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Hydromedusan photoproteins responsible for the bioluminescence of a variety of marine jellyfish and hydroids are a unique biochemical system recognized as a stable enzyme-substrate complex consisting of apoprotein and preoxygenated coelenterazine, which is tightly bound in the protein inner cavity. The binding of calcium ions to the photoprotein molecule is only required to initiate the light emission reaction. Although numerous experimental and theoretical studies on the bioluminescence of these photoproteins were performed, many features of their functioning are yet unclear. In particular, which ionic state of dioxetanone intermediate decomposes to yield a coelenteramide in an excited state and the role of the water molecule residing in a proximity to the N1 atom of 2-hydroperoxycoelenterazine in the bioluminescence reaction are still under discussion. With the aim to elucidate the function of this water molecule as well as to pinpoint the amino acid residues presumably involved in the protonation of the primarily formed dioxetanone anion, we constructed a set of single and double obelin and aequorin mutants with substitutions of His, Trp, Tyr, and Ser to residues with different properties of side chains and investigated their bioluminescence properties (specific activity, bioluminescence spectra, stopped-flow kinetics, and fluorescence spectra of Ca2+-discharged photoproteins). Moreover, we determined the spatial structure of the obelin mutant with a substitution of His64, the key residue of the presumable proton transfer, to Phe. On the ground of the bioluminescence properties of the obelin and aequorin mutants as well as the spatial structures of the obelin mutants with the replacements of His64 and Tyr138, the conclusion was made that, in fact, His residue of the Tyr-His-Trp triad and the water molecule perform the "catalytic function" by transferring the proton from solvent to the dioxetanone anion to generate its neutral ionic state in complex with water, as only the decomposition of this form of dioxetanone can provide the highest light output in the light-emitting reaction of the hydromedusan photoproteins. [ABSTRACT FROM AUTHOR]

Coelenterazine‐v (CTZ‐v), a synthetic derivative with an additional benzyl ring, yields a bright bioluminescence of Renilla luciferase and its "yellow" mutant with a significant shift in the emission spectrum toward longer wavelengths, which makes it the substrate of choice for deep tissue imaging. Although Ca2+‐regulated photoproteins activated with CTZ‐v also display red‐shifted light emission, in contrast to Renilla luciferase their bioluminescence activities are very low, which makes photoproteins activated by CTZ‐v unusable for calcium imaging. Here, we report the crystal structure of Ca2+‐regulated photoprotein obelin with 2‐hydroperoxycoelenterazine‐v (obelin‐v) at 1.80 Å resolution. The structures of obelin‐v and obelin bound with native CTZ revealed almost no difference; only the minor rearrangement in hydrogen‐bond pattern and slightly increased distances between key active site residues and some atoms of 2‐hydroperoxycoelenterazine‐v were found. The fluorescence quantum yield (ΦFL) of obelin bound with coelenteramide‐v (0.24) turned out to be even higher than that of obelin with native coelenteramide (0.19). Since both obelins are in effect the enzyme‐substrate complexes containing the 2‐hydroperoxy adduct of CTZ‐v or CTZ, we reasonably assume the chemical reaction mechanisms and the yields of the reaction products (ΦR) to be similar for both obelins. Based on these findings we suggest that low bioluminescence activity of obelin‐v is caused by the low efficiency of generating an electronic excited state (ΦS). In turn, the low ΦS value as compared to that of native CTZ might be the result of small changes in the substrate microenvironment in the obelin‐v active site. PDB Code(s): 7O3U; 1QV0, 1QV1, 1EJ3, 4NQG, 3KPX, 1UHK, 1UHJ, 1UHI, 1UHH, 5ZAB, 1EL4, 4MRX. [ABSTRACT FROM AUTHOR]

