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The mechanism of bioluminescence in the luminous land snails remains largely unknown. Here, we analyzed basic biochemical properties of the luminous land snail, Quantula weinkauffiana. The luminescence activity was extracted from the light organ located near the mouth using a neutral buffer containing detergent. The reaction of the crude buffer extract was triggered by the addition of only hydrogen peroxide (H2O2). These results are inconsistent with the single precedent report on the bioluminescence in the Quantula striata by Shimomura and Haneda in 1986, in which the luminescence of the buffer extract (without detergent) from the light organ was induced by the coaddition of three indispensable components H2O2, ferrous ion, and 2‐mercaptoethanol. Based on the present findings, we suggested that an insoluble photoprotein is involved in the bioluminescence of the luminous land snails and the luminescence reaction is simply triggered by H2O2. [ABSTRACT FROM AUTHOR]

We used site‐specific mutagenesis by targeting E179 and F190 on the structure of photoprotein Mnemiopsin 2 (Mn2) from Mnemiopsis leidyi. The tertiary structure of E179S and F190L mutants was made by the MODELLER program. Far‐ultraviolet circular dichroism data showed that the overall secondary structural content of photoprotein is not changed upon mutation, however the helicity and stabilizing interactions in helical structure decreases in mutants as compared with the wild‐type (WT) photoprotein. Fluorescence spectra data revealed that the tertiary structure of the mutants is more compact than that of WT Mn2. According to the heat‐induced denaturation experiments data, the melting temperature (Tm) for the unfolding of tertiary structure of the F190L variant increases by 3°C compared with that of the WT and E179S mutant. Interestingly, the conformational enthalpy of the F190L mutant (86 kcal mol−1) is considerably lower than those in the WT photoprotein (102 kcal mol−1) and E179S mutant (106 kcal mol−1). The significant difference in the enthalpy of the thermal unfolding process could be explained by considering that the thermally denatured state of the F190L mutant is structurally less expanded than the WT and E179S variants. Bioluminescence activity data showed that the maximum characteristic wavelengths of the mutants undergo blue shift as compared with the WT protein. Initial intensity of the F190L and E179S variants was recorded to be 137.5% and 55.9% of the WT protein, respectively. [ABSTRACT FROM AUTHOR]

Approximately 80% of luminous organisms live in the oceans, and considerable diversity of life dependence on bioluminescence has been observed in marine organisms. Among vertebrates, luminous fish species are the only group of vertebrates that have the ability to emit bioluminescent light. Meanwhile, the lantern fish family (Myctophidae), with 33 genera all of which have the ability to emit light, is considered the most prominent family among the luminous fish of the deep oceans and seas. Lantern fish Benthosema pterotum has bioluminescence properties due to the presence of photophores scattered in its ventral-lateral region. However, no research has been performed on its bioluminescence system and light emission mechanism. The present research aimed to assess the type of bioluminescence, pigment, photoprotein, or luciferin-luciferase system in B. pterotum. In order to determine the type of light-emitting system in B. pterotum species, several specific experiments were designed and performed. It was shown that the light emission system in B. pterotum species is categorized into the luciferin-luciferase type. Conducting this research was not only innovative, but it also could be the beginning of further research in the field of marine biochemistry and production of the recombinant active forms of enzymes for industrial, commercial, medical, and pharmaceutical purposes. [ABSTRACT FROM AUTHOR]

