Recombinant aequorin and recombinant semi-synthetic aequorins. Cellular Ca2+ ion indicators.

Properties of a recombinant aequorin were investigated in comparison with those of natural aequorin. In chromatographic behaviour the recombinant aequorin did not match any of ten isoaequorins tested, although it was very similar to aequorin J. Its sensitivity to Ca2+ was found to be higher than that of any isoaequorin except aequorin D. The recombinant aequorin exhibited no toxicity when tested in various kinds of cells, even where samples of natural aequorin had been found to be toxic. Properties of four recombinant semi-synthetic aequorins (fch-, hcp-, e- and n-types), prepared from the recombinant apo-aequorin and synthetic analogues of coelenterazine, were approximately parallel with those of corresponding semi-synthetic aequorins prepared from natural apo-aequorin. Both recombinant e-aequorin and natural e-aequorin J luminesced with high values of the luminescence intensity ratio I400/I465, although the ratios were not pCa-dependent. The recombinant aequorin and recombinant semi-synthetic aequorins are highly suited for monitoring cellular Ca2+.     

Preparation and handling of aequorin solutions for the measurement of cellular Ca2+

Main characteristics of the various types of aequorin presently available for measuring cellular Ca2+, i.e. heterogeneous aequorin, isoaequorins, recombinant aequorin, fluorescein-labeled aequorin and semi-synthetic aequorins, are summarized. Basic techniques of preparing and handling the solutions of those aequorins for measuring Ca2+, including such techniques as concentrating aequorin solutions, freeze-drying, changing buffer composition, and the regeneration of active aequorin, are described.     

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, expression, purification and characterization of an isotype of clytin, a calcium-binding photoprotein from the luminous hydromedusa Clytia gregarium

The cDNA for an isotype of clytin, a calcium-binding photoprotein from the luminous jellyfish Clytia gregarium, was identified and named clytin-II. The histidine-tagged apoprotein of clytin-II expressed into the periplasmic space of Escherichia coli cells was isolated by nickel chelate affinity chromatography. Recombinant clytin-II regenerated from apoprotein by incubation with coelenterazine was purified. The yield of purified clytin-II was 13 mg from 2 l of cultured cells with purity >95%. The luminescence properties of clytin-II were characterized by comparison with clytin-I and aequorin, and semi-synthetic clytin-II was also prepared. The initial luminescence intensity of clytin-II triggered by Ca(2+) was 4.5 times higher than that of clytin-I and aequorin, but the luminescence capacity was close to clytin-I and aequorin. Thus, clytin-II is a useful protein, showing high sensitivity in the signal-to-noise ratio of luminescence intensity.     

Calcium-sensitive photoproteins

The recombinant Ca2+ sensitive photoprotein aequorin was the first probe used to measure specifically the Ca2+ concentration, [Ca2+], inside the intracellular organelles of intact cells. Aequorin-based methods offer several advantages: (i) targeting of the probe is extremely precise, thus permitting a selective intracellular distribution; (ii) the use of wild-type and low Ca2+-affinity aequorins allows covering a large dynamic range of [Ca2+], from 10(-7) to 10(-3)M; (iii) aequorin has a low Ca2+ buffering effect and it is nearly insensitive to changes in Mg2+ or pH; (iv) it has a high signal-to-noise ratio; (v) calibration of the results in [Ca2+] is made straightforward using a simple algorithm; and (vi) the equipment required for luminescence measurements in cell populations is simple and low-cost. On the negative side, this technique has also some disadvantages: (i) the relatively low amount of emitted light makes difficult performing single-cell imaging studies; (ii) reconstitution of aequorin with coelenterazine is necessary to generate the functional photoprotein and this procedure requires at least 1h; (iii) in the case of aequorin targeted to high Ca2+ compartments, because of the high rate of aequorin consumption at steady-state, only relatively brief experiments can be performed and, because of the steepness of the Ca2+-response curve, the calibrated [Ca2+] values may not reflect the real mean in cells or compartments with dyshomogeneous behavior; and (iv) expression of targeted aequorins requires previous transfection or infection to introduce the appropriate DNA construct, or alternatively the use of stable cell clones.     

Semi-synthetic aequorins with improved sensitivity to Ca2+ ions.