Site‐directed mutagenesis is a powerful tool to investigate the structure–function relationship of proteins and a function of certain amino acid residues in catalytic conversion of substrates during enzymatic reactions. Hence, it is not surprising that this approach was repeatedly applied to elucidate the role of certain amino acid residues in various aspects of photoprotein bioluminescence, mostly for aequorin and obelin, and to design mutant photoproteins with altered properties (modified calcium affinity, faster or slower bioluminescence kinetics, different emission color) which would either allow the development of novel bioluminescent assays or improvement of characteristics of the already existing ones. This information, however, is scattered over different articles. In this review, we systematize the findings that were made using site‐directed mutagenesis studies regarding the impact of various amino acid residues on bioluminescence of hydromedusan Ca2+‐regulated photoproteins. All key residues that have been identified are pinpointed, and their influence on different aspects of photoprotein functioning such as active photoprotein complex formation, bioluminescence reaction, calcium response and light emitter formation is discussed. Site‐directed mutagenesis is a powerful tool to investigate the structure–function relationship of proteins and a function of certain amino acid residues in catalytic conversion of substrates during enzymatic reactions. In this review, we systematize the findings that were made using a site‐directed mutagenesis with regard to the impact of various amino acid residues on bioluminescence of hydromedusan Ca2+‐regulated photoproteins. All key residues that have been identified are pinpointed and their influence on different aspects of photoprotein functioning such as active photoprotein complex formation, bioluminescence reaction, calcium response and light emitter formation are discussed. [ABSTRACT FROM AUTHOR]

Nowadays, physicochemical approach to understanding toxic effects remains underdeveloped. A proper development of such mode would be concerned with simplest bioassay systems. Coelenteramide-Containing Fluorescent Proteins (CLM - CFPs) can serve as proper tools for study primary physicochemical processes in organisms under external exposures. CLM - CFPs are products of bioluminescent reactions of marine coelenterates. As opposed to Green Fluorescent Proteins, the CLM - CFPs are not widely applied in biomedical research, and their potential as colored biomarkers is undervalued now. Coelenteramide, fluorophore of CLM - CFPs, is a photochemically active molecule; it acts as a proton donor in its electron-excited states, generating several forms of different fluorescent state energy and, hence, different fluorescence color, from violet to green. Contributions of the forms to the visible fluorescence depend on the coelenteramide microenvironment in proteins. Hence, CLM - CFPs can serve as fluorescence biomarkers with color differentiation to monitor results of destructive biomolecule exposures. The paper reviews experimental and theoretical studies of spectral-luminescent and photochemical properties of CLM-CFPs, as well as their variation under different exposures – chemicals, temperature, and ionizing radiation. Application of CLM-CFPs as toxicity bioassays of a new type is justified. [ABSTRACT FROM AUTHOR]

Ca2+-regulated photoproteins genetically fused with biospecific polipeptides have been extensively used in intracellular Ca2+ measurements and in the development of binding assays. Gene fusions to aequorin have been limited to its N-terminus, since as previous studies indicated the protein loses bioluminescent activity upon modification of its C-terminus. To investigate this in regard to another photoprotein - obelin (OL) the one was elongated at its C-terminus with tyrosine (Y) and then fused with green fluorescent protein Clytia gregaria (cgreGFP) through the flexible 31 aa linker. Both proteins (OL-Y and OL-cgreGFP) were isolated and investigated. The OL-Y was found to form a stable photoprotein comlex, possessing 75 % of WT-OL bioluminescent activity. OL-cgreGFP activity preserves 46 % of WT-OL activity and demonstrates an effective resonance energy transfer, where OL-partner and cgreGFP-partner are energy donor and acceptor respectively. Thus, it was shown that the labels on the base of Ca2+-regulated photoprotein obelin may be obtained by fusing with biospecific polypeptides regardless its termini.

Coelenteramide-containing fluorescent proteins are products of bioluminescent reactions of marine coelenterates. They are called ‘discharged photoproteins’. Their light-induced fluorescence spectra are variable, depending considerably on external conditions. Current work studies a dependence of light-induced fluorescence spectra of discharged photoproteins obelin, aequorin, and clytin on excitation energy. It was demonstrated that photoexcitation to the upper electron-excited states (260–300 nm) of the discharged photoproteins initiates a fluorescence peak in the near UV region, in addition to the blue-green emission. To characterize the UV fluorescence, the light-induced fluorescence spectra of coelenteramide (CLM), fluorophore of the discharged photoproteins, were studied in methanol solution. Similar to photoproteins, the CLM spectra depended on photoexcitation energy; the additional peak (330 nm) in the near UV region was observed in CLM fluorescence at higher excitation energy (260–300 nm). Quantum chemical calculations by time depending method with B3LYP/cc-pVDZ showed that the conjugated pyrazine-phenolic fragment and benzene moiety of CLM molecule are responsible for the additional UV fluorescence peak. Quantum yields of CLM fluorescence in methanol were 0.028 ± 0.005 at 270–340 nm photoexcitation. A conclusion was made that the UV emission of CLM might contribute to the UV fluorescence of the discharged photoproteins. The study develops knowledge on internal energy transfer in biological structures – complexes of proteins with low-weight aromatic molecules. [ABSTRACT FROM AUTHOR]

