Determination and evaluation of secondary structure content derived from calcium-induced conformational changes in wild-type and mutant mnemiopsin 2 by synchrotron-based Fourier-transform infrared spectroscopy

Mnemiopsin 2 from a luminous ctenophore with two functional EF-hand motifs is a calcium-regulated photoprotein that is responsible for emitting a bright blue bioluminescence upon reacting with coelenterazine and calcium ions in Mnemiopsis leidyi. Synchrotron radiation-based Fourier-transform infrared (SR-FTIR) spectroscopy was applied to analyze the distribution of secondary structures, the conformational changes resulting from calcium binding and the structural stabilities in wild-type mnemiopsin 2, as well as its mutant type that possesses three EF-hand motifs. The distribution of secondary structures of these proteins indicates that mutant apo-mnemiopsin 2 has a more stable secondary structure than the wild-type. Analyses of the SR-FTIR spectra revealed that the conformational changes at the secondary structures of both mnemiopsin 2 depend on the calcium concentrations, such that the most noticeable changes in structures of wild-type and mutant mnemiopsin 2 occur at optimum concentrations 6 and 2 mM of calcium chloride, respectively. The addition of calcium to both proteins increases the proportions of their secondary structures in the amide I and II regions. The major amide I bands in the IR spectra of both mnemiopsin‑calcium complexes shift towards smaller wavenumbers, whereas their main amide II bands are identified at larger wavenumbers.     

Binding of murine monoclonal antibodies to the active and inactive configurations of aequorin

Murine monoclonal IgG1 antibodies (MAb), designated Aq-11 and Aq-12, were prepared against the photoprotein aequorin from jelly fish. Aequorin is a calcium-sensitive photoprotein which consists of a single polypeptide chain, apoaequorin, and a functional chromophore, coelenterazine. Native aequorin consists of two species with molecular masses of 25 and 23.5 kDa. MAb Aq-12 was found by immunoblot analysis to bind specifically to the 25 kDa species, while MAb Aq-11 reacted with the 23.5 kDa protein. Activation of apoaequorin with coelenterazine was associated with a shift of the 23.5 kDa molecule to the 25 kDa species. In contrast, treatment with calcium ions induced a shift back to the 23.5 kDa form. These changes between the active and inactive forms were identified by reactivity with MAbs Aq-11 and Aq-12. The results thus indicate that these MAbs should be useful in monitoring activation of this photoprotein.     

Overexpression and purification of the recombinant Ca2+-binding protein, apoaequorin

The small, monomeric Ca2+-binding photoprotein, aequorin, emits blue light by an intramolecular reaction when mixed with Ca2+. The photoprotein is made up of coelenterazine and molecular oxygen, bound noncovalently to apoaequorin (apoprotein). The chemical steps leading to light emission, involving the oxidative degradation of coelenterazine, have been studied extensively, but little is known about the active site and how the molecule catalyzes the oxidation of coelenterazine. The three-dimensional structure of the protein has not been determined and therefore answers to these questions have remained unavailable. The present paper describes a procedure for preparing fairly large amounts of apoaequorin and aequorin for X-ray crystallographic studies. It consists of fusing the apoaequorin cDNA to the signal peptide coding sequence of the outer membrane protein A of Escherichia coli, which is under the control of the lipoprotein promoter. When the cDNA was expressed in E. coli, a large excess of the recombinant protein was produced and released into the culture medium. Purification of the protein was accomplished by acid precipitation and DEAE-cellulose chromatography. The procedure yielded 7.4 mg of recombinant apoaequorin with a purity greater than 95% from 200 ml of culture medium. On regeneration with coelenterazine, the recombinant aequorin was fully active with Ca2+.     

Semi-synthetic aequorin. An improved tool for the measurement of calcium ion concentration.

