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Bioluminescent Properties of Semi-Synthetic Obelin and Aequorin Activated by Coelenterazine Analogues with Modifications of C-2, C-6, and C-8 Substituents - PubMed

️Wed Jan 01 2020

Bioluminescent Properties of Semi-Synthetic Obelin and Aequorin Activated by Coelenterazine Analogues with Modifications of C-2, C-6, and C-8 Substituents

Elena V Eremeeva et al. Int J Mol Sci. 2020.

Abstract

Ca²⁺-regulated photoproteins responsible for bioluminescence of a variety of marine organisms are single-chain globular proteins within the inner cavity of which the oxygenated coelenterazine, 2-hydroperoxycoelenterazine, is tightly bound. Alongside with native coelenterazine, photoproteins can also use its synthetic analogues as substrates to produce flash-type bioluminescence. However, information on the effect of modifications of various groups of coelenterazine and amino acid environment of the protein active site on the bioluminescent properties of the corresponding semi-synthetic photoproteins is fragmentary and often controversial. In this paper, we investigated the specific bioluminescence activity, light emission spectra, stopped-flow kinetics and sensitivity to calcium of the semi-synthetic aequorins and obelins activated by novel coelenterazine analogues and the recently reported coelenterazine derivatives. Several semi-synthetic photoproteins activated by the studied coelenterazine analogues displayed sufficient bioluminescence activities accompanied by various changes in the spectral and kinetic properties as well as in calcium sensitivity. The poor activity of certain semi-synthetic photoproteins might be attributed to instability of some coelenterazine analogues in solution and low efficiency of 2-hydroperoxy adduct formation. In most cases, semi-synthetic obelins and aequorins displayed different properties upon being activated by the same coelenterazine analogue. The results indicated that the OH-group at the C-6 phenyl ring of coelenterazine is important for the photoprotein bioluminescence and that the hydrogen-bond network around the substituent in position 6 of the imidazopyrazinone core could be the reason of different bioluminescence activities of aequorin and obelin with certain coelenterazine analogues.

Keywords: aequorin; analogues; coelenterazine; obelin; photoprotein.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

**Figure A1**
Ca²⁺ concentration–effect curves for semi-synthetic aequorins (left panel) and obelins (right panel) activated with native CTZ (circles), A6 (triangles up), B14 (triangles down), and B1 coelenterazine analogues (squares) with (*open*) and without (*filled*) 1 mM Mg²⁺. All photoproteins were pre-equilibrated with 1 mM of Mg²⁺ for 1 h before measurements. L and L_int are the peak bioluminescence intensity for a given [Ca²⁺] and the total bioluminescence recorded for a saturating Ca²⁺ dose for the same sample, respectively.

**Figure 1**
Coelenterazine analogues used in this study.

**Figure 2**
Fluorescent emitters of coelenteramide [30] (A), bioluminescence (B), and fluorescence (C) spectra of obelins activated with native coelenterazine and some coelenterazine analogues.

**Figure 3**
Ca²⁺ concentration-effect curves for semi-synthetic aequorins (left panel, black) and obelins (right panel, gray) activated with native CTZ (circles) and CTZ analogues A6 (triangles up), B14 (triangles down), and B1 (squares). L, light intensity at the particular Ca²⁺ concentration; L_int, total light intensity at saturating Ca²⁺ concentration.

**Figure 4**
Synthesis of the A-series CTZ analogues.

**Figure 5**
Synthesis of the B-series CTZ analogues.

**Figure 6**
Synthesis of the T-series CTZ analogues.

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Bioluminescent Properties of Semi-Synthetic Obelin and Aequorin Activated by Coelenterazine Analogues with Modifications of C-2, C-6, and C-8 Substituents - PubMed