A bioluminescent immunoassay system was developed to determine serine/threonine protein kinase activity using an aequorin-labeled monoclonal antibody and a synthetic peptide as the substrate. A monoclonal antibody against the synthetic phosphorylated serine peptide (K9P peptide) of histone H3 (19 amino acid residues), referred to as the H3S10P antibody, was chemically conjugated to maleimide-activated aequorin to prepare aequorin-labeled H3S10P (AQ-S-H3S10P). For the serine/threonine kinase assay, a non-phosphorylated serine peptide (K9C peptide) coated on a microplate was incubated with serine/threonine protein kinase in the presence of ATP and Mg2+. The resulting phosphorylated K9C peptides (K9P peptide) were identified using AQ-S-H3S10P. Thus, after the removal of unbound AQ-S-H3S10P though washing, the serine/threonine kinase activity was determined by the luminescence activity of aequorin from AQ-S-H3S10P bound to the K9P peptide. This assay system, in combination with the K9C peptide and AQ-S-H3S10P, could be used to screen inhibitors of various serine/threonine protein kinases in general. [Display omitted] • The K9C peptide of histone H3 is used as a substrate for Ser/Thr protein kinase. • A monoclonal antibody against phosphorylated K9C peptide was labeled with aequorin. • Phosphorylated K9C peptide is detected using an aequorin-labeled antibody. • Ser/Thr kinase activity was determined with luminescence activity of aequorin. • Inhibition on Ser/Thr kinase by chemicals is determined by aequorin activity. [ABSTRACT FROM AUTHOR]

Mnemiopsin 1 (Mn1) and Mnemiopsin 2 (Mn2) are photoproteins found in Mnemiopsis leidyi. We have tried to answer the question of whether the structural features of photoproteins can explain the observed activity data. According to the activity measurements data, they have the same characteristic wavelength. However, the initial intensity of Mn2 is significantly higher than that of Mn1, and decay time of Mn1 (0.92 s−1) is lower than that of Mn2 (1.46 s−1). The phylogenetic analysis demonstrates that, compared with Obelin and Aequorin from Obelia longissima and Aequorea victoria, respectively, a gene modification event may have caused the expansion of the N‐terminal side of all photoproteins from M. leidyi. An in silico study has shown that the stability of the photoprotein–substrate complex of Mn2 is higher than that of Mn1, indicating a higher affinity of the substrate for Mn2 compared with Mn1. It was revealed that the active EF‐hand loops 1 and III in Mn2 is locally more rigid compared with those in Mn1. We concluded that different stability of the photoprotein complexes leads to different initial intensity. While different patterns of the local dynamics of loops I and III may influence the decay rate. [ABSTRACT FROM AUTHOR]

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]

The bright bioluminescence of ctenophores inhabiting the oceans worldwide is caused by light-sensitive Ca2+-regulated photoproteins. By now, the cDNAs encoding photoproteins from the four different ctenophore species have been cloned and the recombinant proteins have been characterized to some extent. In this work, we report on the specific activity and the quantum yield of bioluminescence reaction as well as the absorbance characteristics of high-purity recombinant berovin. To determine those, we applied the amino acid composition analysis to accurately measure berovin concentration and the recombinant aequorin as a light standard to convert relative light units to quanta. The extinction coefficient of 1% berovin solution at 435 nm was found to be 1.82. The one can be employed to precisely determine the protein concentration of active photoproteins from other ctenophore species. The specific activity and the bioluminescence quantum yield were respectively found to be 1.98 × 1015 quanta/mg and 0.083. These values appeared to be several times lower than those of the cnidarian photoproteins, which is obviously due to differences in amino acid environments of the substrate in active sites of these photoproteins. [Display omitted] • Berovin specific activity in quanta/mg and reaction quantum yield were determined. • Extinction coefficient at 435 nm for high-purity berovin was determined. • Specific activity and quantum yield are lower than those of cnidarian photoproteins. • Distinctions of these parameters are caused by differences in active site structures. [ABSTRACT FROM AUTHOR]

We designed two mutants of photoprotein Mnemiopsin 2 (Mn2) including M52I and V144I, where the mutations were applied in the EF-hand loops I and III. Far-UV CD measurements demonstrated that the stability of the helices in the wild-type (WT) protein is greater compared with the mutants. Heat-induced denaturation experiments in the apo-form of photoproteins showed that WT Mn2 has higher value of the enthalpy change for the unfolding process, indicating that it has more stabilizing interaction compared with mutants. According to the activity measurement data, both mutants, particularly V144I have lower initial intensity as well as slower decay rate as compared with the WT photoprotein. Importantly, it was found that V144I variant shows 25 nm of red shift in the characteristic wavelengths as compared with the WT photoprotein. This finding can be considered as an advantage for in vivo application of photoprotein for imaging purposes. It concluded that this position on loop III of Mn2 is a hotspot point for characteristic wavelength determination. However, further research on this mutant is needed for making stable variants of Mn2 with novel optical features. [ABSTRACT FROM AUTHOR]

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.