Luminous organisms use different protein-mediated strategies to modulate light emission color. Here, we report the transient-state kinetic studies of the interaction between photoprotein clytin from Clytia gregaria and its antenna protein, cgre GFP. We propose that cgre GFP forms a transient complex with Ca2+-bound clytin before the excited singlet state of the coelenteramide product is formed. From the spectral distribution and donor-acceptor separation distance, we infer that clytin reaction intermediates may interact only with the middle side part of cgre GFP. [ABSTRACT FROM AUTHOR]

Light-sensitive photoprotein berovin accounts for a bright bioluminescence of ctenophore Beroe abyssicola . Berovin is functionally identical to the well-studied Ca 2 + -regulated photoproteins of jellyfish, however in contrast to those it is extremely sensitive to the visible light. Berovin contains three EF-hand Ca 2 + -binding sites and consequently belongs to a large family of the EF-hand Ca 2 + -binding proteins. Here we report the spatial structure of apo-berovin with bound Mg 2 + determined at 1.75 Å. The magnesium ion is found in each functional EF-hand loop of a photoprotein and coordinated by oxygen atoms donated by the side-chain groups of aspartate, carbonyl groups of the peptide backbone, or hydroxyl group of serine with characteristic oxygen-Mg 2 + distances. As oxygen supplied by the side-chain of the twelfth residue of all Ca 2 + -binding loops participates in the magnesium ion coordination, it was suggested that Ca 2 + -binding loops of berovin belong to the mixed Ca 2 + /Mg 2 + rather than Ca 2 + -specific type. In addition, we report an effect of physiological concentration of Mg 2 + on bioluminescence of berovin (sensitivity to Ca 2 + , rapid-mixed kinetics, light-sensitivity, thermostability, and apo-berovin conversion into active protein). The different impact of physiological concentration of Mg 2 + on berovin bioluminescence as compared to hydromedusan photoproteins was attributed to different affinities of the Ca 2 + -binding sites of these photoproteins to Mg 2 + . [ABSTRACT FROM AUTHOR]

Calcium ion is a ubiquitous intracellular messenger, performing this function in many eukaryotic cells. To understand calcium regulation mechanisms and how disturbances of these mechanisms are associated with disease states, it is necessary to measure calcium inside cells. Ca-regulated photoproteins have been successfully used for this purpose for many years. Here we report the results of comparative studies on the properties of recombinant aequorin from Aequorea victoria, recombinant obelins from Obelia geniculata and Obelia longissima, recombinant mitrocomin from Mitrocoma cellularia, and recombinant clytin from Clytia gregaria as intracellular calcium indicators in a set of identical in vitro and in vivo experiments. Although photoproteins reveal a high degree of identity of amino acid sequences and spatial structures, and, apparently, have a common mechanism for the bioluminescence reaction, they were found to differ in the Ca concentration detection limit, the sensitivity of bioluminescence to Mg, and the rates of the rise of the luminescence signal with a sudden change of Ca concentration. In addition, the bioluminescence activities of Chinese hamster ovary cells expressing wild-type photoproteins also differed. The light signals of cells expressing mitrocomin, for example, slightly exceeded the background, suggesting that mitrocomin may be hardly used to detect intracellular Ca without modifications improving its properties. On the basis of experiments on the activation of endogenous P2Y receptor in Chinese hamster ovary cells by ATP, we suggest that wild-type aequorin and obelin from O. longissima are more suitable for calcium detection in cytoplasm, whereas clytin and obelin from O. geniculata can be used for calcium measurement in cell compartments with high Ca concentration. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Ca2+-regulated photoproteins are responsible for the bioluminescence of a variety of marine coelenterates. All hydromedusan photoproteins are a single-chain polypeptide to which 2-hydroperoxycoelenterazine is tightly but non-covalently bound. Bioluminescence results from oxidative decarboxylation of 2-hydroperoxycoelenterazine, generating protein-bound coelenteramide in an excited state. The bioluminescence spectral maxima of recombinant photoproteins vary in the range 462-495 nm, despite a high degree of identity of amino acid sequences and spatial structures of these photoproteins. Based on studies of obelin and aequorin mutants with substitution of Phe to Tyr and Tyr to Phe, respectively [Stepanyuk GA et al. (2005) FEBS Lett 579, 1008-1014], it was suggested that the spectral differences may be accounted for by an additional hydrogen bond between the hydroxyl group of a Tyr residue and an oxygen atom of the 6-( p-hydroxyphenyl) substituent of coelenterazine. Here, we report the crystal structures of two conformation states of the F88Y obelin mutant that has bioluminescence and product fluorescence spectra resembling those of aequorin. Comparison of spatial structures of the F88Y obelin conformation states with those of wild-type obelin clearly shows that substitution of Phe to Tyr does not affect the overall structures of either F88Y obelin or its product following Ca2+ discharge, compared to the conformation states of wild-type obelin. The hydrogen bond network in F88Y obelin being due to the Tyr substitution clearly supports the suggestion that different hydrogen bond patterns near the oxygen of the 6-( p-hydroxyphenyl) substituent are the basis for spectral modifications between hydromedusan photoproteins. [ABSTRACT FROM AUTHOR]

