Coelenterazine-v ligated to Ca2+-triggered coelenterazine-binding protein is a stable and efficient substrate of the red-shifted mutant of Renilla muelleri luciferase

It has been shown that the coelenterazine analog, coelenterazine-v, is an efficient substrate for a reaction catalyzed by Renilla luciferase. The resulting bioluminescence emission maximum is shifted to a longer wavelength up to 40 nm, which allows the use of some “yellow” Renilla luciferase mutants for in vivo imaging. However, the utility of coelenterazine-v in small-animal imaging has been hampered by its instability in solution and in biological tissues. To overcome this drawback, we ligated coelenterazine-v to Ca²⁺-triggered coelenterazine-binding protein from Renilla muelleri, which apparently functions in the organism for stabilizing and protecting coelenterazine from oxidation. The coelenterazine-v bound within coelenterazine-binding protein has revealed a greater long-term stability at both 4 and 37 °C. In addition, the coelenterazine-binding protein ligated by coelenterazine-v yields twice the total light over free coelenterazine-v as a substrate for the red-shifted R. muelleri luciferase. These findings suggest the possibility for effective application of coelenterazine-v in various in vitro assays.     

Ca(2+)-triggered coelenterazine-binding protein from Renilla as an enzyme-dependent label for binding assay

The recombinant Ca²⁺-triggered coelenterazine-binding protein (CBP) from Renilla muelleri was investigated as a biospecifically labeled molecule for in vitro assay applications. The protein was shown to be stable in solutions in the frozen state, as well as stable under heating and to chemical modifications. Conjugates with biotin, oligonucleotide, and proteins were obtained and applied as biospecific molecules in a solid-phase microassay. CBP detection was performed with intact (no modifications were made) Renilla luciferase in the presence of calcium, and the detection limit was found to be 75 amol. Model experiments indicate that this approach shows much promise, especially with regard to the development of multianalytical systems.     

Bioluminescent properties of obelin and aequorin with novel coelenterazine analogues

The main analytical use of Ca²⁺-regulated photoproteins from luminous coelenterates is for real-time non-invasive visualization of intracellular calcium concentration ([Ca²⁺]_i) dynamics in cells and whole organisms. A limitation of this approach for in vivo deep tissue imaging is the fact that blue light emitted by the photoprotein is highly absorbed by tissue. Seven novel coelenterazine analogues were synthesized and their effects on the bioluminescent properties of recombinant obelin from Obelia longissima and aequorin from Aequorea victoria were evaluated. Only analogues having electron-donating groups (m-OCH₃ and m-OH) on the C6 phenol moiety or an extended resonance system at the C8 position (1-naphthyl and α-styryl analogues) showed a significant red shift of light emission. Of these, only the α-styryl analogue displayed a sufficiently high light intensity to allow eventual tissue penetration. The possible suitability of this compound for in vivo assays was corroborated by studies with aequorin which allowed the monitoring of [Ca²⁺]_i dynamics in cultured CHO cells and in hippocampal brain slices. Thus, the α-styryl coelenterazine analogue might be potentially useful for non-invasive, in vivo bioluminescence imaging in deep tissues of small animals.     

Characterization of hydromedusan Ca2+-regulated photoproteins as a tool for measurement of Ca2+concentration

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. Ca²⁺-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 Ca²⁺ concentration detection limit, the sensitivity of bioluminescence to Mg²⁺, and the rates of the rise of the luminescence signal with a sudden change of Ca²⁺ 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 Ca²⁺ without modifications improving its properties. On the basis of experiments on the activation of endogenous P2Y₂ 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 Ca²⁺ concentration. Figure

Hydromedusan photoproteins differ in Ca²⁺ concentration detection limit, sensitivity of bioluminescence to Mg²⁺, and rates of rise of luminescence signal with a sudden change of [Ca²⁺] despite a high degree of identity of their amino acid sequences and spatial structures, and, apparently, a common mechanism for the bioluminescence reaction.     

Interaction energies of histidine with cations (H<Superscript>+</Superscript>, Li<Superscript>+</Superscript>, Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>, Mg<Superscript>2+</Superscript>, Ca<Superscript>2+</Superscript>)

The imidazol side group of histidine has two nitrogen atoms capable of being protonated or participating in metal binding. Hence, histidine can take on various metal-bound and protonated forms in proteins. Because of its variable structural state, histidine often functions as a key amino acid residue in enzymatic reactions. Ab initio (HF and MP2) calculations were done in modeling the cation (H⁺, Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺) interaction with side chain of histidine. The region selectivity of metal ion complexation is controlled by the affinity of the side of attack. In the imidazol unite of histidine the ring nitrogen has much higher metal ion (as well as proton) affinity. The complexation energies with the model systems decrease in the following order: Mg²⁺ > Ca²⁺ > Li⁺ > Na⁺ > K⁺. The variation of the bond lengths and the extent of charge transfer upon complexation correlate well with the computed interaction energies.     

