesis of Arg and Lys. The Arg, moreover, had to be there otherwise we would have had 6 meaningless triplets. It seems quite reasonable to add to the list of 12 also these two amino acids and so we have 14 amino acids present in prebiotic era.
Let us now consider the synthesis of Met and Cys.
As we have seen in the review of Miller, adding H2S (hydrogen sulphide) to the primitive-atmosphere model (CH4, NH3, H2O, H2) you get the Met, while the Cys was obtained from Khane and Sagan under ultraviolet radiation of a mixture of: CH4, C2H6, NH3, H2O, H2S. Now, at first glance the formation of Met seems chemically simpler than Cys.
Yet the Cys is one of nine amino acid components of the Ferrodoxine, ancient proteins appeared during the process that gave rise to life. If Cys formed, definitely also Met had to be formed. We actually have no valid element that we can do to exclude the
 Met. Even these two amino acids should therefore be added to the twelve already listed. However, since no researcher has ever suggested that the earth's atmosphere contained H2S at the global level, we must assume that these two amino acids are formed only in places where H2S was present that is in proximity to volcanic areas.
According to Christian de Duve in "Origine della vita" in 2010, tryptophan (Trp), and Histidine (Hys) appeared later in the development of life. In fact, they have not been found in meteorites and also Histidine has never been synthesized in prebiotic chemistry experiments.
Recalling again Miller’s experiment, Phe (Phenylalanine), Tyr (Tyrosine), Trp (Tryptophan) were obtained through pyrolysis processes and reactions in solution. The pyrolysis processes, ie demolition of compounds by heat, are processes that occur at high temperature. In the laboratory we get good amounts as we have well-controlled processes. These three amino acids have a relatively complex molecular structure and the Trp is the most complex of the three. But de Duve does not exclude the Trp for the
 complexity of its molecule but only because it was not identified in meteorites. This does not
seem a discriminating reason. And so, either we exclude all three of them, or we imagine that, in the prebiotic era, modest amounts of these compounds were formed in particular areas of the earth's crust, for example in the presence of cooling lava.
The analysis done until now it suggest two conclusions.
A) The amino acids present in prebiotic era were 14:
Gly, Ala, Val, Leu, Ileu, Pro, Asp, Glu, Ser, Thr, Gln, Asn, Arg, Lys
the other 6 were absent.
B) The 20 existing amino acids were all present but the first group, spread over the entire surface of the planet, consists of 14 amino acids listed from above:
Gly, Ala, Val, Leu, Ileu, Pro, Asp, Glu, Ser, Thr, Gln, Asn, Arg, Lys
A second localized group, meaning that they formed in particular areas of the planet probably in the proximity of volcanic areas, consists of 6 amino acids.

Met, Cys, Phe, Tyr, Trp, Hys

Now, let us follow a different argument.
As we have already said, Ageno does not elaborate the consequences of the fact that the pair of letters being “active” or read were in the 1st and 2nd position.
And then, we start from the structure of the genetic code and in particular by a necessary condition: there can’t be meaningless triplets. This condition can be satisfied if the 1st and 2nd position are used for encoding at least one amino acid and the third position as spacing. Ageno has already admitted this possibility when he writes: 'However, at the beginning not all three positions were read: maybe the first two and the third had the spacing function". Now, if the third position is reduced to spacing, the number of amino acids that the genetic code can encode using only the 1st and the 2nd position is reduced to 24 = 16. This means that in the prebiotic era we should have 16 amino acids, otherwise we would have been in the presence of meaningless triplets.
By the genetic code, however, Ser and Arg occupy the triplets in the 1st and 2nd position CC and CG respectively and the triplets AG in 1st and 2nd position. In short, two amino acids occupy three positions and so to not have meaningless triplets 15 amino acids are sufficient.
Furthermore, also the Leu is encoded by triplets in 1st and 2nd position, CU and UU. The Leu occupies 2 positions alone and the necessary amino acids goes down to 14.
In conclusion, in order not to have meaningless hat-tricks, 14 amino acids were necessary.
We are still missing 6 amino acids.
Meanwhile, it is remarkable that these numbers coincide exactly with the conclusions on Miller’s experiment reported in A) and B).
Let us follow the structure of the genetic code and try to identify any 6 missing amino acids.
As can be seen from the genetic code, there are pairs of amino acids that are encoded by the same nucleotide pairs in 1st and 2nd position, they are:
UU- (Phe, Leu),          AU-(Ileu, Met),           CA-(His, Gln),            AA- (Asn, Lys),              GA- (Asp, Glu),               UG- (Cys, Trp),                       AG- (Ser, Arg)
Furthermore UA- (Tyr, t), were the end signal.
In order not to have meaningless triplets, in the prebiotic era, at least one of these amino acids had to be present.
Meanwhile Asp and Glu were certainly present because they have both been identified in ancient proteins and by amino acids of certain prebiotic origin as in Miller’s analysis.

