Bioluminescence in biotechnology
It is alive and glows green
"Nobel" for the living light
Bioluminescence is the phenomenon of light emission by a living organism.
Corals, fireflies, jellyfish, worms, mushrooms glow. These are very dissimilar organisms representing different kingdoms of nature. The taxonomic (classification) diversity of organisms that are able to glow is very high. A good example for its characterization is given by Osamu Shimomura, one of the main experts in the field of bioluminescence, winner of the 2008 Nobel Prize in Chemistry.
If you take a poster with the evolutionary tree of all living organisms – animals, plants, fungi, and bacteria – printed on it and splash paint on it, it will approximately show the distribution of luminous creatures along the tree.
Accordingly, the mechanisms of luminescence are also dissimilar. According to modern estimates, the phenomenon of bioluminescence has up to 30 different biochemical mechanisms. Of these 30 mechanisms, Shimomura managed to decipher four, and only six were deciphered. Thus, there is no connection between luminous organisms either systematically or biochemically, and the biological mechanism of this phenomenon is known only by 15-20%.
Luminescence itself – luminescence – is a form of energy release during a chemical reaction – oxidation. But since this reaction is biochemical, in addition to an oxidizer (oxygen) and a reducing agent (substrate), an enzyme also participates in it, with which the body can control the glow.
The most famous and detailed case of bioluminescence is the green glow of the jellyfish Aequorea victoria, the study of which brought Shimomura the Nobel Prize.
Here the glow is even more complicated: in addition to the equorin enzyme, an additional protein is involved in its occurrence – GFP (green fluorescent protein), which is responsible for the green glow itself, forming a complex with equorin. If you take equorin and oxygen separately, then only a flash of blue color will occur. However, in the body of the jellyfish, equorin forms a complex with GFP, and non-radiative energy transfer occurs between them.
A quantum of blue light is transmitted to the GFP, which absorbs it and emits green light already. This mechanism has been studied in detail, however, what role in the nature of this phenomenon is still completely unclear. It is not clear why the jellyfish needs to emit light at all, nor why the light is green and not blue. Meanwhile, it is difficult to write off this as an accident: it is quite expensive for the jellyfish itself, because it is very energy-consuming to produce additional proteins in large quantities.
It should be noted that Shimomura's work on GFP was done back in the 60s. He isolated GFP and characterized it, also trying by the methods that were available at that time to determine the structure of a special group of atoms that determines the color of this protein – chromophore (Greek. "carrier color"). As is clear from the very word "proteins", usually these substances are white, colorless. A small part of them – GFP and related ones – are colored.
It is now known that coloring occurs due to the fact that a chemical reaction occurs inside the protein molecule, which is catalyzed by itself. That is, usually a protein catalyzes the interaction of other substances, and in this case, a reaction within itself. The protein has the shape of a barrel, in the very middle of which there are three amino acids that react with each other to form a chromophore. Moreover, in this case, the chromophore is able not only to absorb light of a certain wavelength (that is, to acquire color), but also to emit it (that is, to glow).
Initially, Shimomura's work did not become a sensation, and for a while GFP was nothing more than an interesting incident. However, then the methods of genetic engineering and molecular biology were greatly developed. Using these methods, it was possible to sequence GFP, that is, to obtain its amino acid sequence, then translate it into the DNA sequence that encodes it, then find this sequence by PCR (polymerase chain reaction) in a jellyfish.
It turned out that the fluorescent glow of the jellyfish is completely unique: it is encoded by only one gene. This simplicity made it possible to clone this gene and artificially endow cells and living organisms with this feature by embedding the gene in their DNA. That is, the jellyfish gene can be embedded in a bacterium that reads this gene, produces the necessary proteins, including GFP, and begins to fluoresce.
The same was done with nematode worms, clearly confirming that the phenotypic, external sign of fluorescence can be encoded by just one gene. This has already caused a real explosion of interest in GFP as a promising biochemical label.
Fluorescence can be observed either with the naked eye or with the help of fluorescent microscopes, and when such an easily observable trait is encoded by just one gene, it can be transmitted to almost any organism. Cloning one gene is easier than cloning ten genes. So about 25 years after the discovery of GFP, it turned out that this is a very useful thing.
Now there are hundreds of different methods of its application. They all boil down to using fluorescence to observe various phenomena that occur in cells or whole organisms: gene expression, tissue development, stem cells, the behavior of cancer tumors, the interaction of proteins with each other, the fate of cellular organelles.