The Ca2+-Regulated Photoprotein Obelin as a Tool for SELEX Monitoring and DNA Aptamer Affinity Evaluation

Bioluminescent solid-phase analysis was proposed to monitor the selection process and to determine binding characteristics of the aptamer–target complexes during design and development of the specific aptamers. The assay involves Ca²⁺-regulated photoprotein obelin as a simple, sensitive and fast reporter. Applicability and the prospects of the approach were exemplified by identification of DNA aptamers to cardiac troponin I, a highly specific early biomarker for acute myocardial infarction. Two structurally different aptamers specific to various epitopes of troponin I were obtained and then tested in a model bioluminescent assay.     

Violet and greenish photoprotein obelin mutants for reporter applications in dual-color assay

Two kinds of Ca²⁺-regulated photoprotein obelin with altered color of bioluminescence were obtained by active-center amino acid substitution. The mutant W92F-H22E emits violet light (λ_max = 390 nm) and the mutant Y139F emits greenish light (λ _max = 498 nm), with small spectral overlap, both display high activity and stability and thus may be used as reporters. For demonstration, the mutants were applied in dual-color simultaneous immunoassay of two gonadotropic hormones—follicle-stimulating hormone and luteinizing hormone. Bioluminescence of the reporters was simultaneously triggered by single injection of Ca²⁺ solution, divided using band-pass optical filters and measured with a two-channel photometer. The sensitivity of simultaneous bioluminescence assay was close to that of a separate radioimmunoassay. Figure Two kinds of Ca²⁺-regulated photoprotein obelin with altered color of bioluminescence were obtained and applied in dual-color simultaneous immunoassay of two gonadotropic hormones.     

1,2-Dichlorobenzene affects the formation of the phosphoenzyme stage during the catalytic cycle of the Ca<Superscript>2+</Superscript>-ATPase from sarcoplasmic reticulum

Background

1,2-Dichlorobenzene (1,2-DCB) is a benzene-derived molecule with two Cl atoms that is commonly utilized in the synthesis of pesticides. 1,2-DCB can be absorbed by living creatures and its effects on naturally-occurring enzymatic systems, including the effects on Ca²⁺-ATPases, have been poorly studied. Therefore, we aimed to study the effect of 1,2-DCB on the Ca²⁺-ATPase from sarcoplasmic reticulum (SERCA), a critical regulator of intracellular Ca²⁺ concentration.

Results

Concentrations of 0.05–0.2 mM of 1,2-DCB were able to stimulate the hydrolytic activity of SERCA in a medium-containing Ca²⁺-ionophore. At higher concentrations (0.25–0.75 mM), 1,2-DCB inhibited the ATP hydrolysis to ~80 %. Moreover, ATP hydrolysis and Ca²⁺ uptake in a medium supported by K-oxalate showed that starting at 0.05 mM,1,2-DCB was able to uncouple the ratio of hydrolysis/Ca²⁺ transported. The effect of this compound on the integrity of the SR membrane loaded with Ca²⁺ remained unaffected. Finally, the analysis of phosphorylation of SERCA by [γ-³²P]ATP, starting under different conditions at 0° or 25 °C showed a reduction in the phosphoenzyme levels by 1,2-DCB, mostly at 0 °C.

Conclusions

The temperature-dependent decreased levels of phosphoenzyme by 1,2-DCB could be due to the acceleration of the dephosphorylation mechanism – E₂P?·?Ca₂ state to E₂ and P_i, which explains the uncoupling of the ATP hydrolysis from the Ca²⁺ transport.

