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Ca2+-Triggered Coelenterazine-Binding Protein Renilla: Expected and Unexpected Features
Alexander N Kudryavtsev1, Vasilisa V Krasitskaya1, Maxim K Efremov2
1Institute of Biophysics, Federal Research Center "Krasnoyarsk Science Center SB RAS", 660036 Krasnoyarsk, Russia.
International Journal of Molecular Sciences
|February 11, 2023
Summary
Coelenterazine-binding protein (CBP) offers a stable, water-soluble substrate for bioluminescence assays. While furimazine is optimal for NanoLuc, CBP provides a viable, Ca2+-triggered alternative for in vitro applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Assay Development
Background:
- Coelenterazine-binding protein (CBP) is a stable, soluble complex of coelenterazine and apoprotein, enhancing Renilla bioluminescence.
- CBP offers advantages over free coelenterazine, including improved stability in aqueous environments and higher signal output.
- There is a need for similar stable substrate forms for other luciferase systems, such as NanoLuc.
Purpose of the Study:
- To evaluate a furimazine-apoCBP complex as a potential substrate for the NanoLuc luciferase system.
- To compare the performance of furimazine-apoCBP with other substrates (furimazine, coelenterazine, CBP) for NanoLuc.
- To assess the suitability of CBP as a substrate for NanoLuc in in vitro analytical applications.
Main Methods:
- Preparation and verification of a furimazine-apoCBP complex.
- Testing the stability of furimazine-apoCBP in solution and under storage conditions (frozen, lyophilized).
- Measuring bioluminescence generated by NanoLuc with four different substrates: furimazine, coelenterazine, CBP, and furimazine-apoCBP, analyzing signal efficiency and Ca2+ dependence.
Main Results:
- Furimazine-apoCBP demonstrated relative stability in solution and when stored frozen or lyophilized.
- Unlike CBP, the bioluminescence reaction of furimazine-apoCBP with NanoLuc was independent of Ca2+.
- NanoLuc effectively utilized all tested substrates. CBP-NanoLuc and coelenterazine-NanoLuc pairs showed comparable bioluminescence efficiency.
- The furimazine-apoCBP-NanoLuc pair generated bioluminescence with approximately half the efficiency of the furimazine-NanoLuc pair.
- The CBP-NanoLuc pair's integral signal was only 22% lower than that of furimazine-NanoLuc.
Conclusions:
- Furimazine remains the most efficient substrate for NanoLuc.
- CBP is a recommended substrate for NanoLuc in vitro assays due to its water solubility, stability, and Ca2+-triggering characteristic.
- The development of apo-complexes offers versatile substrate options for different luciferase systems.
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