Coelenterazine-dependent luciferases.

Bioluminescence is a widespread natural phenomenon. Luminous organisms are found among bacteria, fungi, protozoa, coelenterates, worms, molluscs, insects, and fish. Studies on bioluminescent systems of various organisms have revealed an interesting feature - the mechanisms underlying visible light emission are considerably different in representatives of different taxa despite the same final result of this biochemical process. Among the several substrates of bioluminescent reactions identified in marine luminous organisms, the most commonly used are imidazopyrazinone derivatives such as coelenterazine and Cypridina luciferin. Although the substrate used is the same, bioluminescent proteins that catalyze light emitting reactions in taxonomically remote luminous organisms do not show similarity either in amino acid sequences or in spatial structures. In this review, we consider luciferases of various luminous organisms that use coelenterazine or Cypridina luciferin as a substrate, as well as modifications of these proteins that improve their physicochemical and bioluminescent properties and therefore their applicability in bioluminescence imaging in vivo.

Preparation and X-ray crystallographic analysis of recombinant obelin crystals diffracting to beyond 1.1 A.

Crystals of recombinant obelin, the Ca(2+)-regulated photoprotein from the marine hydroid Obelia longissima, have been grown from a solution containing PEG 8000 and potassium phosphate. Hexaminecobalt trichloride was used as an additive to increase the chance of crystallization. The crystals grow in a light yellow cubic form (0.5 x 0.5 x 0.45 mm) which diffracts to beyond 1.1 A resolution. The crystals belong to the space group C2, with unit-cell parameters a = 83.43, b = 54.92, c = 52.99 A, beta = 112.00 degrees. The asymmetric unit contains one molecule. Crystals exposed to calcium ion before and after X-ray irradiation emit light, confirming that the crystals consist of an active photoprotein.

Structural basis for the emission of violet bioluminescence from a W92F obelin mutant.

Mutation of the Trp92 that is known to lie within the active site of the photoprotein obelin from Obelia longissima, results in a shift of the bioluminescence color from blue (lambda(max)=485 nm) to violet. The corrected spectrum shows a new band with lambda(max)=410 nm now contributing equally to the one at longer wavelength. The crystal structure of this W92F obelin determined at 1.72 A resolution shows that there is no significant change in the dimensions of the active site between WT obelin (recombinant Ca2+-regulated photoprotein from Obelia longissima) and the mutant. It is proposed that the bioluminescence spectral shift results from removal of a hydrogen bond from the indole of W92 nearby a hydroxyl belonging to the 6-phenyl substituent of the substrate coelenterazine. Propagation of this change through a conjugated bond system in the excited state of the product coelenteramide affects the coupling of the N1-position and the hydrogen-bonded Y138.

Structure of the Ca2+-regulated photoprotein obelin at 1.7 A resolution determined directly from its sulfur substructure.

The crystal structure of the photoprotein obelin (22.2 kDa) from Obelia longissima has been determined and refined to 1.7 A resolution. Contrary to the prediction of a peroxide, the noncovalently bound substrate, coelenterazine, has only a single oxygen atom bound at the C2-position. The protein-coelenterazine 2-oxy complex observed in the crystals is photo-active because, in the presence of calcium ion, bioluminescence emission within the crystal is observed. This structure represents only the second de novo protein structure determined using the anomalous scattering signal of the sulfur substructure in the crystal. The method used here is theoretically different from that used for crambin in 1981 (4.72 kDa) and represents a significant advancement in protein crystal structure determination.

Preparation and preliminary study of crystals of the recombinant calcium-regulated photoprotein obelin from the bioluminescent hydroid Obelia longissima.

Crystals of recombinant obelin, the Ca(2+)-regulated photoprotein from the marine hydroid Obelia longissima, have been grown from sodium citrate solutions. Crystals grow as hexagonal light-yellow rods (0.1 x 0.1 x 1.0 mm) which diffract to beyond 1.8 A with synchrotron radiation of 1.0 A wavelength. The crystals have a primitive hexagonal lattice with unit-cell parameters a = 81.55, c = 86.95 A. The asymmetric unit contains two molecules. This represents the successful preparation of single crystals of a photoprotein obelin which have promising diffraction properties.

Cotranslational formation of active photoprotein obelin in a cell-free translation system: direct ultrahigh sensitive measure of the translation course.

Translation of apoobelin mRNA in a cell-free wheat germ translation system in the presence of coelenterazine and molecular oxygen results in cotranslational formation of active photoprotein. Active obelin formation is recorded by its luminescence, either direct in the translation mixture in the presence of coelenterazine and calcium ions or in aliquots from the translation mixture. In the second case translation is carried out with coelenterazine and EGTA. Registration of the translation course by luminescence of the synthesized product in both cases allows use of apoobelin mRNA at very low concentrations as an internal marker for immediate measure of protein biosynthesis activity of in vitro translation systems. It is shown that the simultaneous translation of any other mRNA does not affect translation of photoprotein mRNAs under standard conditions. Continuous registration of luminescence in a cuvette of a liquid scintillation counter in photon-counting mode varies the time of signal accumulation in a wide temporal range, thus increasing the numerical values of the recorded signals. Registration of photoprotein luminescence during translation can be used to obtain additional information about the translation process, for example codon reading speed, about protein folding, and about the formation of active proteins on ribosomes.