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]

The mechanism of bioluminescence in the luminous land snails remains largely unknown. Here, we analyzed basic biochemical properties of the luminous land snail, Quantula weinkauffiana. The luminescence activity was extracted from the light organ located near the mouth using a neutral buffer containing detergent. The reaction of the crude buffer extract was triggered by the addition of only hydrogen peroxide (H 2 O 2 ). These results are inconsistent with the single precedent report on the bioluminescence in the Quantula striata by Shimomura and Haneda in 1986, in which the luminescence of the buffer extract (without detergent) from the light organ was induced by the coaddition of three indispensable components H 2 O 2 , ferrous ion, and 2-mercaptoethanol. Based on the present findings, we suggested that an insoluble photoprotein is involved in the bioluminescence of the luminous land snails and the luminescence reaction is simply triggered by H 2 O 2 ., (© 2024 John Wiley & Sons Ltd.)

During a search for bioluminescent marine annelids around the island of Carrie Bow Cay, Belize, we discovered bioluminescence in the sipunculan worm Nephasoma pellucidum. The identification of N. pellucidum was verified by DNA barcoding of ∼650 bp of the mitochondrial cytochrome c oxidase I gene, using sequences of the same species from previous studies as references. Our study constitutes the first record of a bioluminescent sipunculan species. Green luminescence was detected in four of the five individuals of N. pellucidum collected from coral rubble from a shallow reef flat and one deeper (14 m) site. To test for bioluminescent activity, all collected annelids (including sipunculans) were sorted into morphotypes and identified to the lowest taxonomic level possible. After several hours of dark adaptation, they were mechanically stimulated, and luminescent reactions were noted and recorded by photography and/or video whenever possible. Of a total of nine examined sipunculan species, N. pellucidum was the only one that showed bioluminescent activity. The underlying biochemical processes for the bioluminescence are so far unknown, but transcriptome data indicate the presence of proteins with similarities to Renilla-type luciferases. We did not find similarities to any known photoproteins. Our findings broaden the known diversity of bioluminescent annelid taxa and support the notion that bioluminescence arose multiple times in annelids. The bioluminescent properties of N. pellucidum add a new reason to pursue this species as a model species, as has been previously proposed.

Bioluminescence is widespread throughout the phylum Annelida and occurs in terrestrial and marine lineages. Among marine taxa, bioluminescence has been documented in eight families and anecdotally reported in six additional families. Although new bioluminescent systems have been recently described in annelids, there are still many other families whose light emission mechanisms have not been sufficiently studied. Some of these include luminescent species belonging to the Polynoidae family, also known as scale worms, whose iterations of dorsal elytra (scales) have the ability to emit intense light when stimulated. Depending on the degree of stimulation, some polynoids can autotomize these luminous elytra and posterior segments, which could potentially give them an advantage in evading attacks by predators. It is believed that Polynoidae bioluminescence is associated with a membrane enzyme known as “polynoidin,” which was isolated during the early 1980s from Malmgrenia lunulata. However, the characterization and properties of this enzyme, as well as the chemical nature of its substrate or additional potential cofactors, have never been fully described and remain largely unknown. As such, this paper seeks to revisit previous research involving bioluminescence studies in Polynoidae, as well as the morphological, phylogenetic and ecological aspects related to this emission of light.