The photoprotein aequorin isolated from the jellyfish Aequorea emits blue light in the presence of Ca2+ by an intramolecular process that involves chemical transformation of the coelenterazine moiety into coelenteramide and CO2. Because of its high sensitivity to Ca2+, aequorin has widely been used as a Ca2+ indicator in various biological systems. We have replaced the coelenterazine moiety in the protein with several synthetic coelenterazine analogues, providing semi-synthetic Ca2+-sensitive photoproteins. One of the semi-synthetic photoproteins, derived from coelenterazine analogue (II) (with an extra ethano group), showed highly promising properties for the measurement of Ca2+, namely (1) the rise time of luminescence in response to Ca2+ was shortened by approx. 4-fold compared with native aequorin and (2) the luminescence spectrum showed two peaks at 405 nm and 465 nm and the ratio of their peak heights was dependent on Ca2+ concentration in the range of pCa 5-7, thus allowing the determination of [Ca2+] directly from the ratio of two peak intensities. Coelenterazine analogue (I) (with a hydroxy group replaced by an amino group) was also incorporated into apo-aequorin, yielding a Ca2+-sensitive photoprotein, which indicates that an electrostatic interaction between the phenolate group in the coelenterazine moiety and some cationic centre in apo-aequorin is not important in native aequorin, contrary to a previous suggestion.     

The intrinsic fluorescence of apo-obelin and apo-aequorin and use of its quenching to characterize coelenterazine binding

The intrinsic fluorescence of two apo-photoproteins has been characterized and its concentration-dependent quenching by coelenterazine has been for the first time applied to determine the apparent dissociation constants for coelenterazine binding with apo-aequorin (1.2 ± 0.12 μM) and apo-obelin (0.2 ± 0.04 μM). Stopped-flow measurements of fluorescence quenching showed that coelenterazine binding is a millisecond-scale process, in contrast to the formation of an active photoprotein complex taking several hours. This finding evidently shows that the rate-limiting step of active photoprotein formation is the conversion of coelenterazine into its 2-hydroperoxy derivative.     

Blue fluorescent protein from the calcium-sensitive photoprotein aequorin: catalytic properties for the oxidation of coelenterazine as an oxygenase

Blue fluorescent protein from the calcium-binding photoprotein aequorin (BFP-aq) is a complex of Ca2+ -bound apoaequorin and coelenteramide, and shows luminescence activity like a luciferase, catalyzing the oxidation of coelenterazine with molecular oxygen. To understand the catalytic properties of BFP-aq, various fluorescent proteins (FP-aq) have been prepared from semi-synthetic aequorin and characterized in comparison with BFP-aq. FP-aq has luciferase activity and could be regenerated into native aequorin by incubation with coelenterazine. The results from substrate specificity studies of FP-aq using various coelenterazine analogues have suggested that the oxidation of coelenterazine by BFP-aq in the luciferase reaction and the regeneration process to aequorin might involve the same catalytic site of BFP-aq.     

Coelenterazine is a superoxide anion-sensitive chemiluminescent probe: its usefulness in the assay of respiratory burst in neutrophils.

The oxidation of free coelenterazine by superoxide anion was analyzed and compared to the oxidation by the semisynthetic photoprotein obelin, prepared by incorporation of synthetic coelenterazine into apoobelin. The oxidation of bound coelenterazine was triggered upon binding of calcium to the reconstituted photoprotein. The oxidation of free synthetic coelenterazine, in the absence of the apoprotein, was triggered by superoxide anion. The production of reactive oxygen metabolites by fMet-Leu-Phe- and 4b-phorbol 12b-myristate 13a-acetate-stimulated neutrophils was studied by means of the luminescence of synthetic coelenterazine. The features of this chemiluminescent probe were compared with those of luminol and are summarized as follows: (a) coelenterazine-dependent chemiluminescence was inhibited by superoxide dismutase; (b) coelenterazine was as sensitive as luminol in detecting the oxidative burst of neutrophils; (c) azide failed to inhibit coelenterazine chemiluminescence; (d) in contrast with luminol, which requires the catalytic removal of hydrogen peroxide, coelenterazine chemiluminescence did not depend on the activity of cell-derived myeloperoxidase. These results indicate the usefulness of coelenterazine as a very sensitive and specific chemiluminescence probe of superoxide anion.     

