Krasnoyarsk scientists have synthesized a photoprotein obelin mutant that is twice as sensitive to the intracellular calcium concentration changes as the natural protein. This makes it possible to detect weak calcium signals, previously unnoticed, and opens up new opportunities for studying cellular processes. The results of the study have been published in the International Journal of Biological Macromolecules.

Photoproteins emit light in the presence of calcium ions. They are found in nature in many organisms, including bioluminescent jellyfish, and have long been used to track calcium signals within cells under the laboratory conditions, since calcium regulates key cellular processes. An important advantage of photoproteins is that the intensity of their luminescence is directly proportional to the amount of calcium: the higher the calcium level, the brighter the signal. The reaction requires neither oxygen nor additional substances; the protein and calcium ions are sufficient. These properties make photoproteins perfect indicators for measuring the intracellular calcium concentrations.

A research team from the Krasnoyarsk Scientific Center, Siberian Branch of the Russian Academy of Sciences, has improved the properties of the natural luminescent protein obelin by ​​replacing amino acid isoleucine with other amino acid variants in its structure. In one of the mutant forms obtained, isoleucine has been replaced by leucine. This variant responds to the intracellular calcium changes dozens of times more efficiently as compared with the natural protein and could underlie a new generation of biotests.

The synthesized mutant proved stable in living cells as well, where it was found to be twice as sensitive to calcium as the natural protein form. This means it can track cytoplasmic calcium concentration changes with twice the precision of the natural protein and can detect even weak calcium signals within the cell.

"Protein engineering is an effective approach to creating proteins with enhanced properties. In natural obelin, the substrate molecule responsible for luminescence is tightly packed within the protein and the rest of the molecule structure is arranged around it. We hypothesize that substitution of leucine for isoleucine alters the position of this molecule inside the protein. This creates structures with a higher affinity for calcium ions, thereby enabling luminescence at much lower calcium concentrations. Interestingly, similar substitutions we performed for comparison in related luminescent proteins, aequorin and clytin, also increased the calcium sensitivity, but the signal enhancement was much lower than that observed with the obelin mutant," says the study’s leading researcher Evgenii S. Vysotskii, Cand. Sc. in Biology, Head of the Photobiology Laboratory of the Institute of Biophysics.

"Thanks to its high sensitivity, the synthesized protein makes it possible to detect minor changes in the intracellular calcium concentration. It holds promise as a tool for studying cellular processes, in which calcium acts as a signaling molecule. In addition, it can be used to investigate the effects of drugs and to identify new therapeutic approaches. For instance, calcium monitoring allows for more precise tracking of a drug's impact on the cell, enabling the selection of the most promising compounds based on this data," explains the study’s co-author Natalia Malikova, Cand. Sc. in Biology, Senior Researcher of the Institute of Biophysics.

The study was supported by the Russian Science Foundation,  project no.24-44-00009.