We used site-specific mutagenesis by targeting E179 and F190 on the structure of photoprotein Mnemiopsin 2 (Mn2) from Mnemiopsis leidyi. The tertiary structure of E179S and F190L mutants was made by the MODELLER program. Far-ultraviolet circular dichroism data showed that the overall secondary structural content of photoprotein is not changed upon mutation, however the helicity and stabilizing interactions in helical structure decreases in mutants as compared with the wild-type (WT) photoprotein. Fluorescence spectra data revealed that the tertiary structure of the mutants is more compact than that of WT Mn2. According to the heat-induced denaturation experiments data, the melting temperature (T m ) for the unfolding of tertiary structure of the F190L variant increases by 3°C compared with that of the WT and E179S mutant. Interestingly, the conformational enthalpy of the F190L mutant (86 kcal mol -1 ) is considerably lower than those in the WT photoprotein (102 kcal mol -1 ) and E179S mutant (106 kcal mol -1 ). The significant difference in the enthalpy of the thermal unfolding process could be explained by considering that the thermally denatured state of the F190L mutant is structurally less expanded than the WT and E179S variants. Bioluminescence activity data showed that the maximum characteristic wavelengths of the mutants undergo blue shift as compared with the WT protein. Initial intensity of the F190L and E179S variants was recorded to be 137.5% and 55.9% of the WT protein, respectively., (© 2024 John Wiley & Sons Ltd.)

Светоизлучающие белки – люциферазы и фотопротеины – широко используются как репортеры для высокочувствительного молекулярного анализа in vitro и in vivo. Соединение (конъюгирование) люциферазы с молекулами, обладающими аффинностью к определенным мишеням (иммуноглобулинами, олигонуклеотидами, гаптенами и пр.), обеспечивает специфичность анализа. Такие молекулы (биоспецифические биолюминесцентные метки) получают химическим синтезом на основе различных коммерчески доступных реагентов, которые взаимодействуют с функциональными группами на поверхности молекул. Однако такой синтез не может обеспечить получение гомогенного аддукта, вызывает потерю (иногда значительную) функциональной активности «сшиваемых» молекул. Как правило, при получении конъюгатов химическим способом проводят длительные поиски условий синтеза (соотношение реагентов, рН и температура среды, время проведения анализа и т.п.), которые позволяют максимально уменьшить негативные последствия влияния химических реагентов и при этом иметь удовлетворительный выход целевых конъюгатов. Другим способом получения биоспецифических меток является создание гибридных молекул с помощью генетической инженерии. Для этого получают генетическую конструкцию, в которой в одной рамке считывания последовательно находятся гены, кодирующие биоспецифический и репортерный белки, а также последовательности, кодирующие соединительный линкер, и часто различные вспомогательные полипептиды. Генетический фьюзинг обеспечивает доступную бактериальную экспрессию, гомогенность гибридных молекул, простые способы очистки с помощью вспомогательных полипептидных фрагментов и т.д. В данной работе описан ряд гибридов «люцифераза–биоспецифический белок», созданных в лаборатории фотобиологии Института биофизики СО РАН, их дизайн, получение и применение в качестве биоспецифических биолюминесцентных меток в микроанализе

Terebelliformia is a benthic group of marine annelid worms. The bioluminescence of several species has been reported in taxonomical and histological literature, but very little information is known about the biochemical aspects of this phenomenon. In this study, we examined the basic properties of the luminescence system using an extract of the Japanese terebelliform worm, Thelepus japonicus. The bioluminescence extract was soluble in water, and emitted blue‐green light at λmax 508 nm following the addition of divalent cations. This triggering action was highly specific to Fe2+ and addition of ATP, H2O2 or coelenterazine did not enhance activity. The bioluminescence was inactivated by heat treatment and organic solvents, indicating the involvement of a protein component. These results suggested that Thelepus worm produces light using a novel system that differs from that in other known luminescent annelids. [ABSTRACT FROM AUTHOR]

Abstract To validate the use of recombinant aequorin (reAequorin) as a light emission standard, the protein concentrations of highly purified reAequorin were determined by amino acid composition analysis, and the presence of active reAequorin was confirmed by the ratio of absorbance peak at 460 nm to that at 280 nm. The high correlation of the luminescence intensity with the protein concentration showed that reAequorin could be used for a light emission standard to study the luminescence properties of luciferases and to evaluate the detection sensitivity of luminometers. The specific activity of Gaussia luciferase with I max was 7.5-fold higher than that of reAequorin and was calculated to be 3.8 × 1016 quanta/mg protein. Highlights • Recombinant aequorin (reAequorin) was evaluated for use as a light emission standard. • reAequorin protein concentration was determined by amino acid composition analysis. • The presence of active reAequorin was confirmed by the absorption peak ratio at 280/460 nm. • The I max value of Gaussia luciferase was 7.5-fold higher than that of reAequorin. • reAequorin can generally be used as a light emission standard. [ABSTRACT FROM AUTHOR]

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.