Thirty-seven coelenterazine analogues were synthesized and incorporated into apo-aequorin, yielding 30 semi-synthetic aequorins that have the capacity to emit a significant amount of light in the presence of Ca2+. The properties of resultant photoproteins were investigated. The most prominent feature of those photoproteins was the wide range in their sensitivities to Ca2+ concentration. The relative intensity of Ca2+-triggered luminescence of the photoproteins ranged from 0.01 to 190 when compared with natural aequorin (relative intensity 1.0) at pCa 6 for the cases where the relative intensity is less than 1 and at pCa 7 for the cases where the relative intensity is higher than 1. Eight of the semi-synthetic aequorins belonged to the class of e-aequorin. With two of those photoproteins, the degree of dependence of the luminescence intensity ratio I400/I465 on pCa was greater than that with e-aequorin, suggesting that these two photoproteins are possibly superior to e-aequorin in measuring Ca2+ concentration by the ratio method.     

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.     

One-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography

A hexahistidine tag was fused to the N-terminus of apoaequorin. A suitable vector encoding the fusion protein was constructed and used for transformation of Escherichia coli JM109 cells. Apoaequorin was overexpressed under the control of tac promoter. It was found, however, that most of the protein existed in the form of inclusion bodies. Inclusion bodies were solubilized with urea, followed by purification and refolding of (His)(6)-apoaequorin in a single chromatographic step by immobilized metal-ion affinity chromatography using Ni(2+)-nitrilotriacetic acid agarose. The purity, as determined by SDS-PAGE analysis, was greater than 80%. The yield was 0.7-1 mg apoaequorin from a 50 ml bacterial culture. The kinetics of light emission of purified aequorin upon addition of Ca(2+) was typical of the commercial aequorin. The luminescence of the purified aequorin was a linear function of its concentration extending over six orders of magnitude. As low as 0.5 attomoles purified aequorin gave a signal-to-noise ratio of 1.8.     

Photoinduced electron-transfer kinetics of singly labeled ruthenium bis(bipyridine) dicarboxybipyridine cytochrome c derivatives

Cytochrome c derivatives labeled at specific lysine amino groups with ruthenium bis(bipyridine) dicarboxybipyridine [RuII(bpy)2(dcbpy)] were prepared by using the procedure described previously [Pan, L. P., Durham, B., Wolinska, J., & Millett, F. (1988) Biochemistry 27, 7180-7184]. Four additional singly labeled derivatives were purified, bringing the total number to 10. These derivatives have a strong luminescence emission centered at 662 nm arising from the excited state, RuII*. Transient absorption spectroscopy was used to directly measure the rate constants for the photoinduced electron-transfer reaction from RuII* to the ferric heme group (k1) and for the thermal back-reaction from the ferrous heme group to RuIII (k2). The rate constants were found to be k1 = 14 X 10(6) s-1 and k2 = 24 X 10(6) s-1 for the derivative modified at lysine 72, which has a distance of 8-16 A between the ruthenium and heme groups. Similar rate constants were found for the derivatives modified at lysines 13 and 27, which have distances of 6-12 A separating the ruthenium and heme groups. The rate constants were significantly slower for the derivatives modified at lysine 25 (k1 = 1 X 10(6) s-1, k2 = 1.5 X 10(6) s-1) and lysine 7 (k1 = 0.3 X 10(6) s-1, k2 = 0.5 X 10(6) s-1), which have distances of 9-16 A. Transients due to photoinduced electron transfer could not be detected for the remaining derivatives, which have larger distances between the ruthenium and heme groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Halistaurin, phialidin and modified forms of aequorin as Ca2+ indicator in biological systems.

Two kinds of aequorin-type photoproteins, i.e., halistaurin and phialidin, and four kinds of modified forms of aequorin, i.e., products of acetylation, ethoxycarbonylation, fluorescamine-modification and fluorescein labelling, were prepared. The modified forms of aequorin were more sensitive to Ca2+ than was aequorin in their Ca2+-triggered luminescence reactions, whereas halistaurin and phialidin were less sensitive. The emission maxima of luminescence were all within a wavelength range 450-464 nm, except for fluorescein-labelled aequorin, which emitted yellowish light (lambda max. 520 nm). A new technique of measuring Ca2+ concentration is suggested.     

Effects of halothane, enflurane, and isoflurane on measurements of Ca(2+) by calcium electrode and aequorin luminescence

We assessed the possible effects of the volatile halogenated anesthetics halothane, enflurane, and isoflurane on Ca(2+) electrode measurements and on the Ca(2+) sensitivity of the bioluminescent protein aequorin. In Ca(2+)-EGTA buffers of different pCa values (7. 870, 6.726, 6.033, 4.974, 4.038, and 2.995) and in serial Ca(2+) dilutions (10(-4), 10(-3), and 10(-2) M), halothane, enflurane, and isoflurane each caused a concentration-dependent and reversible increase in the absolute value of the negative electrode potential. Isoflurane and enflurane had larger effects than halothane. Neither of these anesthetics changed aequorin luminescence at any pCa tested in the range 2-8. There was no potentiation or inactivation of aequorin luminescence over a period of up to 2 h. These results suggest that (1) halothane, enflurane, and isoflurane interfere with Ca(2+) electrode measurements, most likely by changing the physicochemical properties of the membrane; (2) these anesthetics do not inactivate or otherwise modify the characteristics of the reaction of Ca(2+) with aequorin; and (3) these anesthetics do not change the apparent affinity of EGTA for Ca(2+).     

Obelin from the bioluminescent marine hydroid Obelia geniculata: cloning, expression, and comparison of some properties with those of other Ca2+-regulated photoproteins

A cDNA encoding the Ca2+-regulated photoprotein of the bioluminescent marine hydroid Obelia geniculata was cloned and sequenced. The cDNA is a 774 bp fragment containing two overlapping open reading frames, one of which contained 585 bp encoding a 195 amino acid polypeptide which obviously has the primary structure of the apoprotein of a calcium-regulated photoprotein. Many of the residues are identical to those in other Ca2+-regulated photoproteins: 86% compared with that from Obelia longissima, 76% with that from Clytia (Phialidium), 64% with that from Aequorea, and 64% with that from Mitrocoma(Halistaura). The obelin from O. geniculata was overexpressed in Escherichia coli, refolded from inclusion bodies, and purified. The yield of highly purified recombinant protein was 55-80 mg/L of LB medium. O. geniculata obelin has absorption maxima at 280 and 460 nm and a shoulder at approximately 310 nm. The calcium-discharged protein loses visible absorption but exhibits a new absorption maximum at 343 nm. The bioluminescence of the obelin from O. geniculata is blue (lambda(max) = 495 nm). In contrast, the fluorescence of the calcium-discharged protein is yellow-green (lambda(max) = 520 nm; excitation at 340 nm). This is in sharp contrast to aequorin in which the bioluminescence and fluorescence emission spectra of the calcium-discharged protein are almost identical (lambda(max) = 465 nm). The Ca2+ concentration-effect curve for O. geniculata obelin is similar to those of many other photoproteins: at [Ca2+] below approximately 10(-8) M, calcium-independent luminescence is observed, and at [Ca2+] approximately 10(-3) M, the luminescence reaches a maximum. Between these extremes, the curve spans a vertical range of almost 8 log units with a maximum slope on a log-log plot of about 2.5. In the absence of Mg2+ the rate constant for the rise of bioluminescence determined by the stopped-flow technique is about 450 s(-1). The effects of Mg2+ on the kinetics of bioluminescence are complicated, but at all concentrations studied they are relatively small compared to the corresponding effects on aequorin luminescence. At least with respect to speed and sensitivity to Mg2+, the obelins from both O. longissima and O. geniculata would appear to be more suitable than aequorin for use as intracellular Ca2+ indicators.     

Calcium measurement in the periphery of an axon

Aequorin was microinjected into squid giant axons, the axons were stimulated, and the change in light emission was followed. This response was compared with that found when the axon, in addition to being microinjected with aequorin, is also injected with the dye phenol red. Large concentrations of phenol red injected into axons result in a high probability that photons emitted by aequorin, when it reacts with Ca in the core of the axoplasm, will be absorbed before they escape from the axon; photons produced by the aequorin reaction at the periphery of the axoplasm are much less likely to be absorbed. This technique thus favors observing changes in Cai taking place in the periphery of the axon. Stimulation in 50 mM Ca seawater of an aequorin-phenol red-injected axon at 180 s-1 for 1 min produces a scarcely detectable change in Cai; the addition of 2 mM cyanide (CN) to the seawater produces an easily measureable increase in Cai, suggesting that mitochondrial buffering in the periphery is substantial. Making the pH of the axoplasm of a normal axon alkaline with 30 mM NH4+ -50 mM Ca seawater, reduces the resting glow of the axon but results in an even more rapid increase in Cai with stimulation. In a phenol red-injected axon, this treatment results in a measureable response to stimulation in the absence of CN.     

Purification of histidine-tagged aequorin with a reactive cysteine residue for chemical conjugations and its application for bioluminescent sandwich immunoassays

Highly purified histidine-tagged aequorin with a reactive cysteine residue (His-Cys4-aequorin) was obtained from the periplasmic space of Escherichia coli cells by nickel-chelate affinity chromatography and hydrophobic chromatography. The procedure yielded 40.3mg of His-Cys4-aequorin from 2L of cultured cells with over 95% purity. The chemical conjugates of His-Cys4-aequorin with maleimide-activated streptavidin and maleimide-activated biotin were prepared without significant loss of luminescence activity and were applied to the bioluminescent sandwich immunoassay for α-fetoprotein (AFP) as a model analyte. The measurable range of AFP by these conjugates was 0.01-100 ng/ml and the sensitivities were similar to that using aequorin-labeled specific antibody and amino-biotinylated aequorin.     

High-level expression and purification of apoaequorin.

A fairly rapid and improved method for producing large amounts of highly pure apoaequorin, the apoprotein of aequorin which emits light on binding Ca2+, is described. The method consists of fusing the gene of the outer membrane protein A (ompA) secretion signal peptide of Escherichia coli to the apoaequorin gene and expressing the fused gene in the bacterium. The expressed protein is correctly cleaved in the process of being secreted across the cell membrane into the culture medium. The apoaequorin is subsequently purified by acid precipitation and DEAE-cellulose chromatography, yielding a product of greater than 95% purity. The availability of pure apoaequorin makes possible detailed studies of the physical-chemical properties of this Ca2(+)-binding protein and allows for the preparation of pure aequorin for use in highly specific and sensitive assays for Ca2+.     

Toxoid of Clostridium botulinum type F: purification and immunogenicity studies.

Toxin from Clostridium botulinum type F was recovered from dialysis cultures and partially purifed by: (i) ammonium sulfate and ethanol precipitation; (ii) O-(diethylaminoethyl)-cellulose chromatography; or (iii) diethylaminoethyl-cellulose chromatography followed by O-(carboxymethyl)-cellulose chromatography. Toxin purities as reflected by specific activity were 1.83 X 10(6), 9.8 X 10(6), and 2.0 X 10(7) mouse 50% lethal doses (LD50)/mg of N, respectively, for toxins purified by the three methods. The toxins were converted to toxoids by incubation at 35 C in the presence of 0.3 to 0.45% formalin for 21 to 35 days. Toxoids were immunogenic in guinea pigs, as demonstrated by serum antitoxin response and the immunized animals' resistance to challenge by type F botulinal toxin. The immune response to type F toxoids was lower when toxoids of serotypes A, B, C, D, and E were combined with the type F toxoid than when the type F toxoid only was administered. The toxoid prepared from the most highly purified toxin (method [iii]) conferred the highest immunity in guinea pigs at a given dose level. A relation between serum antitoxin level and resistance to challenge was observed. At least 50% of the groups of guinea pigs with 0.015 antitoxin units or more per ml survived challenge by 10(5) mouse LD50 of type F botulinal toxin. A dose of 3.75 mug of N of the most highly purified type F toxoid in combination with the other five serotypes of botulinal toxoid invoked an immune response in guinea pigs comparable to that considered adequate for the other toxoids.     

Excitation-contraction coupling in cardiac Purkinje fibers. Effects of cardiotonic steroids on the intracellular [Ca2+] transient, membrane potential, and contraction

The [Ca2+]-activated photoprotein aequorin was used to measure [Ca2+] in canine cardiac Purkinje fibers during the positive inotropic and toxic effects of ouabain, strophanthidin, and acetylstrophanthidin. The positive inotropic effect of these substances was associated with increases in the two components of the aequorin signal, L1 and L2. On the average, strophanthidin at 10(-7) M produced steady, reversible increases in L1, L2, and peak twitch tension of 20, 91, and 240%, respectively. This corresponds to increases in the upper-limit spatial average [Ca2+] from 1.9 X 10(-6) M to 2.1 X 10(-6) M at L1 and from 1.4 X 10(-6) M to 1.8 X 10(-6) M at L2. Elevation of diastolic luminescence above the control level was not detected. At higher concentrations (5 X 10(-7) M), strophanthidin produced aftercontractions, diastolic depolarization, and transient depolarizations, all of which were associated with temporally similar changes in [Ca2+]. During these events, diastolic [Ca2+] rose from the normal level of approximately 3 X 10(-7) M up to 1-2 X 10(-6) M. The negative inotropic effect of 5 X 10(-7) M strophanthidin was not associated with a corresponding decrease in the [Ca2+] transient but was associated with a change in the relationship between [Ca2+] and tension. Assuming the Na+-lag mechanism of cardiotonic steroid action, we conclude the following: at low concentrations of drug, increased Ca2+ uptake by the sarcoplasmic reticulum prevents a detectable rise in cytoplasmic [Ca2+] during diastole, but this increased Ca2+ uptake results in increased release of Ca2+ during the action potential. At higher drug concentrations, observable [Ca2+] changes during diastole activate tension and membrane conductance changes.     

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.     

Preparation and characterization of singly labeled ruthenium polypyridine cytochrome c derivatives

A novel two-step procedure has been developed to prepare cytochrome c derivatives labeled at specific lysine amino groups with ruthenium bis(bipyridine) dicarboxybipyridine [RuII(bpy)2(dcbpy)]. In the first step, cytochrome c was treated with the mono-N-hydroxysuccinimide ester of 4,4'-dicarboxy-2,2'-bipyridine (dcbpy) to convert positively charged lysine amino groups to negatively charged dcbpy-lysine groups. Singly labeled dcbpy-cytochrome c derivatives were then separated and purified by ion-exchange chromatography. In the second step, the individual dcbpy-cytochrome c derivatives were treated with RuII(bpy)2CO3 to form singly labeled RuII(bpy)2(dcbpy-cytochrome c) derivatives. The specific lysine labeled in each derivative was determined by reverse-phase chromatography of a tryptic digest. All of the derivatives had a strong luminescence emission centered at 662 nm, but the luminescence decay rates were increased relative to that of a non-heme protein control, RuII(bpy)2(dcbpy-lysozyme), which was 1.8 X 10(6) s-1. The luminescence decay rates were found to be 21, 16, 7.2, 5.7, 4.3, 4.3, and 3.5 X 10(6) s-1 for derivatives singly labeled at lysines 13, 72, 25, 7, 39, 86, and 87, respectively. There was an inverse relationship between the luminescence decay rates and the distances between the ruthenium labels and the heme group. The increased luminescence decay rates observed in the cytochrome c derivatives might be due to electron transfer from the excited triplet state of ruthenium to the ferric heme group. However, it is also possible that an energy-transfer mechanism might contribute to the luminescence quenching.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of calcium chelators on the Ca2+-dependent luminescence of aequorin.

The luminescence of aequorin, a useful tool for studying intracellular Ca2+, was recently found to be inhibited by the free EDTA and EGTA that are present in calcium buffers. In the present study we have examined the effect of the free forms of various chelators in the calibration of [Ca2+] with aequorin. Free EDTA and EGTA in low-ionic-strength solutions strongly inhibited the Ca2+-triggered luminescence of aequorin, causing large errors in the calibration of [Ca2+] (approx. 2 pCa units), whereas in solutions containing 150mM-KCl, errors were relatively small (0.2-0.3 pCa units). Citric acid in low-ionic-strength solutions and [(carbamoylmethyl)imino]diacetic acid in high-ionic-strength solutions showed no inhibition and did not cause detectable error in the calibration of [Ca2+], indicating that they are better chelators than EDTA and EGTA for use with aequorin.