coelenterazine and obelin

Ca

Topics: Aequorin; Animals; Calcium; Hydrogen Bonding; Imidazoles; Luminescent Proteins; Mutagenesis, Site-Directed; Protein Conformation; Pyrazines

Changes in the intracellular concentration of free ionized calcium ([Ca(2+)]i) control a host of cellular processes as varied as vision, muscle contraction, neuronal signal transmission, proliferation, apoptosis etc. The disturbance in Ca(2+)-signaling causes many severe diseases. To understand the mechanisms underlying the control by calcium and how disorder of this regulation relates to pathological conditions, it is necessary to measure [Ca(2+)]i. The Ca(2+)-regulated photoproteins which are responsible for bioluminescence of marine coelenterates have been successfully used for this purpose over the years. Here we report the results on comparative characterization of bioluminescence properties of aequorin from Aequorea victoria, obelin from Obelia longissima, and clytin from Clytia gregaria charged by native coelenterazine and coelenterazine analogues f, i, and hcp. The comparison of specific bioluminescence activity, stability, emission spectra, stopped-flow kinetics, sensitivity to calcium, and effect of physiological concentrations of Mg(2+) establishes obelin-hcp as an excellent semisynthetic photoprotein to keep track of fast intracellular Ca(2+) transients. The rate of rise of its light signal on a sudden change of [Ca(2+)] is almost 3- and 11-fold higher than those of obelin and aequorin with native coelenterazine, respectively, and 20 times higher than that of the corresponding aequorin-hcp. In addition, obelin-hcp preserves a high specific bioluminescence activity and displays higher Ca(2+)-sensitivity as compared to obelin charged by native coelenterazine and sensitivity to Ca(2+) comparable with those of aequorin-f and aequorin-hcp.

Topics: Aequorin; Animals; Calcium; Calcium Signaling; Cations, Divalent; Hydrozoa; Imidazoles; Luminescent Agents; Luminescent Measurements; Luminescent Proteins; Pyrazines

Previous studies have stated that aequorin loses most of its bioluminescence activity upon modification of the C-terminus, thus limiting the production of photoprotein fusion proteins at its N-terminus. In the present work, we investigate the importance of the C-terminal proline and the hydrogen bonds it forms for photoprotein active complex formation, stability and functional activity. According to the crystal structures of obelin and aequorin, two Ca(2+)-regulated photoproteins, the carboxyl group of the C-terminal Pro forms two hydrogen bonds with the side chain of Arg21 (Arg15 in aequorin case) situated in the first α-helix. Whereas, deletion or substitution of the C-terminal proline could noticeably change the bioluminescence activity, stability or the yield of an active photoprotein complex. Therefore, modifications of the first α-helix Arg has a clear destructive effect on the main photoprotein properties. A C-terminal hydrogen-bond network is proposed to be important for the stability of photoprotein molecules towards external disturbances, when taking part in the formation of locked protein conformations and isolation of coelenterazine-binding cavities.

Topics: Aequorin; Arginine; Crystallization; Escherichia coli; Hydrogen Bonding; Imidazoles; Kinetics; Luminescent Measurements; Luminescent Proteins; Mutation; Proline; Protein Stability; Protein Structure, Secondary; Pyrazines; Recombinant Proteins

The main analytical use of Ca(2+)-regulated photoproteins from luminous coelenterates is for real-time non-invasive visualization of intracellular calcium concentration ([Ca(2+)]i) dynamics in cells and whole organisms. A limitation of this approach for in vivo deep tissue imaging is the fact that blue light emitted by the photoprotein is highly absorbed by tissue. Seven novel coelenterazine analogues were synthesized and their effects on the bioluminescent properties of recombinant obelin from Obelia longissima and aequorin from Aequorea victoria were evaluated. Only analogues having electron-donating groups (m-OCH3 and m-OH) on the C6 phenol moiety or an extended resonance system at the C8 position (1-naphthyl and α-styryl analogues) showed a significant red shift of light emission. Of these, only the α-styryl analogue displayed a sufficiently high light intensity to allow eventual tissue penetration. The possible suitability of this compound for in vivo assays was corroborated by studies with aequorin which allowed the monitoring of [Ca(2+)]i dynamics in cultured CHO cells and in hippocampal brain slices. Thus, the α-styryl coelenterazine analogue might be potentially useful for non-invasive, in vivo bioluminescence imaging in deep tissues of small animals.