Background: Nowadays photoproteins are excellent reporter systems as they have virtually no background. The most commonly studied photoprotein is aequorin. But because of it’s low quantum yield and very fast reaction kinetic this photoprotein assay don’t adapt for High-Throughput Screening (HTS). Consequently, some researchers developed three improved photoproteins optimized for the generation of precise Ca2+ mobilization assays photina, i-photina and c-photina. Although the total light release of these photoproteins is greater and their reaction kinetic is slower compared to other existing photoproteins, opening new opportunities for the application of flash luminescence assays in HTS. Recently, the three photoproteins by several companies such as PerkinElmer and Axxam have been commercialized. So, we selected i-photina which has the highest luminescence signal and good stability compared with two other photoproteins. Materials and Methods : Codon usage of i-Photina was optimized for both E. coli and mammalian cells. The encoding gene of i-photina was synthesized and overexpressed in E. coli. Then i-Photina was characterized and compared with aequorin. Results: i-Photina showed about a 14-fold higher bioluminescence signal than aequorin. Ca2+ sensivity of i-Photina was found to be slightly less than aequorin. In respect to other measured properties such as degree of stability, bioluminescence spectrum and decay half-life time, these two photoproteins were almost the same. Conclusion: i-Photina is an improved version of photoprotein that in many ways is superior to use of other Ca2+ indicators for HTS assays and can also be considered as a potential candidate for creating genetic mutations improved the bioluminescence propertises.

Mnemiopsin 1 (Mn1) and Mnemiopsin 2 (Mn2) are photoproteins found in Mnemiopsis leidyi. We have tried to answer the question of whether the structural features of photoproteins can explain the observed activity data. According to the activity measurements data, they have the same characteristic wavelength. However, the initial intensity of Mn2 is significantly higher than that of Mn1, and decay time of Mn1 (0.92 s -1 ) is lower than that of Mn2 (1.46 s -1 ). The phylogenetic analysis demonstrates that, compared with Obelin and Aequorin from Obelia longissima and Aequorea victoria, respectively, a gene modification event may have caused the expansion of the N-terminal side of all photoproteins from M. leidyi. An in silico study has shown that the stability of the photoprotein-substrate complex of Mn2 is higher than that of Mn1, indicating a higher affinity of the substrate for Mn2 compared with Mn1. It was revealed that the active EF-hand loops 1 and III in Mn2 is locally more rigid compared with those in Mn1. We concluded that different stability of the photoprotein complexes leads to different initial intensity. While different patterns of the local dynamics of loops I and III may influence the decay rate., (© 2023 John Wiley & Sons Ltd.)

Abstract Photoproteins in their functional form are complexed noncovalently with 2-hydroperoxycoelenterazine. A conformational change upon coordination of Ca+2 ions with their EF-hand loops leads to oxidation of substrate and emission of light. In all photoproteins, EF-hand loops Ι, ΙΙΙ and ΙV have standard sequence for binding to Ca+2 ion, however the second one is not able for Ca+2 coordination. Sequence analysis of Mnemiopsin 2 and other known photoproteins shows that Glutamate (Glu) is occurred in the 6th position of its first EF-hand loop, but this position in other loops of mnemiopsin 2 and all functional loops of other photoproteins is occupied by Glycine (Gly). Here we designed and made single and double mutants where Gly residue at the 6th positions of loops ΙΙΙ and ΙV of mnemiopsin 2 was replaced with Glu. According to the activity measurements, wild-type (WT) and G142E variants have more initial luminescence intensity than G176E and double mutants; while WT and G176E have higher values of half decay time when compared with G142E and double mutants. According to the isothermal denaturation experiments, all protein variants are structurally more stable than WT mnemiopsin 2 and that the stabilizing effects of single mutants are paired resulting in more stability of double mutant against urea denaturation. We concluded that simultaneous occurrence of Gly in the 6th position of loops ΙΙΙ and ΙV is essential for evolutionary adjustment of initial intensity and decay rate of luminescence emission via affecting the interaction of the core structure of photoprotein with coelenteramide and binding affinity of Ca+2 to the corresponding loops, respectively. Highlights • Glu residue is occurred in the 6th position of the first EF-hand loop of mnemiopsin. • This position in other known photoproteins is occupied by Gly. • Occurrence of Gly in loops ΙΙΙ and ΙV is essential for adjustment of initial intensity and decay rate. [ABSTRACT FROM AUTHOR]