     

Free Ca<Superscript>2+</Superscript> as an early intracellular biomarker of exposure of cyanobacteria to environmental pollution

Calcium functions as a versatile messenger in a wide variety of eukaryotic and prokaryotic cells. Cyanobacteria are photoautotrophs which have a great ecological impact as primary producers. Our research group has presented solid evidence of a role of calcium in the perception of environmental changes by cyanobacteria and their acclimation to these changes. We constructed a recombinant strain of the freshwater cyanobacterium Anabaena sp. PCC 7120 that constitutively expresses the calcium-binding photoprotein apoaequorin, enabling in-vivo monitoring of any fluctuation in the intracellular free calcium concentration of the cyanobacterium in response to any environmental stimulus. The “Ca²⁺ signature” is the combination of changes in all Ca²⁺ signal properties (magnitude, duration, frequency, source of the signal) produced by a specific stimulus. We recorded and analyzed the Ca²⁺ signatures generated by exposure of the cyanobacterium to different groups of environmental pollutants, for example cations, anions, organic solvents, naphthalene, and pharmaceuticals. We found that, in general, each group of tested chemicals triggered a specific calcium signature in a reproducible and dose-dependent manner. We hypothesize that these Ca²⁺ signals may be related to the cellular mechanisms of pollutant perception and ultimately to their toxic mode of action. We recorded Ca²⁺ signals triggered by binary mixtures of pollutants and a signal induced by a real wastewater sample which could be mimicked by mixing its main constituents. Because Ca²⁺ signatures were induced before toxicity was evident, we propose that intracellular free Ca²⁺ may serve as an early biomarker of exposure to environmental pollution.     

Cation binding by 2<Superscript>1</Superscript>,3<Superscript>1</Superscript>-diphenyl-l <Superscript>2</Superscript>,4<Superscript>2</Superscript>-dioxo-7,10,13-trioxa-1,4(3,1)-diquinoxalina-2 (2,3),3(3,2)-diindolizinacyclopentadecaphane and its acyclic analog

The binding of cations Li⁺, Na⁺, K⁺, Cs⁺, Ag⁺, Zn²⁺, Ni²⁺, Co²⁺, NH₄ ⁺ (group I), H⁺, Mg²⁺, Al³⁺, Ga³⁺ (group II), and Ca²⁺, Pb²⁺ (group III) by 2¹,3¹-diphenyl-l ²,4²-dioxo- 7,10,13-trioxa-l,4(3,1)-diquinoxalina-2(2,3),3(3,2)-diindolizinacyclopentadecaphane (1), which contains two indolizine and two quinoxaline fragments and 3,6,9-trioxaundecanes spacer, and by its acyclic analog (2) was studied using cyclic voltammetry in MeCN/0.1 M Bu₄NBF₄. It was concluded that the ions of group I are not bound by these compounds, the ions of group II exhibit the reversible redox-switched binding by the carbamoyl groups of the quinoxaline fragments, whereas the ions of group III are bound not only by the initial compounds and radical cations 1 and 2, but also by dication 1. This binding of the Ca²⁺ and Pb²⁺ ions stabilizes dication 1.     

Formation of coordination compounds with aniline in the interlayer space of Ca<Superscript>2+</Superscript>-, Cu<Superscript>2+</Superscript>- and Fe<Superscript>3+</Superscript>-exchanged montmorillonite

The influence of Ca²⁺-, Cu²⁺- and Fe³⁺-exchanged montmorillonite (MMT) on the type of interaction with aniline in the interlayer space of MMT has been studied by means of X-ray powder diffraction and infrared spectra. Results of X-ray diffraction showed that aniline was successfully intercalated into the interlayer space of MMT. Based on IR spectra evaluation, aniline was indirectly coordinated through a water-bridge in Ca²⁺- and Fe³⁺-MMT and it was indirectly coordinated through a water-bridge as well as protonated in Cu²⁺-MMT (the spectrum of protonated aniline showed deformation and changes in the NH ₃ ⁺ absorption at approximately 1521 cm^?1). It is important to point out that Cu²⁺-MMT indirect coordination and protonation occur simultaneously.     

Specific features of absorption of Cu<Superscript>2+</Superscript>, Zn<Superscript>2+</Superscript>, Co<Superscript>2+</Superscript>, and Pb<Superscript>2+</Superscript> cations from aqueous solutions by calcium phosphates

Kinetic characteristics of the interaction of trisubstituted calcium phosphate Ca₃(PO₄)₂ · xH₂O with Cu²⁺, Zn²⁺, Co²⁺, and Pb²⁺ ions in aqueous solutions were studied.     

