Photosensitive enzyme

Squirrels have become obedient
Alexandra Borisova, Newspaper.RuRegulation of protein activity in the body is one of the important tasks of bioorganic chemistry.

For example, the control of transcription factors – proteins responsible for the transfer of information from the DNA molecule to MRNA, will allow to influence the morphology of cells controlled by them. Proteins can be used to control the body's response to extracellular signals and environmental changes, as well as the processes of cell division and cellular aging processes. However, studies are complicated by the fact that there are obvious additional requirements for in vivo control of protein function – the acting agents must be safe for the living organism as a whole. For this reason, relatively simple chemical reactions that are well carried out with proteins in vitro are not suitable for promising medical use – the "small" molecules used in them (for example, formaldehyde) are poisonous.

Because of this, the activation-deactivation of enzymes with the help of light is the preferred, "gentle" method of controlling the function of proteins. A study of the mechanism of modulating the activity of restriction enzymes using ultraviolet or blue light is published by the Proceedings of the National Academy of Sciences.

Restrictases are enzymes of the hydrolase class that catalyze the destruction of phosphodiester bonds (these bonds play a key role in all biological systems, forming the backbone of DNA and RNA nucleic acids) of foreign DNA in most prokaryotic organisms – unicellular ones that do not have a decorated cell nucleus, such as bacteria. Thus, restrictases perform a kind of "immune" function. Recognition of foreign DNA is carried out in specific nucleotide sequences (sites), which are "marked" by certain enzymes in the cell's own DNA.

I told the correspondent of the newspaper about the results of the work.En" one of its leaders, head of the Department of Nucleic Acid Chemistry of the Institute of Physico-Chemical Biology named after A.N. Belozersky (Research Institute of the Moscow State University), Doctor of Chemical Sciences, Professor Tatiana Oretskaya: "One of the possible ways to regulate the activity of proteins is based on the introduction of a "photo switch" into the structure of the latter, which allows you to change the activity of the protein under the action of light with a certain wavelength. This allows you to change the activity of the protein in the cell without adding chemicals, which can also negatively affect cellular processes. It is too early to talk about the introduction into practice of our proposed approaches to regulating the activity of restriction enzymes when using azobenzene derivatives as "photo switches". However, in the future, the creation of reagents that are able to modulate the activity of cellular proteins may lead to the development of biomedical drugs of a new generation."

By cross-linking the residues of cysteine (an amino acid that plays an important role in detoxification processes) in the enzyme with a bifunctional azobenzene derivative, which can have both cis and trans configurations depending on the wavelength of light with which it is irradiated (UV or blue), the activity of the enzyme can be reversibly controlled. When irradiated with blue light, azobenzene is in the trans configuration (phenyl substituents are located on opposite sides of the nitrogen-nitrogen double bond), and in UV light - in the cis configuration (substituents are on one side of the bond). This makes it possible to change the packing motif of conformationally mobile chains. The distance between the SH fragments of cysteine varies significantly (see Figure), which makes it possible to modulate the function of the enzyme. Thus, the function of the cross-linked cysteine residues varies with the change of illumination from UV to blue, showing the so-called "photo-switch effect".

To find out which residues react most significantly to the change in light, more than 30 variants of single-stranded restriction endonuclease Pvu II were synthesized. Then they were modified with azobenzene and their ability to cleave DNA was tested. Usually, single crosslinking in an enzyme causes only small changes in its action, and significant effects are observed only with a large number of crosslinking. Some modified enzymes carry a cross-linked fragment in the immediate vicinity of the active center. It is they who are best able to "switch" with the help of light – the intensity of DNA splitting changed 16 times, while remaining reversible. At the same time, the enzyme activity changes in a matter of seconds.

"Our work was carried out within the framework of the International Research Training Group project "Enzymes and Multi-Enzyme Complexes acting on Nucleic Acids" ("Enzymes and multi-enzyme complexes interacting with nucleic acids"). Our team is the first working under this program in Russia and Germany. On the German side, two universities participate in it – Justus Liebig University (Giessen) and Philip University (Marburg). On the Russian side, our team consists of seven teams – five of them from the Lomonosov Moscow State University (Faculty of Chemistry and the Belozersky Research Institute of the Russian Academy of Sciences) and two from the Institute of Gene Biology of the Russian Academy of Sciences. The program involves 20 graduate students and young candidates of sciences from Russia. We have been working in such a large team for the third year, and this publication was the result of one of our scientific contacts. As for the program itself, on the German side it is funded by the DFG (Deutsche Forschungsgemeinschaft, German Research Society), and on the Russian side – by the Russian Foundation for Basic Research (RFBR)," explained Professor Oretskaya.

Eternal Youth Portal www.vechnayamolodost.ru15.12.2009