cf3 -Aequorin is one of the semi-synthetic aequorins that was produced by replacing 2-peroxycoelenterazine (CTZ-OOH) in native aequorin with a 2-peroxycoelenterazine analog, and it was prepared using the C2-modified trifluoromethyl analog of coelenterazine (cf3 -CTZ) and the histidine-tagged apoaequorin expressed in Escherichia coli cells. The purified cf3 -aequorin showed a slow luminescence pattern with half-decay time of maximum intensities of luminescence of 5.0 s. This is much longer than that of 0.9 s for native aequorin, and its luminescence capacity was estimated to be 72.8% of that of native aequorin. The crystal structure of cf3 -aequorin was determined at 2.15 Å resolution. The light source of 2-peroxytrifluoromethylcoelenterazine (cf3- CTZ-OOH) was stabilized by the hydrogen-bonding interactions at the C2-peroxy moiety and the p -hydroxy moiety at the C6-phenyl group. In native aequorin, three water molecules contribute to stabilizing CTZ-OOH through hydrogen bonds. However, cf3 -aequorin only contained one water molecule, and the trifluoromethyl moiety at the C2-benzyl group of cf3 -CTZ-OOH interacted with the protein by van der Waals interactions. The slow luminescence kinetics of cf3 -aequorin could be explained by slow conformational changes due to the bulkiness of the trifluoromethyl group, which might hinder the smooth cleavage of hydrogen bonds at the C2-peroxy moiety after the binding of Ca2+ to cf3 -aequorin. [ABSTRACT FROM AUTHOR]

Objective: Assessment of caries lesion activity is usually performed using visual-tactile criteria. A new approach is to use a system consisting of a photoprotein, which is specific for free calcium ions, along with an integrated camera that visualizes the elevated calcium ions on the lesion as a light signal (bioluminescence). This study aimed to evaluate the reproducibility and diagnostic accuracy of a newly developed bioluminescence system to assess caries lesion activity on occlusal surfaces in vitro.Materials and Methods: Ninety-four extracted permanent teeth with sound surfaces and both active and inactive caries lesions were included. The extent and activity of the investigation sites were classified visually according to the International Caries Detection and Assessment System. Digital images of the teeth were produced by the bioluminescence system and the images were analyzed for the presence of caries activity (the bioluminescent areas at the investigation sites). The images were reanalyzed after 4 weeks. Teeth were hemisected and methyl red dye was applied on the sections to validate lesion activity histologically.Results: Agreement between the bioluminescence readings was shown by κ values of 0.802-0.917. A significant positive correlation (p < 0.001) was found across all methods (rs 0.618-0.811). Sensitivity and specificity for activity assessment using histology as the gold standard (cut-off: active/inactive) were, respectively, 83.6 and 85.0% for the visual assessment, and 92.5 and 90.0% for the bioluminescence method. Comparison of areas under the receiver-operating characteristic curves showed no significant difference between the visual and bioluminescence systems (p = 0.094).Conclusion: The bioluminescence system demonstrated high in vitro reproducibility and good diagnostic accuracy for activity assessment of caries lesions on occlusal surfaces which were not significantly different from the values obtained in the visual assessment. [ABSTRACT FROM AUTHOR]

