Ca2+-Triggered Coelenterazine-Binding Protein Renilla: Expected and Unexpected Features

Alexander N Kudryavtsev; Vasilisa V Krasitskaya; Maxim K Efremov; Sayana V Zangeeva; Anastasia V Rogova; Felix N Tomilin; Ludmila A Frank

doi:10.3390/ijms24032144

Ca²⁺-Triggered Coelenterazine-Binding Protein Renilla: Expected and Unexpected Features

Int J Mol Sci. 2023 Jan 21;24(3):2144. doi: 10.3390/ijms24032144.

Authors

Alexander N Kudryavtsev¹, Vasilisa V Krasitskaya¹, Maxim K Efremov², Sayana V Zangeeva², Anastasia V Rogova^{2

3}, Felix N Tomilin^{2

3}, Ludmila A Frank^{1

2}

Affiliations

¹ Institute of Biophysics, Federal Research Center "Krasnoyarsk Science Center SB RAS", 660036 Krasnoyarsk, Russia.
² School of Fundamental Biology and Biotechnology, School of Non-Ferrous Metals and Material Science, Siberian Federal University, pr. Svobodny 79, 660041 Krasnoyarsk, Russia.
³ Kirensky Institute of Physics, Federal Research Center "Krasnoyarsk Science Center SB", 660036 Krasnoyarsk, Russia.

Abstract

Ca²⁺-triggered coelenterazine-binding protein (CBP) is a natural form of the luciferase substrate involved in the Renilla bioluminescence reaction. It is a stable complex of coelenterazine and apoprotein that, unlike coelenterazine, is soluble and stable in an aquatic environment and yields a significantly higher bioluminescent signal. This makes CBP a convenient substrate for luciferase-based in vitro assay. In search of a similar substrate form for the luciferase NanoLuc, a furimazine-apoCBP complex was prepared and verified against furimazine, coelenterazine, and CBP. Furimazine-apoCBP is relatively stable in solution and in a frozen or lyophilized state, but as distinct from CBP, its bioluminescence reaction with NanoLuc is independent of Ca²⁺. NanoLuc turned out to utilize all the four substrates under consideration. The pairs of CBP-NanoLuc and coelenterazine-NanoLuc generate bioluminescence with close efficiency. As for furimazine-apoCBP-NanoLuc pair, the efficiency with which it generates bioluminescence is almost twice lower than that of the furimazine-NanoLuc. The integral signal of the CBP-NanoLuc pair is only 22% lower than that of furimazine-NanoLuc. Thus, along with furimazine as the most effective NanoLuc substrate, CBP can also be recommended as a substrate for in vitro analytical application in view of its water solubility, stability, and Ca²⁺-triggering "character".

Keywords: B3LYP; Ca2+-triggered coelenterazine-binding protein; DFTB3; TDDFT; coelenterazine; fragmented molecular orbitals method; furimazine; luciferase NanoLuc.

MeSH terms

Animals
Calcium / metabolism
Carrier Proteins* / metabolism
Luciferases / metabolism
Luminescent Measurements*
Renilla

Substances

Carrier Proteins
coelenterazine
Luciferases
Calcium

Grants and funding

This research was supported by the state budget allocated to the fundamental research at the Russian Academy of Sciences, project No. 0287-2022-0002 and the Interagency Supercomputer Center of the Russian Academy of Sciences, MVS-100K and MVS-10P.