A Novel Catalytic Function of Synthetic IgG‐Binding Domain (Z Domain) from Staphylococcal Protein A: Light Emission with Coelenterazine

The synthetic IgG‐binding domain (Z domain) of staphylococcal protein A catalyzes the oxidation of coelenterazine to emit light like a coelenterazine‐utilizing luciferase. The Z domain derivatives (ZZ‐gCys, Z‐gCys and Z‐domain) were purified and the luminescence properties were characterized by comparing with coelenterazine‐utilizing luciferases, including Renilla luciferase, Gaussia luciferase and the catalytic 19 kDa protein of Oplophorus luciferase. Three Z domain derivatives showed luminescence activity with coelenterazine and the order of the initial maximum intensity of luminescence was ZZ‐gCys (100%) > Z‐gCys (36.8%) > Z‐domain (1.1%) > bovine serum albumin (BSA; 0.9%) > staphylococcal protein A (0.1%) and the background value of coelenterazine (0.1%) in our conditions. The luminescence properties of ZZ‐gCys showed the similarity to that of Gaussia luciferase, including the luminescence pattern, the emission spectrum, the stimulation by halogen ions and nonionic detergents and the substrate specificity for coelenterazine analogues. In contrast, the luminescence properties of Z‐gCys were close to the catalytic 19 kDa protein of Oplophorus luciferase. The catalytic region of the Z domain for the luminescence reaction might be different from the IgG‐binding region of the Z domain.     

Solution <Superscript>1</Superscript>H n.m.r. of paramagnetic Co<Superscript>2+</Superscript>complexes with dioxobis(carboxymethyl)tetraazamacrocycles

Paramagnetic shifts in the ¹H n.m.r. spectra were observed for high-spin Co²⁺ complexes with 12--14-membered tetraazamacrocycles incorporating two amide groups and two pendant carboxymethyl groups. The pseudocontact term due to the dipolar interaction between the metal ion and the resonant protons was calculated on the basis of X-ray structures, and the Fermi contact term due to spin delocalization was determined. The ethylenediamine moiety of the ligand molecule is coordinated in an unsymmetric manner, even in solution, and internal motion (involving conformational change of the macrocyclic frame and exchange of coordinate bonds) is much slower than the n.m.r. observation frequency. In the 12-membered macrocyclic complex Co(L12), three of the eight CH₂ groups fix their orientation in the n.m.r. time scale; in Co(L13) only one CH₂ group fixes its orientation; in Co(L14) all CH₂ groups undergo a rapid reorientation. The facility in internal motion increases with the ring size, and shows a correlation with the chemical stabilities of these Co²⁺ complexes against oxidation; to atmospheric oxygen, Co²⁺(L12) is the most resistant and Co²⁺(L14) is the most susceptible.     

Extraction and <Emphasis Type="Italic">ab initio</Emphasis> calculation studies on the complexation of Ca<Superscript>2+</Superscript> with valinomycin

From extraction experiments and γ-activity measurements, the exchange extraction constant corresponding to the equilibrium Ca²⁺ (aq)+1·Sr²⁺ (nb) ⇆ 1·Ca²⁺ (nb) + Sr²⁺ (aq) taking place in the two-phase water-nitrobenzene system (1 = valinomycin; aq = aqueous phase, nb = nitrobenzene phase) was evaluated as log K _ex (Ca²⁺, 1·Sr²⁺) = 2.4±0.1. Further, the stability constant of the valinomycin-calcium complex (abbrev. 1·Ca²⁺) in nitrobenzene saturated with water was calculated for a temperature of 25 °C: log β _nb (1·Ca²⁺) = 8.3±0.1. By using quantum mechanical DFT calculations, the most probable structure of the 1·Ca²⁺·2H₂O complex species was predicted. In this complex, the “central” Ca²⁺ cation is bound by strong bonds to two oxygen atoms of the respective water molecules and to four ester carbonyl oxygens of the parent valinomycin ligand 1. Finally, the calculated binding energy of the considered complex 1·Ca²⁺·2H₂O is −319.2 kcal/mol, which confirms the relatively high stability of this cationic complex species.     

An Isotope (<Superscript>18</Superscript>O, <Superscript>15</Superscript>N,and <Superscript>2</Superscript>H) Technique to Investigate the Metal Ion Interactions Between the Phosphoryl Group and Amino Acid Side Chains by Electrospray Ionization Mass Spectrometry