For example, a cancer or stem cell placed in the test organism will no longer be able to "mix in the crowd" – it will be visible wherever it turns out to be. Moreover, her "offspring", when she begins to reproduce, will also be the carrier of the "luminosity gene", and doctors can trace the entire emerging "population". By the way, fluorescent aquarium fish are also the result of GFP cloning, they do not exist in nature.
A fluorescent label is like a label that can be "attached" to anything in a cell or an organism. After that, it becomes convenient to observe what happens to the marked object. Proteins with a far-red spectrum are especially in demand, because in this area living tissues are transparent even in mammals.
In this way, you can see through a living organism and see the tumor inside it. That is, a tumor whose cells are genetically modified so that they fluoresce in this region of the spectrum is placed in a test animal, and then the development of the tumor and its reaction, for example, to chemotherapy, are visually observed in a living organism.
Another option is in the embryo at the stage when the cells are not yet specialized, they mark a certain part of them and look at which cells have turned out of them, which tissues have developed. This is not so easy to do in the usual ways: standard biochemical staining methods require multiple manipulations, as a result of which the result is often not clearly observed.
Once again, we emphasize that the peculiarity of GFP is that it is not injected from the outside, by injection, like some other fluorescent labels, but is initially encoded in the genome of a cell or organism. In particular, this allows us to study the functioning of the genome itself, and this is a very difficult task even now. Although it is already quite easy to "read" the genome, it is still not so easy to understand the "meaning of what you read": how the genome works, what is the mechanism of realization of genetic information in the form of signs in the body itself. Now a new field is engaged in this – functional genomics, which actively uses the GFP cloning capabilities.
Yellow, Red, BlueIn 1999, Sergey Lukyanov (now an academician of the Russian Academy of Sciences) began searching in nature for proteins similar to GFP.
The search was successful: at first, proteins similar to GFP were found in colored corals that are well known to diving enthusiasts in the Red Sea. And only recently, about 10 years ago, it became known that all this diverse beauty is due to the presence of such GFP proteins. However, if GFP emits only green light, then the proteins found in the corals turned out to be of very different colors.
At the same time, all of them, as well as GFP, are encoded by only one gene. This has brought additional opportunities for the use of fluorescent labels in the genome: since the "labels" have become multicolored, their arsenal has expanded. That is, today they can be used to observe three or four objects at the same time. Plus, you can observe the interaction of proteins with each other in space due to the transfer of energy, that is, by changing the color, you can see the convergence of proteins. The search for new fluorescent proteins and ways to use them continues in our laboratory.
In the course of this search, in particular, proteins were found whose luminosity can be switched using light of different wavelengths (that is, different colors). Under the influence of one light, they fluoresce, the other – they stop. This is a new feature – to turn on and off the label.
Fluorescent labels – chemical and genetic – have replaced isotopic labels that were previously popular in biochemistry and molecular biology. Fluorescent paints are safer, cheaper, and better stored. It is already a huge market, and in molecular biology there is always a demand for new dyes with new properties.
Instead of a heart , a fiery chromophoreAnother important task was to find out why proteins similar to GFP glow in different colors.
It turned out that they have different chemical structures of chromophores. The field of chemistry related to the study of chromophores of luminous proteins boils down to two questions – what is their structure and how they are obtained in nature. It is not always possible to understand exactly which chromophore is in a protein, even with the help of X-ray diffraction: against the background of a huge molecule of tens of thousands of atoms, it is not easy to accurately determine even the composition, not to mention the spatial structure of a small substituent.
And often it is such subtle effects that determine the whole phenomenon of fluorescence. To resolve this issue, chromophores can be synthesized artificially. Comparing the data of various methods, it is established which group of atoms is responsible for the fluorescence and color of a particular protein. The synthesis of chromophores often provides answers to questions about their work that cannot be obtained by studying a whole protein. A small piece of it, a model compound, can say a lot about the fluorescence process.
Our latest work related to the synthesis of chromophores has allowed us to solve the riddle of the chromophore of GFP itself – the ancestor of luminous proteins. It consists in the fact that when this small "brick" is in the composition of the protein, it is a very bright fluorescent paint, but the synthetic analog itself, completely repeating the structure of the natural chromophore, while in solution, almost does not fluoresce.
We found out that the point here is precisely in the spatial structure – the mutual arrangement of atoms. In protein, it is fixed, "clamped" by neighboring amino acids, and in solution it is free and bends so that it loses its fluorescent properties. However, if an additional element of rigidity is introduced that restricts the rotation of the molecule, fluorescence occurs at approximately the same high level that is characteristic of the protein.
Thus, based on the natural structure, we have at our disposal a new promising fluorescent dye, which, we hope, will find practical application.
Portal "Eternal youth" http://vechnayamolodost.ru06.04.2012