Coelenterazine-binding protein of Renilla muelleri: cDNA cloning, overexpression, and characterization as a substrate of luciferase

Abstract

The Renilla bioluminescent system in vivo is comprised of three proteins--the luciferase, green-fluorescent protein, and coelenterazine-binding protein (CBP), previously called luciferin-binding protein (LBP). This work reports the cloning of the full-size cDNA encoding CBP from soft coral Renilla muelleri, its overexpression and properties of the recombinant protein. The apo-CBP was quantitatively converted to CBP by simple incubation with coelenterazine. The physicochemical properties of this recombinant CBP are determined to be practically the same as those reported for the CBP (LBP) of R. reniformis. CBP is a member of the four-EF-hand Ca(2+)-binding superfamily of proteins with only three of the EF-hand loops having the Ca(2+)-binding consensus sequences. There is weak sequence homology with the Ca(2+)-regulated photoproteins but only as a result of the necessary Ca(2+)-binding loop structure. In combination with Renilla luciferase, addition of only one Ca(2+) is sufficient to release the coelenterazine as a substrate for the luciferase for bioluminescence. This combination of the two proteins generates bioluminescence with higher reaction efficiency than using free coelenterazine alone as the substrate for luciferase. This increased quantum yield, a difference of bioluminescence spectra, and markedly different kinetics, implicate that a CBP-luciferase complex might be involved.

The smallest natural high-active luciferase: Cloning and characterization of novel 16.5-kDa luciferase from copepod Metridia longa

“…3B shows the concentration dependence of bioluminescence intensity maximum when MLuc7 produced in insect cells is assayed with coelenterazine. Light intensity reaches a constant value at $10 lM that is twice higher than for MLuc164 produced in E. coli [15] but approximately 10 times less than for coelenterazine-dependent luciferase from Renilla muelleri [20]. The apparent Michaelis constant determined from dependence of initial light intensity on coelenterazine concentrations amounts to 3.2 lM.…”

Protein-protein complexation in bioluminescence

“…When the Ca 2+ concentration rises upon nerve stimulation, Ca 2+ binds to CBP to make coelenterazine available for reaction with luciferase and O 2 . It is proposed that the bioluminescence reaction occurs within a CBP-luciferase complex so that coelenterazine does not necessarily dissociate (Charbonneau and Cormier, 1979;Titushin et al, 2008;Stepanyuk et al, 2009). Following oxygen addition to coelenterazine in the active site of luciferase, the decarboxylation reaction deposits excitation again into the coupled electronic state transitions of the coelenteramide-GFP fluorophore pair, from which S 1 →S 0 radiation as GFP's green fluorescence takes place (509 nm).…”

“…The bioluminescence reaction of Renilla luciferase is triggered either by injection of coelenterazine into a solution of luciferase, or by injection of calcium into a mixture of luciferase and CBP (Matthews et al, 1977a;Titushin et al, 2008). Both reactions have very similar bioluminescence spectra (Fig.…”

“…However, kinetic parameters of the reactions are different, such as luciferase is able to turn over with coelenterazine enclosed within CBP six times faster than with free coelenterazine, and emits with a two-fold higher quantum yield ( Fig. 4D and 4E) (Titushin et al, 2008). Knowing that apo-CBP does not enhance luciferase activity, a higher bioluminescence quantum yield for Renilla luciferase with CBP as its substrate, would be difficult to explain if coelenterazine had to first dissociate from Ca 2+ -loaded CBP into solution.…”

“…Several lines of evidence indicate that delivery of luciferin from the binding protein to the luciferase as well as modulation of bioluminescence spectra as a result of energy transfer to the antenna proteins, involves protein-protein interactions (Morise et al, 1974;Ward andCormier, 1976, 1978;Nicolas et al, 1991;Cutler, 1995;Schultz et al, 2005;Titushin et al, 2008Titushin et al, , 2010Stepanyuk et al, 2009;. The interactions are of a transient nature, with K D in the range of 10 −6 -10 −3 mol/L, which highly impedes direct detection of the complexes, or their structural studies with Xray crystallography or NMR methods.…”