Coelenterazine-binding protein of Renilla muelleri: cDNA cloning, overexpression, and characterization as a substrate of luciferase.
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.
Titushin
Maxim S
MS
Photobiology Lab, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, Russia.
Markova
Svetlana V
SV
Frank
Ludmila A
LA
Malikova
Natalia P
NP
Stepanyuk
Galina A
GA
Lee
John
J
Vysotski
Eugene S
ES
D000595:Amino Acid Sequence; D000818:Animals; D037421:Anthozoa / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D007093:Imidazoles / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011719:Pyrazines / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D007093:Imidazoles; D011506:Proteins; D011719:Pyrazines; C017144:coelenterazine; D008156:Luciferases