Cationic metal ion-coordinated N-diisopropyloxyphosphoryl dipeptides (DIPP-dipeptides) were analyzed by electrospray ionization multistage tandem mass spectrometry (ESI-MS ⁿ ). Two novel rearrangement reactions with hydroxyl oxygen or carbonyl oxygen migrations were observed in ESI-MS/MS of the metallic adducts of DIPP-dipeptides, but not for the corresponding protonated DIPP-dipeptides. The possible oxygen migration mechanisms were elucidated through a combination of MS/MS experiments, isotope (¹⁸O, ¹⁵N, and ²H) labeling, accurate mass measurements, and density functional theory (DFT) calculations at the B3LYP/6-31 G(d) level. It was found that lithium and sodium cations catalyze the carbonyl oxygen migration more efficiently than does potassium and participation through a cyclic phosphoryl intermediate. In addition, dipeptides having a C-terminal hydroxyl or aromatic amino acid residue show a more favorable rearrangement through carbonyl oxygen migration, which may be due to metal cation stabilization by the donation of lone pair of the hydroxyl oxygen or aromatic π-electrons of the C-terminal amino acid residue, respectively. It was further shown that the metal ions, namely lithium, sodium, and potassium cations, could play a novel directing role for the migration of hydroxyl or carbonyl oxygen in the gas phase. This discovery suggests that interactions between phosphorylated biomolecules and proteins might involve the assistance of metal ions to coordinate the phosphoryl oxygen and protein side chains to achieve molecular recognition.     

Tyr72 and Tyr80 are Involved in the Formation of an Active Site of a Luciferase of Copepod Metridia longa

Luciferase of copepod Metridia longa (MLuc) is a naturally secreted enzyme catalyzing the oxidative decarboxylation of coelenterazine with the emission of light. To date, three nonallelic isoforms of different lengths (17–24 kDa) for M. longa luciferase have been cloned. All the isoforms are single‐chain proteins consisting of a 17‐residue signal peptide for secretion, variable N‐terminal part and conservative C‐terminus responsible for luciferase activity. In contrast to other bioluminescent proteins containing a lot of aromatic residues which are frequently involved in light emission reaction, the C‐terminal part of MLuc contains only four Phe, two Tyr, one Trp and two His residues. To figure out whether Tyr residues influence bioluminescence, we constructed the mutants with substitution of Tyr to Phe (Y72F and Y80F). Tyrosine substitutions do not eliminate the ability of luciferase to bioluminescence albeit significantly reduce relative specific activity and change bioluminescence kinetics. In addition, the Tyr replacements have no effect on bioluminescence spectrum, thereby indicating that tyrosines are not involved in the emitter formation. However, as it was found that the intrinsic fluorescence caused by Tyr residues is quenched by a reaction substrate, coelenterazine, in concentration‐dependent manner, we infer that both tyrosine residues are located in the luciferase substrate‐binding cavity.     

Calculations of chemical equilibria in heparin-arginine-H<Subscript>2</Subscript>O-NaCl and MCl<Subscript>2</Subscript>-heparin-arginine-H<Subscript>2</Subscript>O-NaCl systems (M = Ca<Superscript>2+</Superscript>, Mg<Superscript>2+</Superscript>) in a physiological solution

Chemical equilibria in the high-molecular-weight heparin (Na₄hep)-arginine (HArg)-H₂O-NaCl and MCl₂-Na₄hep-HArg-H₂O-NaCl systems of electrolytes (M = Ca²⁺, Mg²⁺) were calculated by the method of mathematical simulation of chemical equilibria from representative planned pH-metric titration experiment at 2.30 ≤ pH ≤ 10.50 in a physiological solution medium in the presence of 0.154 M NaCl as a background electrolyte at 37°C. The initial concentrations of the basic components were n × 10⁻³ M (n ≤ 4).     

Development of Luminescent Coelenterazine Derivatives Activatable by β‐Galactosidase for Monitoring Dual Gene Expression

Two bioluminogenic caged coelenterazine derivatives (bGalCoel and bGalNoCoel) were designed and synthesized to detect β‐galactosidase activity and expression by means of bioluminescence imaging. Our approach addresses the instability of coelenterazine by introducing β‐galactose caging groups to block the auto‐oxidation of coelenterazine. Both probes contain β‐galactosidase cleavable caging groups at the carbonyl group of the imidazo–pyrazinone moiety. One of the probes in particular, bGalNoCoel, displayed a fast cleavage profile, high stability, and high specificity for β‐galactosidase over other glycoside hydrolases. bGalN‐oCoel could detect β‐galactosidase activity in living HEK‐293T cell cultures that expressed a mutant Gaussia luciferase. It was determined that coelenterazine readily diffuses in and out of cells after uncaging by β‐galactosidase. We showed that this new caged coelenterazine derivative, bGalNoCoel, could function as a dual‐enzyme substrate and detect enzyme activity across two separate cell populations.