The properties of mnemiopsin,a bioluminescent and light sensitive protein purified by hollow fiber techniques

Summary A calcium activated photoprotein, termed mnemiopsin, which emits bioluminescence upon the addition of calcium ion, has been isolated from the Ctenophore, Mnemiopsis leidyi, and purified by hollow fiber techniques. The system is similar to aequorin, from the jellyfish Aequorea, except that mnemiopsin can be light-inactivated. Separation of mnemiopsin from the dilute and large volume animal homogenate proved difficult with conventional biochemical techniques. A continuous flow process utilizing large surface area hollow fibers for filtration, concentration, and dialysis was developed which may also be applicable to the purification of other proteins. The resulting mnemiopsin concentrate, after further purification, was judged to be about 90% pure by its gel electrophoretic profile. Estimates by molecular sieve chromatography and SDS gel electrophoresis gave a molecular weight of about 23,000 daltons. The calcium specificity for triggering light emission was studied by comparison of triggering with a variety of cations and anions and by investigating the effects of calcium ionophores and antagonists. The activity of mnemiopsin was characterized with respect to pH, temperature and ionic strength. The stability of mnemiopsin activity after exposure to proteases, denaturants, protein group-specific reagents, detergents, elevated temperatures and light was determined.     

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

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

The crystal structures of semi-synthetic aequorins

The photoprotein aequorin emits light by an intramolecular reaction in the presence of a trace amount of Ca(2+). Semi-synthetic aequorins, produced by replacing the coelenterazine moiety in aequorin with the analogues of coelenterazine, show widely different sensitivities to Ca(2+). To understand the structural basis of the Ca(2+)-sensitivity, we determined the crystal structures of four semi-synthetic aequorins (cp-, i-, br- and n-aequorins) at resolutions of 1.6-1.8 A. In general, the protein structures of these semi-synthetic aequorins are almost identical to native aequorin. Of the four EF-hand domains in the molecule, EF-hand II does not bind Ca(2+), and the loop of EF-hand IV is clearly deformed. It is most likely that the binding of Ca(2+) with EF-hands I and III triggers luminescence. Although little difference was found in the overall structures of aequorins investigated, some significant differences were found in the interactions between the substituents of coelenterazine moiety and the amino acid residues in the binding pocket. The coelenterazine moieties in i-, br-, and n-aequorins have bulky 2-substitutions, which can interfere with the conformational changes of protein structure that follow the binding of Ca(2+) to aequorin. In cp-aequorin, the cyclopentylmethyl group that substitutes for the original 8-benzyl group does not interact hydrophobically with the protein part, giving the coelenterazine moiety more conformational freedom to promote the light-emitting reaction. The differences of various semi-synthetic aequorins in Ca(2+)-sensitivity and reaction rate are explained by the capability of the involved groups and structures to undergo conformational changes in response to the Ca(2+)-binding.     

Cloning,Sequencing, Expression and Structural Investigation of Mnemiopsin from <Emphasis Type="Italic">Mnemiopsis leidyi</Emphasis>: An Attempt Toward Understanding Ca<Superscript>2+</Superscript>-Regulated Photoproteins

A comparison of the two most famous groups of calcium-regulated photoproteins, cnidarians and ctenophores, showed unexpectedly high degree of structural similarity regardless of their low sequence identity. It was suggested these photoproteins can play an important role in understanding the structural basis of bioluminescence activity. Based on this postulate, in this study the cDNA of mnemiopsin from luminous ctenophore Mnemiopsis leidyi was cloned, expressed, purified and sequenced. The purified cDNA, with 621 base pairs, coded a 206 residues protein. Sequence of mnemiopsin showed 93.5 and 51% similarity to other ctenophore proteins and cnidarians, respectively. The cDNA encoding apo-mnemiopsin of M. leidyi was expressed in Escherichia coli. The purified apo-protein showed a single band on SDS-PAGE (molecular weight ~27 kDa). A semi-synthetic mnemiopsin was prepared using coelenterazine and EDTA and its luminescence activity was measured in the presence of CaCl₂. The results showed an optimum pH of 9.0 and lower calcium sensitivity compared to aequorin. Comparison of amino acid residues in substrate binding site indicated that binding pocket of ctenophores contains less aromatic residues than cnidarians. This can lead to a decline in the number of stacking interactions between substrate and protein which can affect the stability of coelenterazine in binding cavity. Structural comparison of photoproteins with low sequence identity and high 3D structural similarity, can present a new insight into the mechanism of light emission in photoproteins.     

