Bright and stable bifotochrome
Scientists with the help of mutations have increased the stability of proteins-"traffic lights"
RNF Press Service
Russian scientists have obtained a stable and bright fluorescent protein moxSAASoti, capable of changing the color and intensity of its own glow. To do this, the authors pointwise changed the sequence of the gene encoding it. Previously, all proteins capable of "switching" color were very sensitive to oxidation and stopped glowing, while the new version of the molecule does not lose its properties.
The results of the study, supported by a grant from the Russian Science Foundation (RNF), are published in the journal Scientific Reports (Marynich et al., First biphotochromic fluorescent protein moxSAASoti stabilized for oxidizing environment).
Fluorescent proteins are molecules that, when irradiated with light of certain wavelengths, are able to glow themselves. Scientists isolate them from living organisms, such as jellyfish and corals, or artificially synthesize them in laboratories. To date, there are many fluorescent proteins that differ in color and intensity of radiation, as well as in variants of color change. So, some of them simply switch from a fluorescent state to a non-fluorescent one, that is, they stop glowing, while others are able to change the color of radiation from green to red. This process occurs when a protein is irradiated with light of a certain wavelength. It can be reversible and irreversible. Some fluorescent proteins are able to change the glow color only once, which occurs as a result of breaking chemical bonds. In this case, the structure of the substance is completely disrupted and does not recover on its own. However, there are exceptions, for example, the SAASoti protein, which is able to repeatedly change the intensity of its glow (turn on and off) and switch from green to red. However, SAASoti has a disadvantage — it is very sensitive to oxidants that disrupt its structure. For example, it is oxidized even in air, which is explained by the high photochemical activity of the amino acid residues of cysteine that make up its composition.
Scientists from Institute of Biochemistry named after A.N. Bach FITZ of Biotechnology of the Russian Academy of Sciences (Moscow) and Lomonosov Moscow State University (Moscow) with the help of mutations in the SAASoti gene, variants of a fluorescent protein with a minimum content of cysteine and even not carrying it at all were created. To do this, the authors obtained DNA with the necessary substitutions and then injected it into E. coli cells. soli. Thus, microorganisms obtained a gene encoding the SAASoti protein and synthesized proteins based on it that differ from the original ones in structure: in them, the sequence of amino acids, which can be compared with a very long word, changed by one, two or more letters. However, it turned out that proteins completely without cysteine remained sensitive to oxidants, as well as the original variants. Each of the introduced mutations (replacing one letter with another) led to an interesting change in the properties of the protein. For example, single point mutations caused its discoloration, and as a result of two-point mutagenesis, a number of proteins with varying degrees of color brightness were obtained. At the same time, the researchers could use mathematical modeling to predict the effect of a particular mutation on the properties of the protein.
As a result of experiments, it was revealed that the most favorable positions for mutations are 105 and 117 amino acids. The corresponding proteins were purified and compared with each other in terms of color intensity, speed of its change, resistance to environmental factors and other physico-chemical properties. Thus, at a light length of 520 nanometers, the variant containing the amino acid threonine at position 117, the moxSAASoti—T protein, turned out to be the brightest. In addition, it changed color eight times faster than other options.
"The fluorescent protein moxSAASoti-T obtained by us has unique properties uncharacteristic of other proteins, such as high resistance to oxidants, rapid and reversible changes in the intensity of luminescence and color. It can be used in modern microscopy, for example in the field of neurobiology, to study the behavior of proteins in various environmental conditions. Biphotochromic properties, that is, the ability to change the color of the glow, make moxSAASoti-T an interesting object for further research," says Alexander Savitsky, head of the RNF grant project, Doctor of Chemical Sciences, Professor, Head of the Laboratory of Physical Biochemistry at the A. N. Bach Institute of Biochemistry, FITZ Biotechnology RAS.
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