Even Ser and Arg had to be present otherwise, as we have montioned before, we would have meaningless triplets.
Finally Leu, Ileu, Gln, Asn, Cys had to be present because they were already identified either among the sure amino acids of prebiotic origins or in the ancient origins of proteins.
The 6 missing amino acids would then be:
Met, Phe, Tyr, Trp, His, Lys.
With Lys in place of Cys these amino acids correspond exactly to the 6 amino group, as we assumed in B), probably they were formed in volcanic areas. It is important here to emphasize that, following two completely different paths, less than one amino acid, we obtained the same results both in quantity and in quality.
So were these 6 amino acids really missing?
For Met, Phe, Tyr, Trp is the same logic that brought us to conclusions A) and B).
Arg (Arginine) and Lys (Lysine) are two molecules with basic side chain, but the molecules of Lys is simpler than the dell'Arg molecule. Therefore, it is not logical to exclude Lys.
Do we really want to exclude His just because it was not detected in the prebiotic experiments?
Then how many and what were the amino acids in the prebiotic era?
Let us start with some of Ageno considerations:
He begins by noting that the introduction of a new amino acid is not the result of a simple mutation but requires the setting of a new metabolic pathway.
Also, if the introduction of new amino acids were easy, why is the genetic code universal? And how the different species did not develop their own particular code?
Ageno admits he does not know the answer to these questions.
Actually an answer to these questions can be given, and the conclusion is already exposed in B):
The current 20 amino acids were all present but are divided into two groups.
A first group, spread over the entire surface of the planet, were formed from amino acids considered for sure by Miller’s abiotic experiment, in addition Arg and Lys with a total of 14:
Gly, Ala, Val, Leu, Ileu, Pro, Asp, Glu, Ser, Thr, Gln, Asn, Arg, Lys
A second localized group, which is formed in particular areas of the planet probably near volcanic areas, consisting of 6 amino acids.
Met, Cys, Phe, Tyr, Trp, Hys
This last conclusion is ultimately obligatory. If the genetic code is universal, it follows that it originated when life did, then the 20 amino acids were all be present.
For the development of life, it would be lethal to create new metabolic pathways due to the new appearances of amino acids and hence change continually the genetic code.
Finally, why just these 20 amino acids and not others?
The matter is dealt by Arthur Weber and Stanley Miller in "Reasons of the Occurrence of the Twenty Coded Protein Amino Acids" in 1981. They report the work of various researchers, for us of no interest, because they start from the assumption that life has it origins in a prebiotic soup. Moreover, the two scientists claim unlikely that a single process can explain the selection of the twenty amino acids.
Here we argues that the prebiotic soup has never existed and that life originated on firm land. We also support that  the experiment on double electrical layers, which is still the only known experiment, with a single process, where a physical agent, quartz, connects deductively the separation between Dexter  amino acids from Levo in the prebiotic era with fundamental theory of physics. We assume, as we have seen in the articles, "Origins of proteins: the molecular asymmetry problem", that the colloidal silica has the same behavior of quartz. Since each amino acid has an electromagnetic field associated with its molecule, of about 60 amino acids present in the prebiotic era only those 20 whose electromagnetic field was compatible with the electrokinetic potential of colloidal silica were chosen.
                                                                                                   Giovanni Occhipinti  

Translated by: Sydney Isae Lukee

Nessun commento:

Posta un commento