Topics: Aequorin; Animals; CHO Cells; Cricetulus; Hydrozoa; Imidazoles; Luminescent Agents; Luminescent Measurements; Luminescent Proteins; Molecular Imaging; Pyrazines

Ca(2+) -regulated photoproteins use a noncovalently bound 2-hydroperoxycoelenterazine ligand to emit light in response to Ca(2+) binding. To better understand the mechanism of formation of active photoprotein from apoprotein, coelenterazine and molecular oxygen, we investigated the spectral properties of the anaerobic apo-obelin-coelenterazine complex and the kinetics of its conversion into active photoprotein after exposure to air. Our studies suggest that coelenterazine bound within the anaerobic complex might be a mixture of N7-protonated and C2(-) anionic forms, and that oxygen shifts the equilibrium in favor of the C2(-) anion as a result of peroxy anion formation. Proton removal from N7 and further protonation of peroxy anion and the resulting formation of 2-hydroperoxycoelenterazine in obelin might occur with the assistance of His175. It is proposed that this conserved His residue might play a key role both in formation of active photoprotein and in Ca(2+) -triggering of the bioluminescence reaction.

Topics: Animals; Calcium; Histidine; Hydrozoa; Imidazoles; Luminescence; Luminescent Proteins; Models, Molecular; Oxygen; Protein Binding; Protons; Pyrazines; Spectrophotometry

Bioluminescence of a variety of marine organisms is caused by monomeric Ca(2+)-regulated photoproteins, to which a peroxy-substituted coelenterazine, 2-hydroperoxycoelenterazine, is firmly bound. From the spatial structure the side chains of Tyr138, His175, Trp179, and Tyr190 of obelin are situated within the substrate-binding pocket at hydrogen bond distances with different atoms of the 2-hydroperoxycoelenterazine. Here we characterized several obelin mutants with substitutions of these residues regarding their bioluminescence, coelenterazine binding, and kinetics of active obelin formation. We demonstrate that Tyr138, His175, Trp179, and Tyr190 are all important for coelenterazine activation; substitution of any of these residues leads to significant decrease of the apparent reaction rate. The hydrogen bond network formed by Tyr138, Trp179 and Tyr190 participates in the proper positioning of coelenterazine in the active site and subsequent stabilization of the 2-hydroperoxy adduct of coelenterazine. His175 might serve as a proton shuttle during 2-hydroperoxycoelenterazine formation.

Topics: Amino Acid Substitution; Apoproteins; Calcium; Imidazoles; Kinetics; Luminescent Proteins; Models, Molecular; Mutation; Protein Binding; Protein Conformation; Pyrazines; Spectrometry, Fluorescence

Many proteins require a non-covalently bound ligand to be functional. How ligand binding affects protein conformation is often unknown. Here we address thermal unfolding of the free and ligand-bound forms of photoprotein obelin. Fluorescence and far-UV circular dichroism (CD) data show that the various ligand-dependent conformational states of obelin differ significantly in stability against thermal unfolding. Binding of coelenterazine and calcium considerably stabilizes obelin. In solution, all obelin structures are similar, except for apo-obelin without calcium. This latter protein is an ensemble of conformational states, the populations of which alter upon increasing temperature.

Topics: Calcium; Circular Dichroism; Imidazoles; Luminescent Proteins; Protein Binding; Protein Folding; Pyrazines; Spectrometry, Fluorescence

Up to now, all reported Ca(2+)-regulated photoproteins, except for mnemiopsin, have been cloned and expressed in Escherichia coli. In this study, the cDNA for an isotype of mnemiopsin, from the ctenophore Mnemiopsis leidyi, has been cloned, sequenced, and functionally expressed. The full length cDNA encoding mnemiopsin of M. leidyi was 624 bp open reading frame encoding a protein of 207 amino acid residues with calculated molecular mass of ∼24 kDa. The deduced amino acid sequence showed 90% and 84% identity to berovine (from ctenophore Beroe abyssicola) and bolinopsin 2 (from the ctenophore Bolinopsis infundibulum) respectively. In contrast to all known EF-hand in photoproteins, a unique EF-hand motif was found in mnemiopsin, in which a conserved glycine is substituted with glutamic acid. According to the results, the optimum pH was 9.0, time course of regeneration was 15 h and its Ca(2+) sensitivity was lower than aequorin. Results of pK(a) calculation for ionizable residues, motif scan and hydrophobic interactions of cavity aromatic residues of mnemiopsin in comparison with aequorin showed different patterns in these two photoproteins. In addition, experimental results are confirmed with the theoretical studies.

