Russian scientists acquired stable and bright fluorescent protein moxSAASoti, that can change color and intensity of its fluorescence. To achieve this aim, the scientists made point changes in the sequence of the coding gene. Previously all SAASoti-type proteins, that can “switch” color, were very sensible to oxidation and lost their fluorescence, while the new variant of the molecule does not lose its characteristics.
Fluorescent proteins constitute a group of molecules, that are able to fluoresce by themselves under of the certain wavelength light exposure. Scientists exclude them from living organisms, for example, jellyfish and corals, or artificially synthesize in laboratories. Nowadays there exist a vast variety of fluorescent proteins, which differ in color and intensity of fluorescence, and also in variants of color changing. Thus, some of them simply switch from fluorescent to non-fluorescent state, which means stop to fluoresce, while others are able to switch the fluorescence color from green to red.
These processes take place when the protein is irradiated by a certain wavelength of light. Switching from fluorescent to non-fluorescent state can be reversible, but changing the color of fluorescence is irreversible because it occurs as a result of the breaking of chemical bonds. In that case, the substance’s structure is completely destroyed and cannot be reestablished by itself. Also, there are proteins that combine these properties: for example, protein SAASoti, which is able to change the intensity of fluorescence (on-to-off switching) many times and change its form from green to red. However, SAASoti has one shortcoming: it is highly sensitive to oxidizing agents, that destroy its structure. For example, it is oxidized even in the air, which is explained by high photochemical activity of amino acid residues of cysteine that it contains.
Scientists from the Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences (Moscow) and the Lomonosov Moscow State University (Moscow) by using site-directed and site-saturated mutagenesis in the SAASoti gene, created variants of the fluorescent protein with a minimal concentration of cysteine or even a cysteine-free protein. To achieve this aim, the authors obtained DNA with necessary changes and then brought it into E. coli cells. Thus, microorganisms obtained a gene, coding SAASoti, and on the base of it synthesized proteins, different from the original SAASoti in structure: in the amino acid sequence looking like a very long word, we changed one, two or more letters (amino acid residues). However, it became clear that cysteine-free proteins remained as sensible to oxidizing agents as the initial variants. Each of made mutations (change of one letter for another) led to interesting change in protein’s characteristics. Thus, for example, single-point mutations caused its decoloring, and as a result of two-point mutagenesis, there was obtained a number of proteins with various degrees of brightness. During this process the researchers with the help of mathematical modeling can predict the influence of the certain mutation on the protein’s characteristics.
As a result of the experiments, it became clear that the most favorable position for mutations were 105 and 117 amino acids. The corresponding proteins were purified and compared with each other in the color intensity, the rate of its change, resistibility to environmental factors and other physicochemical characteristics. At the 520 nm light the brightest variant was the one, containing a threonine residue in the 117th position – protein moxSAASoti-T. Moreover, it changed color eight times faster than other variants.
“The fluorescent protein moxSAASoti-T, that we obtained, has unique non-characteristic for other proteins features, such as high resistibility to oxidizing agents, fast and reversible change of intensity of fluorescence and coloring. It can be used in modern microscopy – for example, in the sphere of neurobiology, for studying proteins’ behavior in different environmental conditions. Biphotochromic characteristics, that is the ability to change color of fluorescence, make moxSAASoti-T interesting for further research”, - tells Alexander Savitsky, Doctor Chemistry, head of the laboratory of physical biochemistry of the Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences