Fig 1.
The luminescence reaction of coelenterazine and molecular oxygen catalyzed by luciferase.
Fig 2.
The luminescence reaction of aequorin triggered by Ca2+ and the formation of blue fluorescent protein (BFP).
Fig 3.
Ca2+-triggered luminescence reaction of aequorin, regenerated from apoAequorin and coelenterazine.
A. Regeneration of aequorin from apoAequorin and coelenterazine, followed by the calcium triggered-luminescence reaction. B. Structures of coelenterazine (CTZ) and its chiral deaza-analogs of (S)- and (R)-deaza-CTZ (daCTZ). C. Structures of (S)-2-peroxycoelenterazine and the chiral deaza-analogs of (S)-2- and (R)-2-hydroxymethyl-deaza-CTZ (HM-daCTZ).
Fig 4.
Synthetic route of the racemic deaza-CTZ (daCTZ) compounds, (R)- and (S)-daCTZ, and hydroxymethyl-deaza-CTZ (HM-daCTZ), (R)-2- and (S)-2-HM-daCTZ, and each enantiomer separation.
Fig 5.
Inhibition of aequorin regeneration from apoAequorin and coelenterazine in the presence of chiral deaza-coelenterazine analog.
A. Time course of aequorin regeneration from apoAequorin and coelenterazine in the presence of (R)-2- and (S)-2-hydroxymethyl deaza-CTZ (HM-daCTZ). B. Time course of aequorin regeneration from apoAequorin and coelenterazine in the presence of (S)- and (R)-deaza-CTZ (daCTZ). The reaction conditions are described in Table 1.
Table 1.
Inhibition of aequorin regeneration form apoAequorin and coelenterazine in the presence of chiral deaza-analogs.
Fig 6.
Absorption spectra of apoAequorin/(S)-HM-daCTZ complex, apoAequorin/(S)-daCTZ complex, and recombinant aequorin.
A. Absorption spectrum of the apoAequorin/(S)-HM-daCTZ complex eluted from the Butyl-Sepharose column. Protein concentration is 0.80 mg/mL in 0.4 M (NH4)2SO4 of 10 mM Tris-HCl (pH 7.6)–2 mM EDTA. B. Absorption spectra of the apoAequorin/(S)-daCTZ complex and aequorin eluted from the Butyl-Sepharose column. Protein concentration of apoAequorin/(S)-daCTZ complex and recombinant aequorin are 0.29 mg/mL and 0.32 mg/mL, respectively, in 1.2 M (NH4)2SO4 of 10 mM Tris-HCl (pH 7.6)–2 mM EDTA. Protein concentration in b is 1.36 mg/mL. The final concentration of Ca2+ in apoAequorin/(S)-daCTZ complex is 50 mM of CaCl2.
Fig 7.
Luminescence properties of the apoAequorin/(S)-HM-daCTZ complex, by comparison with apoAequorin.
A. Comparison of the luciferase-like luminescence reaction of the apoAequorin/(S)-HM-daCTZ/Ca2+ complex with the complex of apoAequorin, coelenteramide (CTMD), and Ca2+. The reaction mixture contains 1 μg of apoAequorin in 100 μL of Tris-HCl (pH 7.6) in the presence of 10 mM CaCl2, 1 μg/μL of CTMD, and/or 1 μg/μL of (S)-HM-daCTZ. The luminescence reaction was started by the addition of 1 μg/μL of coelenterazine at 22°C and the luminescence activity was determined using a luminometer. B. Time course of aequorin regeneration from the apoAequorin/(S)-HM-daCTZ complex with coelenterazine. The regeneration conditions are the same as in Fig 5A. The luminescence activity of 5 μL in the mixture was determined using a luminometer with a 0.23% neutral density filter. The bold line and dashed line indicate apoAequorin and the apoAequorin/(S)-HM-daCTZ complex, respectively. C. Ca2+-triggered luminescence reaction of aequorin regenerated from the apoAequorin/(S)-HM-daCTZ complex and coelenterazine. The mixture includes 1 μg of apoAequorin or the apoAequorin/(S)-HM-daCTZ complex in 1 mL of 30 mM Tris-HCl (pH 7.6)–10 mM EDTA containing 1 μg/μL of coelenterazine and 1 μL of 2-mercaptoethanol and incubated at 4°C. The luminescence activity of 1 μL of regenerated aequorin was determined by injection of 100 μL of 50 mM CaCl2 using a luminometer.
Table 2.
Statistics of data collection and structure refinement.
Fig 8.
Crystal structures of the apoAequorin complex with (S)-daCTZ and (S)-HM-daCTZ.
A. a) Superposition of native aequorin (A form, gray color) and the apoAequorin/(S)-daCTZ complex (A form, blue color). b) Superposition of (S)-2-peroxycoelenterazine (gray color) and (S)-daCTZ (yellow color). B. a) Superposition of native aequorin (A form, gray color) and the apoAequorin/(S)-HM-daCTZ complex (A form, red color). b) Superposition of (S)-2-peroxycoelenterazine (gray color) and (S)-HM-daCTZ (green color). The labeled N and C in the structures indicate the amino and carboxyl terminus, respectively. The numbers 2, 6, and 8 indicate the positions of C2, C6, and C8 in the imidazopyrazinone ring of coelenterazine, respectively.
Fig 9.
Comparison of the hydrogen-bonding networks in the (S)-2-peroxycoelenterazine binding cavity among native aequorin (A), the apoAequorin/(S)-daCTZ complex (B), the apoAequorin/(S)-HM-daCTZ complex (C), and the predicted binding of coelenterazine in apoAequorin (D).
Fig 10.
Proposed mechanism for the carbanion formation of coelenterazine in the regeneration process from apoAequorin, coelenterazine, and O2.