Highlights: [•] Coelenterazine binding and kinetics of active obelin formation are studied. [•] Tyr138, His175, Trp179, and Tyr190 are all important for coelenterazine activation. [•] The proper hydrogen bond network over coelenterazine is required for its activation. [•] His175 might serve as a proton shuttle during 2-hydroperoxycoelenterazine formation. [Copyright &y& Elsevier]

Abstract: Photoprotein obelin, the enzyme–substrate complex of polypeptide with 2-hydroperoxycoelenterazine, is responsible for bioluminescence of marine hydroid Obelia longissima. Addition of Ca2+ to the photoprotein triggers the bioluminescent reaction with light emission. The product of the bioluminescent reaction – enzyme-bound coelenteramide – is a fluorescent protein called ‘discharged’ obelin. It is stable and highly fluorescent. The paper considers dependence of its light-induced fluorescence on Ca2+ concentration. Increase of Ca2+ concentration enhanced the fluorescence intensity of discharged obelin; the dependence was found as linear in double logarithmic coordinates at Ca2+ concentration range 10−7–10−6 М, both in excitation and emission spectra. The spectra were divided into components; contributions of the components to experimental excitation and emission spectra depended on Ca2+ concentration. The data suggest enzymatic conformational transition in discharged obelin at ∼5×10−7 M of Ca2+ concentration. Spectra variations were attributed to acidity changes of discharged obelin chromophore (coelenteramide) in its fluorescent state . [ABSTRACT FROM AUTHOR]

Abstract: We constructed a vector for soluble protein expression in the cytoplasm of Escherichia coli cells using the cold induced system. The vector, named pCold-ZZ-P-X, consists of a histidine tag sequence, IgG binding domain of protein A (ZZ domain), the cleavage site of human rhinovirus 3C protease followed by the multiple cloning sites under the controlled of the cold shock protein A (cspA) promoter and the lac operator. Using this expression vector, the calcium binding photoprotein mitrocomin from luminous jellyfish was successfully expressed as a soluble ZZ fusion protein and purified. After removing the ZZ domain by protease digestion, recombinant apomitrocomin was obtained and then regenerated to mitrocomin by incubation with coelenterazine. The luminescence properties of recombinant mitrocomin were characterized and compared to other photoproteins including aequorin, clytin-I and clytin-II. [Copyright &y& Elsevier]

Abstract: The effective new variant of “sandwich” bioluminescent enzyme immunoassay (BEIA) for the sensitive detection of glycoprotein B (gB) of pseudorabies virus (PrV) was presently developed. The high affinity interaction of barnase–barstar protein pair and photoprotein obelin as bioluminescent marker were for the first time successfully applied to BEIA development. Preliminary the two monoclonal antibodies, 11/5 and 34/2, were raised against gB for ELISA PrV detection. Presently we used the same immuno-“sandwich” principle for BEIA. To do this the two different bioconjugates were elaborated. Recombinant barnase was chemically conjugated with monoclonal anti-PrV''s gB IgG, and also barstar was fused in frame to obelin. The characteristics of BEIA method have been compared to ELISA PrV detection. We have shown the proposed here gB-BEIA was 40-fold more sensitive as opposed to gB-ELISA test. The construction might have a broad promise in multiple potential immunological applications. [Copyright &y& Elsevier]

