Proteins from non-natural amino acids: the beginning of the path

A four-letter word Anton Chugunov, "Biomolecule"

One of the most amazing discoveries in biology of the XX century was the decoding of the genetic code, and it was especially difficult to understand that such a code exists. Perhaps the most striking feature of this "language" is its universality – with the exception of some "dialects", it is the same for all domains of life on Earth. At the beginning of the XXI century, scientists managed to "rewire" the genetic code by adding to the standard amino acids an unnatural link encoded by a stop codon in the matrix RNA and read with the participation of "orthogonal" tRNAs. (However, at the same time there can be only one non-standard link in the protein.) Now things are turning towards fully "customizable" proteins: English researchers have managed to create a ribosome that reads not three, but four nucleotides at a time, which potentially allows using more than 250 non-natural amino acids for the design of biopolymers.

Deciphering the genetic code has become one of the most impressive manifestations of the work of scientific thought in the XX century. It took more than a century to realize the very fact of the existence of the genetic code – that is, the unambiguous correspondence of DNA sequences and the proteins encoded in it – while it did not take decades to decipher it. Intermediate milestones on this path were the principle of matrix duplication, belonging to N. K. Koltsov, the ideas of E. Schrodinger about encoding information in the chemical structure of genes, the model of the double helix of DNA by J. Watson and F. Crick and the unexpected contribution of physicist G. A. Gamov to the formulation of the triplet genetic code problem. F. crowned this chain of work . A cry with employees who experimentally discovered the textbook qualities of the genetic code – triplet, fusion, orientation and degeneracy [1].

Amazingly, the genetic code is almost the same for all organisms known on Earth, and this continues to be confirmed with the appearance of more and more new genomic information. Of course, this code is not something static: it continues to evolve, which makes it possible to explain the small differences observed in different organisms (archaebacteria, mycoplasmas, some protozoa) or even for individual subcellular organelles (mitochondria) [2]. However, it is not enough for a person to study how life works: trying on the role of the creator, he seeks to reproduce or even create life "from scratch" [3], in connection with which scientists have already earned a considerable dose of righteous indignation from less inquisitive groups of citizens.

The newfangled paradigm, called synthetic biology, approaches the study of the structure of life without taking it apart, but, on the contrary, trying to reproduce it on the basis of elementary blocks, actually erasing the boundary between living and inanimate matter. Proponents of this approach are extremely interested in obtaining protein molecules with new properties (including those that include non-natural amino acids), not to mention more "mundane" pharmacologists and biotechnologists, for whom such proteins can literally and figuratively become a gold mine. (All this does not mean at all, however, that the potential of natural proteins and proteins built from natural amino acids has been exhausted.)

The number of different non–natural amino acids that can be "embedded" into a protein by genetic (rather than synthetic) means is naturally limited by the number of available codons - three-letter "words" that make up the language of the genetic code. Although only twenty (22, if we count pyrrolysine and selencysteine) natural amino acids are encoded by as many as 64 codons, the high degeneracy of the code leads to the fact that all triplets are "busy", and even the use of synonymous codons for inserting non-standard amino acids is undesirable, since this can disrupt the operation of the entire system as a whole.

Due to the "busy" codons, the UAG (amber) stop codon is usually used to insert non-standard links, recognized by a special "orthogonal" tRNA introduced into the system (see Expanded genetic code). The word "orthogonal" will be used here to refer to externally added tRNAs and the corresponding aminoacyl-tRNA synthetases that do not interact with other cellular components other than the ribosome. The most widespread in this area is a pair of tyrosine tRNA and tyrosyl-tRNA synthetase from the archaebacterium Methanococcus jannaschii, which allows you to "reprogram" the amber stop codon in other bacteria (for example, E. Coli) to tyrosine or – for which everything was started – an unnatural amino acid.

British researchers from Cambridge are following the path of "ribosome liberation": their task was to expand the repertoire of non-natural amino acids that can be simultaneously introduced into the synthesized protein. The latest achievement of Jason Chin's laboratory is the creation of modified ribosomes capable of reading the genetic code not in triplets, but in quadruplets (4 bases each), which potentially allows encoding up to 256 (!) amino acids [4]. By molecular selection, scientists "derived" a mutant variant of the ribosome (it was called Q1 – see Figure 1), which effectively recognizes quadruplets (for the purposes of selection, an antibiotic resistance gene was encoded in the "quadruplet" reading frame). Of course, if all ribosomes in a cell were like this, it would become lethal, but they exist independently of "normal" ribosomes and do not prevent them from synthesizing cellular proteins (that is, they are also orthogonal).

