Temperature Matters: Cold Tolerance of Luciferase Proteins

SibFU scientists have studied two types of luciferase enzymes which are naturally found in the cells of luminous bacteria and are responsible for emitting light. Luciferase proteins synthesized in the laboratory are used in various test systems to test soil, water, and food products for toxicity. Inhibition of the luminescence of the luciferase reaction means the presence of contaminants in the samples. There are several types of bacterial luciferase. The so-called fast enzymes are already used in bioassays, and the properties of slow luciferases are to be studied in order to find practical applications for them in the future.

“Colleagues from other countries study slow luciferases in order to use them to illuminate living human and animal cells. For example, such biological flashlights will help to trace how enzymatic cascades function, or to visualize the activity of various genes. Our bioluminescent biotechnology lab traditionally uses fast luciferases to perform rapid safety tests for drinking water, air, soil, and foodstuffs such as fruits and vegetables. This time we decided to find out how these two types of proteins will behave at high and low temperatures — whether they will retain their activity and whether they will be able to restore their structure if temperature-induced damage occurs,” told Anna Deeva, research co-author, candidate of science in Physics & Mathematics, senior lecturer of Biophysics Department, research fellow of Research Department, SibFU.

According to the biophysicists, this experiment is useful in expanding the range of luciferases already in use. After all, if glowing proteins show their performance in heat or cold, tests based on them can be used in agriculture and near natural water sources in all regions of Russia.

Using stopped-flow methods and differential scanning calorimetry, scientists in the SibFU laboratory and in the laboratory of the Institute of Protein Research of the Russian Academy of Sciences (Pushchino, Moscow Region) conducted a series of experiments, testing the activity of bacterial luciferases at temperatures from 5°C to 45°C. The proteins were placed in a chamber for five minutes, where a certain temperature was maintained, and then checked whether they emit a light signal during the enzymatic reaction. Proteins, as expected, began to denature at high temperatures, which had a negative effect on the luminescence. The experts also conducted a number of experiments with a protein that had already denatured (lost its structure), noting the prospects for its restoration to a working state.

“The protein denatures at 45-50°C, but it begins the reverse process of renaturation at a comfortable temperature of 20°C. Our task was also to find out how much the ability of luciferase to glow in such a situation is restored. We all know very well that, for example, boiled egg protein cannot be returned to its original liquid state, but in our experiment, we are working with a solution in which the protein concentration is not so high. Indeed, protein chains can join with each other at high temperatures, and this prevents them from renaturating when returning to a comfortable temperature. However, bacterial luciferase proteins, in most cases, regain activity,” went on Anna Deeva.

The scientists found that fast luciferases function best at 10-20°C, while slow luciferases work at temperatures up to 35°C, demonstrating a less bright glow in the reaction, but greater resistance to extreme conditions for a protein. By the way, the ability of slow luciferases to work as illumination in living organisms which maintain a constant temperature (36.6°C in humans, up to 39.5°C in animals), is explained precisely by their love for warmth.

The scientists noted that the result obtained for bacterial luciferase under temperature swings is interesting for both theoretical and applied purposes. From the point of view of fundamental science, the influence of high temperatures on the denaturation of luminous proteins and the mechanisms of their return to their original structure will be further investigated. Applied functions of fast luciferases, such as the ability to glow at temperatures from 10°C and restore their structure at 20°C, open up new prospects for rapid toxicity testing. With the development of portable devices, it will be possible to check water, soil and crops with their help almost everywhere — in fields, in forests and in household plots.

The research was supported by the Ministry of Science and Higher Education of the Russian Federation (project No. FS-RZ-2020-0006), the Russian Foundation for Basic Research (RFBR; project no. 20-34-90118) and the RFBR together with the Government of Krasnoyarsk Territory and the Krasnoyarsk Regional Science Foundation (project no. 20-44-243002).

SibFu Press Office, 11 october 2022