Abstract: Fluorescence of the phenolate anion (3(O)−) and the amide anion (5(N)−) of coelenteramide analogues in ion pairs with various counter cations was systematically investigated to elucidate the ionic structure of the light emitter in the bioluminescence of the calcium-activated photoproteins aequorin and obelin. The fluorescent properties of 3(O)− in an ion pair with a conjugate acid of an organic base (BASE–H+) were varied depending on the structural variation of the ion pair and the solvent polarity. In particular, the fluorescence of 3(O)− in the ion pair with the conjugate acid of n-butylamine (NBA–H+) indicates that the singlet-excited state of 3(O)− (1 3(O)−∗) and NBA–H+ make a contact ion pair in which the fluorescence emission maxima of 3(O)− is sensitive to the solvent polarity and the fluorescence quantum yields of 3(O)− increase in a less polar solvent. The results also confirm that 1 3(O)−∗ is a twisted intramolecular charge transfer state. By contrast, the fluorescence of 5(N)− in an ion pair depends little on the BASE–H+ or the solvent polarity. Based on these results, we conclude that the light emitter in aequorin and obelin bioluminescences is the singlet-excited state of coelenteramide phenolate anion 2(O)− (1 2(O)−∗) in a contact ion pair with an imidazolium side chain of a histidine residue, which is located at the less polar active sites of the photoproteins. We also propose a mechanism for the bioluminescence reaction, including the chemiexcitation process to give 1 2(O)−∗. [Copyright &y& Elsevier]

Calcium-regulated photoproteins are bioluminescent proteins that are responsible for the luminescence of marine coelenterates. A photoprotein molecule is a stable enzyme-substrate complex consisting of a single polypeptide chain and an oxygen-preactivated substrate, 2-hydroperoxycoelenterazine, which is tightly but noncovalently bound with the protein. Bioluminescence is triggered by Ca2+ and results from decarboxylation of the substrate bound with the protein. This review considers the current information about the structure of photoproteins, the mechanism of the bioluminescent reaction, the function of particular amino acid residues of the active center in catalysis and the formation of the emitter, and the use of photoproteins in bioluminescent microanalysis. [ABSTRACT FROM AUTHOR]

The crystal structures of calcium-loaded apoaequorin and apo-obelin have been determined at resolutions 1.7 Å and 2.2 Å, respectively. A calcium ion is observed in each of the three EF-hand loops that have the canonical calcium-binding sequence, and each is coordinated in the characteristic pentagonal bipyramidal configuration. The calcium-loaded apo-proteins retain the same compact scaffold and overall fold as the unreacted photoproteins containing the bound substrate, 2-hydroperoxycoelenterazine, and also the same as the Ca2+-discharged obelin bound with the product, coelenteramide. Nevertheless, there are easily discerned shifts in both helix and loop regions, and the shifts are not the same between the two proteins. It is suggested that these subtle shifts are the basis of the ability of these photoproteins to sense Ca2+ concentration transients and to produce their bioluminescence response on the millisecond timescale. A mechanism of intrastructural transmission of the calcium signal is proposed. [ABSTRACT FROM AUTHOR]

The bioluminescence emitted by Aequorea victoria jellyfish is greenish while its single bioluminescent photoprotein aequorin emits blue light. This phenomenon may be explained by a bioluminescence resonance energy transfer (BRET) from aequorin chromophore to green fluorescent protein (GFP) co-localized with it. However, a slight overlapping of the aequorin bioluminescence spectrum with the GFP absorption spectrum and the absence of marked interaction between these proteins in vitro pose a question on the mechanism providing the efficient BRET in A. victoria. Here we report the in vitro study of BRET between homologous Ca2+-activated photoproteins, aequorin or obelin (Obelia longissima), as bioluminescence energy donors, and GFP, as an acceptor. The fusions containing donor and acceptor proteins linked by a 19 aa peptide were purified after expressing their genes in Escherichia coli cells. It was shown that the GFP–aequorin fusion has a significantly greater BRET efficiency, compared to the GFP–obelin fusion. Two main factors responsible for the difference in BRET efficiency of these fusions were revealed. First, it is the presence of Ca2+-induced interaction between the donor and acceptor in the aequorin-containing fusion and the absence of the interaction in the obelin-containing fusion. Second, it is a red shift of GFP absorption toward better overlapping with aequorin bioluminescence induced by the interaction of aequorin with GFP. Since the connection of the two proteins in vitro mimics their proximity in vivo, Ca2+-induced interaction between aequorin and GFP may occur in A. victoria jellyfish providing efficient BRET in this organism. [Copyright &y& Elsevier]

