Bioluminescence and kinetic aspects of double mutated aequorin variants

Aequorin as an old small calcium-sensitive photoprotein is a blue fluorescence protein which converts coelenterazine (a substrate) to coelenteramide with a flash type emission. The decay kinetics and emission properties of this protein can be changed using directed mutagenesis of crucial amino acid residue. In this work, we prepared three double mutants: Y⁸²F/W⁸⁶F, Y⁸²F/D¹⁵³G, and W⁸⁶F/D¹⁵³G. According to our results, it seems that presence of Y⁸²F mutation results in shift of emission to longer wavelengths while the W⁸⁶F mutation shifts the emission to shorter wavelengths. Furthermore, comparison of the variants for light half-life indicated decreased t_1/2 for the two variants of Y⁸²F/D¹⁵³G and W⁸⁶F/D¹⁵³G. But in compared to wild type aequorin, the Y⁸²F/W⁸⁶F variant displayed a 2-fold increase of light half-life. On the other hand, the thermostability properties of double mutants confirmed that only Y⁸²F/D¹⁵³G variant of apoaequorin is higher stability than others. Also, the single W⁸⁶F mutant reached the highest stability against thermal shock. Our data suggest that replacement of single or few point mutations in the binding pocket or active site of aequorin affects its bioluminescence and kinetic properties and so could be used for new reporter production of this photoprotein with the feasibility and limited substitutions.