Figure 1. Molecular "selection" of ribosomes recognizing quadruplet codons [4]. The selection is based on 16S rRNA libraries in the "orthogonal" ribosome ribo-X, previously created in Chin's laboratory [5]; substitutions in rRNA affected a total of 127 nucleotides. Two substitutions located near the tRNA binding site led to the fact that one of the variants (it was called Q1) can effectively decode the quadruplet codons, while retaining the ability to read "standard" triplets. (By the way, the effectiveness of recognizing a quadruplet and the proportion of erroneous readings here is no worse than that of conventional ribosomes.) The selection was based on resistance to the antibiotic chloramphenicol, which occurred only if the ribosome was able to read the resistance gene located "behind the lock" of the AAGA quadruplet codon in a genetic construct, specifically for the purposes of breeding added Wednesday. (They also added a "designer" version of serine tRNA, an anticodon in which the same four nucleotides are long – UCUU.)

The ability of the "quadruplet" ribosome Q1 to read not only natural triplets, but also "four-letter words" (quadruplets) allows it to be used to insert many non-natural amino acids into the protein at the same time, which was impossible before.

To begin with, a variant of the calmodulin protein was obtained, in which two non-natural amino acids containing azide and alkyne groups were introduced. During spatial convergence, these amino acids are connected by the mechanism of synchronous addition of Huesgen and create an additional "stiffening edge" in the protein (Figure 2). The researchers note that this confirms the practical use of quadruplet ribosomes for bioengineering and synthetic biology tasks.

(Synchronous addition reactions are addition reactions in which an attack on both atoms of a multiple bond is carried out simultaneously. Another name for reactions of this type is cycloaddition reactions, since the end product of such reactions are cyclic substrates.
There are two main reactions of this type: joining to conjugated systems – the Diels-Alder reaction, Eng. Diels-Alder reaction, and 1,3-dipolar cycloaddition – Huisgen reaction, Eng. Huisgen reaction.)

Figure 2.
Genetically encoded cyclization of calmodulin by Cu(I)-dependent Huysgen reaction.
Instead of the first and last residue in calmodulin using a quadruplet ribosome
and corresponding orthogonal tRNA and aminoacyl-tRNA synthetases
non-natural amino acids were inserted:
para-azido-phenylalanine and N6-[(2-propinyloxy)carbonyl]-lysine, respectively.
(The AGGA quadruplet codon and the "standard" UAG stop codon were used.)
The deciphered spatial structure shows,
that these two residues are really involved in the "click reaction",
forming a crosslinking, which is difficult to obtain in other ways [4].

However, the creation of a quadruplet ribosome is only part of the task, which can be seen at least from the fact that only one quadruplet codon was used for cyclization of calmodulin, and the second "old–fashioned" was the standard UAG stop codon for these purposes. Apparently, the fact is that the researchers did not find two mutually orthogonal quadruplet pairs of tRNA/tRNA synthetase. "Having received a ribosome capable of reading a lot of codons-"dummy", you wonder where to get tRNA and the corresponding synthetases that would allow you to use this potential?" – asks Chin [7]. However, he has already answered himself in the scientific press – his laboratory published the first work on the creation of pairs of orthogonal tRNA/aminoacyl-tRNA synthetases, introducing mutations into them according to a certain program [6]. This possibility opens the way to a total "tuning" of the protein, into which in the future it will be possible to insert almost any links, not limited to only twenty natural ones, and many scientists have already called these achievements a new era in bioengineering.

It's incredible that it turned out to be possible to take such an unimaginably complex mechanism as protein translation and "flash" it for your own purposes, but this seems to be just the beginning.

Literature

1. V. A. Ratner. (2000). The genetic code as a system. Soros Educational Journal 6, 17-22;
2. biomolecule: Evolution of the genetic code;
3. biomolecule: The meanings of "life";
4. Neumann H., Wang K., Davis L., Garcia-Alai M., Chin J.W. (2010). Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. Nature 464, 441–444;
5. Wang K., Neumann H., Peak-Chew S.Y., Chin J.W. (2007). Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion. Nat. Biotechnol. 25, 770–777;
6. Neumann H., Slusarczyk A.L., Chin J.W. (2010). De novo generation of mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs generation of mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs. J. Am. Chem. Soc. 132, 2142–2144;
7. C&EN – Expanding The Genetic Code.

Portal "Eternal youth" http://vechnayamolodost.ru 23.03.2010