Solid-phase bioluminescent immunoassay of thyroid hormones, human thyrotropin (hTSH) and two forms of thyroxine (T4), whose determinations are vitally important for diagnostics of thyroid diseases and the efficiency of treatment, is described. The recombinant obelin, a Ca2+-regulated photoprotein originally derived from the luminous marine hydroid Obelia longissima, is employed as a bioluminescent label. To produce obelin conjugates with anti-hTSH, anti-T4 immunoglobulins (IgG), and T4, additional SH groups are introduced into the obelin molecule using Traut’s reagent (2-iminothiolane) and then obelin possessing extra SH groups is conjugated with succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate-activated IgGs or T4. The total yield of obelin conjugates determined by luminescent activity is 60–65% after all chemical and purification procedures. The obtained conjugates are stable to lyophilization and in solution for at least 9 months at 4 °C, with loss of activity not exceeding 10%. The application of obelin conjugates for determination of the hTSH, total T4, and free T4 in standard, control, and patient sera displays high sensitivity and reproducibility of results. The results of bioluminescent immunoassays are closely comparable to those obtained by the radioimmunoassay method (R=0.95–0.99). [Copyright &y& Elsevier]

Recombinant plasmids containing genes for the green fluorescent protein (GFP) from Aequorea victoriaand the photoprotein obelin from Obelia longissimalinked in-frame by inserts differing in nucleotides sequences were constructed. The expression of the chimeric genes in Escherichia colicells resulted in synthesis of the GFP–obelin hybrid proteins. These proteins were purified to homogeneity and subjected to limited trypsinolysis. It was shown that the resistance of GFP–obelin hybrid proteins to trypsin depends on the nature of their constituent modules and the amino acid sequences of linkers between the modules. The kinetics of accumulation of full-length hybrid proteins during the growth of bacterial cells does not depend on the structure of the peptide linkers. Most of the full-length product accumulates in cells in the form of inclusion bodies resistant to endogenous proteases. The soluble fraction of the protein undergoes considerable proteolysis regardless of the linker structure. [ABSTRACT FROM AUTHOR]

Using site directed mutagenesis, the conservative residue Cys158 of recombinant apoobelin was substituted for serine (C158S, S-mutant) or alanine (C158A, A-mutant). These point mutations resulted in significant changes in the apoobelin structure accompanied by slowing of photoprotein complex formation, decrease of its stability, and changing of its bioluminescence characteristics. The enzymatic properties of the photoprotein decreased in the series: wild-type protein > S-mutant > A-mutant. This is consistent with rank of nucleophilicity SH > OH > CH3 of cysteine, serine, and alanine side chain functional groups, respectively. Possible mechanisms of the involvement of the apoobelin Cys158 SH-group in the formation of the enzyme–substrate complex are considered. [ABSTRACT FROM AUTHOR]

The crystal structure of the photoprotein obelin (22.2 kDa) from Obelia longissima has been determined and refined to 1.7 + 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. [ABSTRACT FROM PUBLISHER]

Coelenterazine-v (CTZ-v), a synthetic derivative with an additional benzyl ring, yields a bright bioluminescence of Renilla luciferase and its "yellow" mutant with a significant shift in the emission spectrum toward longer wavelengths, which makes it the substrate of choice for deep tissue imaging. Although Ca 2+ -regulated photoproteins activated with CTZ-v also display red-shifted light emission, in contrast to Renilla luciferase their bioluminescence activities are very low, which makes photoproteins activated by CTZ-v unusable for calcium imaging. Here, we report the crystal structure of Ca 2+ -regulated photoprotein obelin with 2-hydroperoxycoelenterazine-v (obelin-v) at 1.80 Å resolution. The structures of obelin-v and obelin bound with native CTZ revealed almost no difference; only the minor rearrangement in hydrogen-bond pattern and slightly increased distances between key active site residues and some atoms of 2-hydroperoxycoelenterazine-v were found. The fluorescence quantum yield (Φ FL ) of obelin bound with coelenteramide-v (0.24) turned out to be even higher than that of obelin with native coelenteramide (0.19). Since both obelins are in effect the enzyme-substrate complexes containing the 2-hydroperoxy adduct of CTZ-v or CTZ, we reasonably assume the chemical reaction mechanisms and the yields of the reaction products (Φ R ) to be similar for both obelins. Based on these findings we suggest that low bioluminescence activity of obelin-v is caused by the low efficiency of generating an electronic excited state (Φ S ). In turn, the low Φ S value as compared to that of native CTZ might be the result of small changes in the substrate microenvironment in the obelin-v active site., (© 2021 The Protein Society.)

