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The staff of the Department of Physical Chemistry of the Faculty of Chemistry of Moscow State University together with scientists from University College London investigated the processes of oxidation of the chromophore of a green fluorescent protein in an aqueous solution. The results will significantly expand the scope of application of one of the most relevant methods for tracking the activity of proteins and will help to create a biosensor for the determination of reactive oxygen species.

Green fluorescent protein is actively used by biologists to monitor processes in living organisms. Its attachment to other molecules as a glowing green label allows you to accurately track their position in the cell.

"We studied the chromophore, the part of the protein responsible for its ability to glow," said Anastasia Bochenkova, head of the Laboratory of Quantum Photodynamics of the Department of Physical Chemistry of the Faculty of Chemistry of Moscow State University, co–author of the work. - After all, it is when the light absorbed by the chromophore is emitted that we see the green glow. However, in some cases, the chromophore can undergo oxidation, that is, give an electron to another molecule. The oxidized protein changes the wavelength of radiation, becoming red, which is promising for the creation of photosensitive redox biosensors. Photooxidation can also lead to irreversible discoloration, in which the protein can no longer glow, which significantly reduces the scope of its practical application."

Until recently, the oxidative properties of the green fluorescent protein chromophore were studied only for molecules in the gas phase - a relatively simple process for conducting and interpreting the results. But since the environment strongly affects the properties of the chromophore, the redox characteristics for the gas phase and for the protein should differ markedly.

"It is very difficult to study the oxidative properties directly in the protein, but our colleagues from England tried to get closer to this: they found a way to study the oxidation of the chromophore in an aqueous solution," Anastasia Bochenkova explained. - From the point of view of the experiment, this is a breakthrough technique, but the results obtained are very difficult to understand. For their correct interpretation, it is necessary to understand in detail the mechanism of photoinduced electron transfer from the chromophore. Therefore, our part of the work was to develop a method for modeling and analyzing photoelectron spectra. We carried out large-scale quantum chemical calculations using the resources of the Lomonosov-2 supercomputer complex."

The combination of multiphoton ultraviolet photoelectron spectroscopy and high-precision quantum chemistry calculations made it possible to obtain the values of the electron separation energy and to evaluate the influence of the environment on the electronic structure of the chromophore, as well as to investigate the nature and role of its electronically excited states in the processes of electron phototransfer. The obtained experimental value of the electron separation energy turned out to be much higher than that measured in the gas phase. The authors also compared it with their calculations for protein. The complete similarity of the two values indicates that the ability of the chromophore to give up an electron when absorbing a photon in a protein environment and an aqueous medium is almost the same.

"In addition to helping experimenters, we were able to verify the calculation method we proposed," explained Anton Boychenko, a graduate student at the Laboratory of Quantum Photodynamics, co–author of the study. "This will help to extend it to other similar systems in the future and will open up the possibility of interpreting the results of complex experimental studies."

"In the future, we want to direct photo–oxidation for good," Anastasia Bochenkova shared. –After all, there are oxidants in the cell, for example, reactive oxygen species. The appearance of a red glow instead of green allows the protein to be used as a biosensor showing the presence of oxidants in the cell. The main problem limiting its use is a small reaction yield. Now we are actively working to eliminate it, using physical approaches we are trying to increase the efficiency of the process with laser irradiation in linear and nonlinear modes."

The work, carried out with the support of the Russian Science Foundation, is published in the journal Nature Communications
PHОTO: The process of photooxidation of the chromophore of a green fluorescent protein © Omri Tau, Anastasia Bochenkova

Source: msu.ru , sci-dig.ru