Glowing jellyfish, luminescence and a molecule called coelenterazine

Luminescence has assumed an important role in analytical biochemistry and molecular biology as an extremely sensitive method for determining the concentration of specific ions and molecules. The luminescent system of the jellyfish Aequorea victoria consists of the photoprotein aequorin, which contains the molecule coelenterazine as a prosthetic group and shows considerable potential in this area.     

Expression, purification and characterization of a photoprotein, clytin, from Clytia gregarium

A novel histidine-tagged secretion vector in Escherichia coli was constructed and large amounts of highly pure clytin, a calcium-binding photoprotein, was prepared. The histidine-tagged apoclytin expressed into the periplasmic space in E. coli was purified by nickel chelate affinity chromatography. Recombinant clytin was regenerated from apoclytin by incubation with coelenterazine in the presence of dithiothreitol and then purified by anion-exchange chromatography and hydrophobic chromatography. The yield of recombinant clytin was 20mg from 2L of cultured cells with purity greater than 95%. Luminescence properties of recombinant clytin were identical to that of native clytin (phialidin). The Ca(2+) sensitivity of recombinant clytin is lower than that of aequorin and clytin is suited for measuring higher concentration of Ca(2+). Semi-synthetic clytins were also prepared with coelenterazine analogues, and the initial intensity, luminescence capacity and half decay time were characterized.     

Bioluminescence and secondary structure properties of aequorin mutants produced for site-specific conjugation and immobilization

Aequorin is one of several photoproteins that emits visible light upon binding to calcium ions. It has been widely used as a Ca(2+)-indicator and as an alternative highly sensitive bioluminescent label in binding assays. The apoprotein of aequorin binds an imidazopyrazine compound (coelenterazine) and molecular oxygen to form a stable photoprotein complex. Upon addition of calcium, the photoprotein undergoes a conformational change leading to the oxidation of the chromophore with the release of CO(2) and blue light. To gain more information of structure-function relationships within the photoprotein that will aid in the design of mutants suitable for site-specific conjugation and immobilization, polymerase chain reaction (PCR)-based site-directed mutagenesis was employed to produce five different aequorin mutants. The five mutants included a cysteine-free mutant and four other mutants with single cysteine residues at selected positions within the protein. The aequorin mutants exhibited different bioluminescence emission characteristics with two mutants showing a decrease in relative light production in comparison to the cysteine-free mutant. Additionally, circular dichroism (CD) spectra revealed that the single amino acid substitutions made for two of the aequorin mutants did alter their secondary structures.     

Blue fluorescent protein from the calcium-sensitive photoprotein aequorin is a heat resistant enzyme, catalyzing the oxidation of coelenterazine

Blue fluorescent protein from the calcium-sensitive photoprotein aequorin (BFP-aq) was prepared and determined to be a heat resistant enzyme, catalyzing the luminescent oxidation of coelenterazine (luciferin) with molecular oxygen as a general luciferase. After treatment with excess ethylenediaminetetraacetic acid to remove Ca2+ from BFP-aq, the blue fluorescence shifted to a greenish fluorescence. This greenish fluorescent protein (gFP-aq) was identified as a non-covalent complex of apoaequorin with coelenteramide (oxyluciferin) in a molar ratio of 1:1. By incubation with coelenterazine in the absence of reducing reagents, gFP-aq was converted to aequorin at 25 degrees C. BFP-aq and gFP-aq possessing both fluorescence and luminescence activities may work as novel reporter proteins.     