As a Ca 2+ -regulated photoprotein, aequorin (Aeq) contains four EF-hand motifs, the second one lacks the standard sequence for Ca 2+ coordination and doesn't bind to Ca 2+ . Here, we replaced this loop with a functional loop. According to structural studies, although the global stability of modified aequorin (4EFAeq) is higher than that of Aeq; increasing the local flexibility accompanied by internal structural rearrangements in 4EFAeq result in its penetrability to urea and acrylamide. A fast decay rate was observed for 4EFAeq. Assuming the presence of intermediate states in the luminescent reaction, this observation indicate that the loop replacement leads to the lowering of the half-life of intermediate states which results in increasing the rate of conformational switching of 4EFAeq to light emitting form. However, considerable reduction in initial luminescence intensity of 4EFAeq suggests that the number of functional complexes is reduced. Our findings demonstrate that the conformational effects of the second loop in Aeq elicit a delicate balance between local flexibility and global stability which may be considered as an important functional parameter in photoproteins. It was also concluded that evolutionary conservation of EF-hand ΙΙ in the current form is a consequence of priority of intensity to decay rate in bioluminescent organisms. [ABSTRACT FROM AUTHOR]

Bioluminescence of a variety of marine coelenterates is determined by Ca 2+ -regulated photoproteins. A strong interest in these proteins is for their wide analytical potential as intracellular calcium indicators and labels for in vitro binding assays. The presently known hydromedusan Ca 2+ -regulated photoproteins contain three (aequorin and clytin) or five (obelin and mitrocomin) cysteine residues with one of them strictly conserved. We have constructed Cys-free aequorin and obelin by substitution of all cysteines to serine residues. Such mutants should be of interest for researchers by the possibility to avoid the incubation with dithiothreitol (or β-mercaptoethanol) required for producing an active photoprotein that is important for some prospective analytical assays in which the photoprotein is genetically fused with a target protein sensitive to the reducing agents. Cys-free mutants were expressed in Escherichia coli , purified, and characterized regarding the efficiency of photoprotein complex formation, functional activity, and conformational stability. The replacement of cysteine residues has been demonstrated to affect different properties of aequorin and obelin. Cys-free aequorin displays a two-fold lower specific bioluminescence activity but preserves similar activation properties and light emission kinetics compared to the wild-type aequorin. In contrast, Cys-free obelin retains only ~ 10% of the bioluminescence activity of wild-type obelin as well as binding coelenterazine and forming active photoprotein much less effectively. In addition, the substitution of Cys residues drastically changes the bioluminescence kinetics of obelin completely eliminating a “fast” component from the light signal decay curve. At the same time, the replacement of Cys residues increases conformational flexibility of both aequorin and obelin molecules, but again, the effect is more prominent in the case of obelin. The values of thermal midpoints of unfolding (T m ) were determined to be 53.3 ± 0.2 and 44.6 ± 0.4 °C for aequorin and Cys-free aequorin, and 49.1 ± 0.1 and 28.8 ± 0.3 °C for obelin and Cys-free obelin, respectively. Thus, so far only Cys-free aequorin is suitable as a partner for fusing with a tag sensitive to reducing agents since the aequorin mutant preserves almost 50% of the bioluminescent activity and can be produced with a substantial yield. [ABSTRACT FROM AUTHOR]

Mnemiopsin, an EF-hand Ca 2+ binding photoprotein isolated from luminous ctenophore Mnemiopsis leidyi , emits blue light from its chromophore, coelenterazine, which is non-covalently bond in its central hydrophobic core. Previous studies have revealed unique biochemical properties for ctenophore photoproteins such as inactivation by light, but only few have focused on photoinactivation process. To understand the nature of photoinactivation process we have investigated the impact of light alone and in the presence of Ca 2+ ion on the structure of this photoprotein. We used UV–Vis, circular dichroism (CD) and fluorescence spectroscopy following Ca 2+ binding assay to analyze the light effects on mnemiopsin conformation in comparison with aequorin at both apo and holo form. Our results showed light induced structural changes which resulted into photoinactivation. These changes include significant modification on secondary structure of mnemiopsin in comparison with aequorin. Our data also revealed that light could influence structure of apo protein regardless of presence of coelenterazine. The comparative studies of Ca 2+ ion binding affinity following light exposure, also showed that light induced structural changes could presumably affect coelenterazine binding or its conformation in binding site in such a way that causes photoinactivation. In conclusion, we have proposed that structural rearrangement of helix 5 and C-terminal motif could be responsible for light induced structural changes. [ABSTRACT FROM AUTHOR]