Topics: Aequorin; Amino Acid Sequence; Animals; Calcium; Ctenophora; Hydrogen-Ion Concentration; Imidazoles; Luminescent Proteins; Molecular Sequence Data; Protein Conformation; Pyrazines

Obelin from the hydroid Obelia longissima and aequorin are members of a subfamily of Ca(2+)-regulated photoproteins that is a part of the larger EF-hand calcium binding protein family. On the addition of Ca(2+), obelin generates a blue bioluminescence emission (lambda(max) = 485 nm) as the result of the oxidative decarboxylation of the bound substrate, coelenterazine. The W92F obelin mutant is noteworthy because of the unusually high speed with which it responds to sudden changes of [Ca(2+)] and because it emits violet light rather than blue due to a prominent band with lambda(max) = 405 nm. Increase of pH in the range from 5.5 to 8.5 and using D(2)O both diminish the contribution of the 405 nm band, indicating that excited state proton transfer is involved. Fluorescence model studies have suggested the origin of the 485 nm emission as the excited state of an anion of coelenteramide, the bioluminescence reaction product, and 405 nm from the excited neutral state. Assuming that the dimensions of the substrate binding cavity do not change during the excited state formation, a His22 residue within hydrogen bonding distance to the 6-(p-hydroxy)-phenyl group of the excited coelenteramide is a likely candidate for accepting the phenol proton to produce an ion-pair excited state, in support of recent suggestions for the bioluminescence emitting state. The proton transfer could be impeded by removal of the Trp92 H-bond, resulting in strong enhancement of a 405 nm band giving the violet color of bioluminescence. Comparative analysis of 3D structures of the wild-type (WT) and W92F obelins reveals that there are structural displacements of certain key Ca(2+)-ligating residues in the loops of the two C-terminal EF hands as well as clear differences in hydrogen bond networks in W92F. For instance, the hydrogen bond between the side-chain oxygen atom of Asp169 and the main-chain nitrogen of Arg112 binds together the incoming alpha-helix of loop III and the exiting alpha-helix of loop IV in WT, providing probably concerted changes in these EF hands on calcium binding. But this linkage is not found in W92F obelin. These differences apparently do not change the overall affinity to calcium of W92F obelin but may account for the kinetic differences between the WT and mutant obelins. From analysis of the hydrogen bond network in the coelenterazine binding cavity, it is proposed that the trigger for bioluminescence reaction in these Ca(2+)-regulated photoproteins may be a shi

Topics: Animals; Binding Sites; Calcium; Color; Crystallography, X-Ray; Hydrozoa; Imidazoles; In Vitro Techniques; Kinetics; Luminescent Measurements; Luminescent Proteins; Magnesium; Models, Molecular; Mutagenesis, Site-Directed; Protein Conformation; Protons; Pyrazines; Spectrophotometry

The spatial structure of the Ca(2+)-regulated photoprotein obelin has been solved to resolution of 1.1A. Two oxygen atoms are revealed substituted at the C2-position of the coelenterazine in contrast to the obelin structure at 1.73A resolution where one oxygen atom only was disclosed. The electron density of the second oxygen atom was very weak but after exposing the crystals to a trace of Ca(2+), the electron densities of both oxygen atoms became equally intense. In addition, one Ca(2+) was found bound in the loop of the first EF-hand motif. Four of the ligands were provided by protein residues Asp30, Asn32, Asn34, and the main chain oxygen of Lys36. The other two were from water molecules. From a comparison of B-factors for the residues constituting the active site, it is suggested that the variable electron densities observed in various photoprotein structures could be attributed to different mobilities of the peroxy oxygen atoms.

Topics: Binding Sites; Calcium; Carbon; Crystallography; Electrons; Imidazoles; Luminescent Proteins; Macromolecular Substances; Nanotechnology; Oxygen; Protein Binding; Protein Conformation; Protein Subunits; Pyrazines; Solutions

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.

Topics: Aequorin; Amino Acid Sequence; Calcium; Crystallography, X-Ray; Imidazoles; Luminescent Proteins; Models, Molecular; Molecular Sequence Data; Oxygen; Protein Binding; Protein Conformation; Protein Structure, Secondary; Pyrazines; Recombinant Proteins; Sequence Homology, Amino Acid; Sulfur

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.

Topics: Cell-Free System; Imidazoles; Luminescent Measurements; Luminescent Proteins; Protein Biosynthesis; Protein Folding; Pyrazines; RNA, Messenger; Time Factors

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.

Topics: Aequorin; Animals; Cnidaria; Dose-Response Relationship, Drug; Free Radical Scavengers; Free Radicals; Hydrogen-Ion Concentration; Imidazoles; Kinetics; Light; Luminescent Measurements; Luminescent Proteins; Manganese; Models, Chemical; Pyrazines; Recombinant Proteins; Sulfhydryl Reagents; Thiosulfates

Topics: Aequorin; Animals; Apoproteins; Biological Evolution; Calcium; Cloning, Molecular; Complement System Proteins; Humans; Imidazoles; In Vitro Techniques; Indicators and Reagents; Liposomes; Luminescent Measurements; Luminescent Proteins; Neutrophils; Pyrazines; RNA, Messenger