Cell-free bioluminescent screening of translation inhibitors.

The possibility of creating new screening methods with a cell-free translation system has been demonstrated with a quantitative determination of diphtheria toxin and some antibiotics (puromycin, kanamycin and tetracycline) as examples. The approach proposed follows from the ability of various substances to inhibit protein synthesis. We used a wheat-germ cell-free translation system stabilized by freeze-drying in the presence of trehalose with the mRNA of the Ca(2+)-activated photoprotein obelin as a reporter template. This freeze-dried cell-free translation system allows prolonged storage of the detecting system before it is required, increases the reproducibility of the results and simplifies the application procedure. The obelin mRNA extends the sensitivity of the method owing to the high sensitivity of detection of the synthesized protein.

Use of proZZ-obelin fusion protein in bioluminescent immunoassay.

Obelin is a photoprotein that emits light by Ca2+-binding. To develop a bioluminescent immunoassay based on the light emission property of obelin, we have expressed the apoobelin fusion protein with ZZ-domain of S. aureus protein A in E. coli by recombinant DNA techniques. The proZZ-obelin expressed was purified by one-step affinity chromatography on IgG-Agarose. The purified proZZ-obelin has both the luminescent activity of obelin and the IgG-binding ability of ZZ-domain. The specific activity of fusion protein was 8.5 X 10(15) photons per mg of protein.

Obelin mRNA--a new tool for studies of translation in cell-free systems.

Obelin mRNA obtained in vitro with the aid of SP6 RNA polymerase was translated in a wheat germ cell-free system. Only the polypeptide with a molecular mass of about 20 kDa was synthesized. The activation of apoobelin with a synthetic coelenterazine revealed a luminescence activity initiated by calcium. The specific activity was 3.6 +/- 0.4 x 10(15)photons per mg of the in vitro synthesized obelin (k=6.9s(-1)). The luminescence of the obelin was in a good correlation with the protein concentration calculated by the incorporation of [14C]Leu. The determination of the amount of de novo synthesized obelin based on measurement of its luminescence is one-thousand times more sensitive than the approach based on the incorporation of labeled amino acid. Thus, obelin mRNA has some advantages for evaluating the efficiency of cell-free translation when compared with standard methods.

Sequence of the cDNA encoding the Ca(2+)-activated photoprotein obelin from the hydroid polyp Obelia longissima.

A cDNA clone encoding the Ca(2+)-activated photoprotein, obelin (Obl), from Obelia longissima was sequenced. The nucleotide (nt) sequence contained two long overlapping open reading frames (ORFs), one of which encoded apoobelin (apoObl). The deduced amino acid (aa) sequence of apoObl revealed that this 195-aa protein has three EF-hand structures that are characteristic for Ca(2+)-binding domains. Strong aa homology was shown among apoObl, apoaequorin and apoclytin. The second ORF present in the obl cDNA consists of 139 codons and encodes a very basic protein with a calculated pI of 10.56 and a molecular mass of 16,153 Da.

Mn(2+)-activated luminescence of the photoprotein obelin.

The light emission of obelin may be initiated by Mn2+ under alkaline conditions. The luminescence takes place in a pH range from 7 to 12 with a sharp optimum at 11.75. The first-order rate constant for Mn(2+)-activated luminescence decay is more than 9 s-1, while that for Ca(2+)-activated luminescence decay is only 6.9 s-1. The Mn2+ concentration-effect curve for obelin determined with simple dilutions of manganese salt is a sigmoid curve. The slope of the curve is moderately dependent on the pH and was not more than 1 within the pH range tested. The maximal light emission, which is initiated by 3.6 x 10(-5) M Mn2+ at pH 11.75 was about 10% of the maximal Ca(2+)-activated luminescence. Mg2+ ions inhibit the Mn(2+)-activated luminescence of obelin. The addition of OH. and O2- scavengers did not influence the Mn(2+)-activated luminescence, but when singlet oxygen quenchers were added, the Mn(2+)-dependent light emission was inhibited. This suggests that the 1O2 might be formed and itself be responsible for chromophore oxidation attended with light emission. NEM and Na2S2O4 inhibit the Mn(2+)-initiated light emission of obelin completely, showing that endogenous hydroperoxide and SH-group(s) of the photoprotein are essential for both Ca(2+)-activated and Mn(2+)-activated light emission of obelin.

Luminescence of Ca(2+)-activated photoprotein obelin initiated by NaOCl and MnCl2.

The luminescence of obelin is initiated by NaOCl in a reaction mixture containing no calcium. The addition of Mn2+ enhances the light emission > 300-fold. Sodium azide and histidine, as singlet oxygen quenchers, inhibit NaOCl-activated obelin luminescence in the presence or absence of Mn2+. This suggests that the addition of NaOCl to the mixture causes singlet oxygen formation (stimulated by Mn2+ ions), and singlet oxygen initiates the light-emitting reaction.