Ca2+-regulated photoproteins responsible for bioluminescence of a variety of marine organisms are single-chain globular proteins within the inner cavity of which the oxygenated coelenterazine, 2-hydroperoxycoelenterazine, is tightly bound. Alongside with native coelenterazine, photoproteins can also use its synthetic analogues as substrates to produce flash-type bioluminescence. However, information on the effect of modifications of various groups of coelenterazine and amino acid environment of the protein active site on the bioluminescent properties of the corresponding semi-synthetic photoproteins is fragmentary and often controversial. In this paper, we investigated the specific bioluminescence activity, light emission spectra, stopped-flow kinetics and sensitivity to calcium of the semi-synthetic aequorins and obelins activated by novel coelenterazine analogues and the recently reported coelenterazine derivatives. Several semi-synthetic photoproteins activated by the studied coelenterazine analogues displayed sufficient bioluminescence activities accompanied by various changes in the spectral and kinetic properties as well as in calcium sensitivity. The poor activity of certain semi-synthetic photoproteins might be attributed to instability of some coelenterazine analogues in solution and low efficiency of 2-hydroperoxy adduct formation. In most cases, semi-synthetic obelins and aequorins displayed different properties upon being activated by the same coelenterazine analogue. The results indicated that the OH-group at the C-6 phenyl ring of coelenterazine is important for the photoprotein bioluminescence and that the hydrogen-bond network around the substituent in position 6 of the imidazopyrazinone core could be the reason of different bioluminescence activities of aequorin and obelin with certain coelenterazine analogues. [ABSTRACT FROM AUTHOR]

Site-directed mutagenesis is a powerful tool to investigate the structure-function relationship of proteins and a function of certain amino acid residues in catalytic conversion of substrates during enzymatic reactions. Hence, it is not surprising that this approach was repeatedly applied to elucidate the role of certain amino acid residues in various aspects of photoprotein bioluminescence, mostly for aequorin and obelin, and to design mutant photoproteins with altered properties (modified calcium affinity, faster or slower bioluminescence kinetics, different emission color) which would either allow the development of novel bioluminescent assays or improvement of characteristics of the already existing ones. This information, however, is scattered over different articles. In this review, we systematize the findings that were made using site-directed mutagenesis studies regarding the impact of various amino acid residues on bioluminescence of hydromedusan Ca 2+ -regulated photoproteins. All key residues that have been identified are pinpointed, and their influence on different aspects of photoprotein functioning such as active photoprotein complex formation, bioluminescence reaction, calcium response and light emitter formation is discussed., (© 2018 The American Society of Photobiology.)

Ca(2+)-regulated photoproteins, which are responsible for light emission in a variety of marine coelenterates, are a highly valuable tool for measuring Ca(2+) inside living cells. All of the photoproteins are a single-chain polypeptide to which a 2-hydroperoxycoelenterazine molecule is tightly but noncovalently bound. Bioluminescence results from the oxidative decarboxylation of 2-hydroperoxycoelenterazine, generating protein-bound coelenteramide in an excited state. Here, the crystal structures of the Y138F obelin mutant before and after bioluminescence are reported at 1.72 and 1.30 Å resolution, respectively. The comparison of the spatial structures of the conformational states of Y138F obelin with those of wild-type obelin gives clear evidence that the substitution of Tyr by Phe does not affect the overall structure of both Y138F obelin and its product following Ca(2+) discharge compared with the corresponding conformational states of wild-type obelin. Despite the similarity of the overall structures and internal cavities of Y138F and wild-type obelins, there is a substantial difference: in the cavity of Y138F obelin a water molecule corresponding to W2 in wild-type obelin is not found. However, in Ca(2+)-discharged Y138F obelin this water molecule now appears in the same location. This finding, together with the observed much slower kinetics of Y138F obelin, clearly supports the hypothesis that the function of a water molecule in this location is to catalyze the 2-hydroperoxycoelenterazine decarboxylation reaction by protonation of a dioxetanone anion before its decomposition into the excited-state product. Although obelin differs from other hydromedusan Ca(2+)-regulated photoproteins in some of its properties, they are believed to share a common mechanism.