The protein expressions of three preferred human codon-optimized Gaussia luciferase genes ( pGLuc , EpGLuc , and KpGLuc ) were characterized in mammalian and bacterial cells by comparing them with those of wild-type Gaussia luciferase gene ( wGLuc ) and human codon-optimized Gaussia luciferase gene ( hGLuc ). Two synthetic genes of EpGLuc and KpGLuc containing the complete preferred human codons have an artificial open-reading frame; however, they had the similar protein expression levels to those of pGLuc and hGLuc in mammalian cells. In bacterial cells, the protein expressions of pGLuc , EpGLuc , and KpGLuc with approximately 65% GC content were the same and showed approximately 60% activities of wGLuc and hGLuc . The artificial open-reading frame in EpGLuc and KpGLuc did not affect the protein expression in mammalian and bacterial cells. [ABSTRACT FROM AUTHOR]

The binding properties of Ca2+ to EF-hand I of aequorin (AQ) were characterized by replacing the loop sequence of EF-hand I (AQ[I]) with other known loop sequences of Ca2+-binding proteins, including photoproteins (aequorin, clytin-I, clytin-II and mitrocomin), Renilla luciferin-binding protein (RLBP) and calmodulin (CaM). For evaluation of the binding affinity of Ca2+ to AQ[I] mutants, the half-decay time of the maximum intensity in the luminescence reaction triggered by Ca2+ was used as an indicator and 22 kinds of AQ[I] mutants were expressed in Escherichia coli cells. AQ[I] mutants replaced with the EF-hand I and EF-hand III from photoproteins showed sufficient luminescence activity, but it was not shown by other EF-hands from RLBP and CaM. An AQ[I] mutant with a lysine or arginine residue at the second position of the non-conserved amino acid residue showed a slow-decay pattern of luminescence, indicating that the Ca2+-binding affinity to aequorin was reduced by a positive charge at the second position of the loop sequence. The specific loop sequence of the EF-hand I motif in aequorin caused the specific Ca2+-triggered luminescence pattern. [ABSTRACT FROM AUTHOR]

Mnemiopsin 1 is a member of photoprotein family, made up of 206 amino acid residues. These Ca 2 + -regulated photoproteins are responsible for light emission in a variety of marine cnidarians and ctenophores. They composed of an apoprotein, a single polypeptide chain of 25 kDa, molecular oxygen and the non-covalently bound chromophore. In this study, we examined whether three mutations, namely R39K, S128G and V183T affect the thermodynamic stability as well as refolding and unfolding kinetics of mnemiopsin 1. Conformational stability measurements using fluorescence and far-UV CD spectroscopies revealed that all variants unfold in multi-step manner in which the secondary and tertiary structures are lost in different steps. However kinetic studies showed that point mutation S128G destabilizes both kinetic intermediate and native conformation; while, these structural elements are stabilized in V183T. We also found that the stability of folded and intermediate states increases in R39K. We concluded that the initial packing of helical segments within the protein structure is more facilitated when Lys with smaller side chain is present in the protein chain. [ABSTRACT FROM AUTHOR]

Abstracts Clytin-II (CL-II) is an isotype of the calcium-binding photoprotein clytin-I (CL-I) from Clytia gregaria . CL-II shows approximately 4.5-fold higher initial luminescence intensity than CL-I with the same luminescence capacity, and is a potential candidate for a G-protein-coupled receptor assay among photoproteins. To investigate the high initial luminescence intensity of CL-II, the chimeric proteins between CL-I and CL-II were prepared and the responsible amino acid residues in CL-II were identified by site-specific mutagenesis of CL-I. The luminescence properties of CL-I were converted to those of CL-II by the replacement of only four amino acids in CL-I, and these amino acids did not interact with 2-peroxycolenterazine. [ABSTRACT FROM AUTHOR]