Spatial structure of the novel light-sensitive photoprotein berovin from the ctenophore Beroe abyssicola in the Ca-loaded apoprotein conformation state

The bright bioluminescence of ctenophores, found in oceans worldwide, is determined by Ca^2 +-regulated photoproteins, functionally identical to and sharing many properties of hydromedusan photoproteins. In contrast, however, the ctenophore photoproteins are extremely sensitive to UV and visible light over the range of their absorption spectrum. The spatial structure of a novel light-sensitive photoprotein from the ctenophore Beroe abyssicola in its apoform bound with three calcium ions is determined at 2.0 Å. We demonstrate that the apoberovin is a slightly asymmetrical compact globular protein formed by two domains with a cavity in the center, which exactly retains the fold architecture characteristic of hydromedusan photoproteins despite their low amino acid sequence identity. However, the structural alignment of these two photoprotein classes clearly shows that despite the high similarity of shape and geometry of their coelenterazine-binding cavities, their interiors differ drastically. The key residues appearing to be crucial for stabilizing the 2-hydroperoxycoelenterazine and for formation of the emitter in hydromedusan photoproteins, are replaced in berovin by amino acid residues having completely different side chain properties. Evidently, these replacements must be responsible for the distinct properties of ctenophore photoproteins such as sensitivity to light or the fact that the formation of active photoprotein from apophotoprotein, coelenterazine, and oxygen is more effective at alkaline pH.     

Molecular mechanism of Symplectoteuthis bioluminescence—Part 4: Chromophore exchange and oxidation of the cysteine residue

Symplectin is one of the few photoproteins, which forms covalent bonds with the dehydro-coelenterazine (DCL) at the binding sites and the active site. This binding takes place through the SH’s of the cysteine residues via conjugate addition reaction. This photoprotein contains the chromophore molecules at the binding cites first, and then moves to the active cite Cys-390 for the luminescence. The current study focuses on these dynamic aspects of the chromophore using the natural photoprotein by analyzing the fluorescence changing of the DCL chromophores analogs with 8-(4′-methoxyphenyl)- or 8-(2′-naphthyl)-group and 2-(2′,4′-difluorophenyl)-group. Exchanges of these chromophores were monitored the fluorescence at slightly acidic media and also from the luminescence function observed at the optimum pH 7.8. The non-fluorescent naphthyl analogs was even proven to make the covalent bond formation at pH 6.0 and evidently to obtain the corresponding luminescent product amide by liquid chromatographic detection from the spent solutions.     

Use of chlorophyll fluorescence to estimate the effect of photoinhibition in outdoor cultures ofSpirulina platensis

Chlorophyll fluorescence measurements were used to evaluate the effect of temperature on photoinhibition inSpirulina platensis cultures grown in tubular reactors outdoors. Cultures grown at 35 °C during the day time showed a lower reduction in the Fv/Fm ratio as compared to cultures grown at 25 °C. It is demonstrated that the lower temperature photoinhibited cells can undergo a complete recovery once transferred to low light and higher temperature. This recovery does not take place when 100 µg ml^-1 chloramphenicol is added to cells. The recovery is light dependent and cells incubated in the dark at low temperature do not show a recovery in the Fv/Fm ratio. The data presented strongly support the hypothesis that photoinhibition takes place in outdoorSpirulina cultures. At the same time it is demonstrated that fluorescence measurements can be used as a fast reliable indication for photoinhibition in outdoor algal cultures.Author for correspondencePublication No. 69 of the Microalgal Biotechnology Laboratory.     

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

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

Application of new semisynthetic aequorins with long half-decay time of luminescence to G-protein-coupled receptor assay

Aequorin is a Ca²⁺-binding photoprotein and consists of an apoprotein (apoaequorin) and a 2-peroxide of coelenterazine. Eight new coelenterazine analogues modified at the C2-position were synthesized and incorporated into recombinant apoaequorin with O₂ to yield different semisynthetic aequorins. The luminescence properties and the sensitivity to Ca²⁺ of these semisynthetic aequorins were characterized. Two semisynthetic aequorins, namely me- and cf3-aequorin, showed a slow decay of the luminescence pattern with less sensitivity to Ca²⁺ and were useful for the cell-based G-protein-coupled receptor (GPCR) reporter assays.