Luminescence of Aequorin Is Triggered by the Binding of Two Calcium Ions

doi:10.1006/bbrc.1995.1821

Biochemical and Biophysical Research Communications

Volume 211, Issue 2, 15 June 1995, Pages 359-363

https://doi.org/10.1006/bbrc.1995.1821 Get rights and content

Abstract

The photoprotein aequorin, capable of emitting light in the presence of a trace amount of Ca²⁺, is a useful indicator for studying intracellular calcium. The primary structure of aequorin indicated the presence of three Ca²⁺-binding sites, whereas log-log plots of the luminescence intensity versus Ca²⁺ concentration gave slopes ranging from 2 to 3 depending on the conditions used, suggesting the involvement of two or three Ca²⁺ ions in the luminescence reaction. Accurate information on the stoichiometry of Ca²⁺ is essential in interpreting the assay results obtained with aequorin. This study clearly shows that aequorin luminescence is triggered by the binding of two Ca²⁺ ions, based on the results of titrating aequorin with Ca²⁺.

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Cited by (58)

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2023, The World of Sea Cucumbers: Challenges, Advances, and Innovations
Bioluminescence, light production by a living organism, is often seen in deep-sea benthic animals, including sea cucumbers. In this chapter, we review holothuroid bioluminescence from taxonomic, behavioral, and biochemical perspectives and describe new observations with an evolutionary analysis. Holothuroids emit light in response to physical stimuli, spontaneous bioluminescence having not been observed. All luminous holothuroids emit light from their body walls and are not known to secrete it extracellularly, although in some species the glowing skin may be shed in the water. The color of emitted light ranges from blue to blue-green, with wavelength peaks between 463 and 505 nm. The chemical basis of light production is unknown, although there is no evidence that bacteria are involved. Out of 249 genera in Holothuroidea, bioluminescence has been reported in 26, including 10 species reported here as discovered to be bioluminescent for the first time. These new observations include the genus Molpadia, which is the first luminous member of the order Molpadida. Using a recent molecular phylogeny, we estimated six independent origins of bioluminescence in the Holothuroidea, suggesting that more than 200 species are possibly bioluminescent. These insights point to the prevalence and ecological importance of bioluminescence in the deep sea, which is susceptible to severe disruption by deep-sea mining and other human activities.
Bioluminescence and kinetic aspects of double mutated aequorin variants
2018, International Journal of Biological Macromolecules
Citation Excerpt :
In the presence of Ca2+, even in trace amounts, conformational changes of aequorin will result in conversion of hydroperoxycoelenterazine (which is formed from coelenterazine and molecular oxygen) to coelenteramide along with release of CO2 [4,5]. Decarboxylation of coelenterazine leads to fast release of photons and subsequently emission of an instant flash of blue light (λmax = 469 nm) in less than 10 s [6–8]. Molecular biology techniques have been extensively used to improve the emission properties of existing photoproteins through production of mutated variants.
Aequorin as an old small calcium-sensitive photoprotein is a blue fluorescence protein which converts coelenterazine (a substrate) to coelenteramide with a flash type emission. The decay kinetics and emission properties of this protein can be changed using directed mutagenesis of crucial amino acid residue. In this work, we prepared three double mutants: Y⁸²F/W⁸⁶F, Y⁸²F/D¹⁵³G, and W⁸⁶F/D¹⁵³G. According to our results, it seems that presence of Y⁸²F mutation results in shift of emission to longer wavelengths while the W⁸⁶F mutation shifts the emission to shorter wavelengths. Furthermore, comparison of the variants for light half-life indicated decreased t_1/2 for the two variants of Y⁸²F/D¹⁵³G and W⁸⁶F/D¹⁵³G. But in compared to wild type aequorin, the Y⁸²F/W⁸⁶F variant displayed a 2-fold increase of light half-life. On the other hand, the thermostability properties of double mutants confirmed that only Y⁸²F/D¹⁵³G variant of apoaequorin is higher stability than others. Also, the single W⁸⁶F mutant reached the highest stability against thermal shock. Our data suggest that replacement of single or few point mutations in the binding pocket or active site of aequorin affects its bioluminescence and kinetic properties and so could be used for new reporter production of this photoprotein with the feasibility and limited substitutions.
Evolutionary conservation of EF-hand ΙΙ loop in aequorin: Priority of intensity to decay rate in bioluminescence emission
2017, Archives of Biochemistry and Biophysics
Citation Excerpt :
Ca2+-regulated photoproteins are mainly found in a variety of marine organisms [9]. Aequorin from the jellyfish Aequorea victoria is one of the Ca2+-regulated photoprotein that was firstly reported in 1962 [10] and as a model photoprotein has been intensively studied [11–14]. It is actually a stable enzyme-substrate complex containing two distinctive units including aequorin with molecular weight of 22 kDa and its substrate known as 2 hydroperoxycoelenterazine which acts as chemiluminescent molecule (luciferin) in bioluminescence activity.
As a Ca²⁺-regulated photoprotein, aequorin (Aeq) contains four EF-hand motifs, the second one lacks the standard sequence for Ca²⁺ coordination and doesn't bind to Ca²⁺. 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.
Characterization of Ca<sup>2+</sup> signaling in the external yolk syncytial layer during the late blastula and early gastrula periods of zebrafish development
2013, Biochimica et Biophysica Acta - Molecular Cell Research
Citation Excerpt :
In addition, during the signalling period, a few (in this case two) rapid Ca2 + spikes are superimposed onto a slower increase in the background level of Ca2 +, after which there is an even more gradual decrease in the overall background level over a period of ~ 60 min with no accompanying rapid Ca2 + spikes. Assuming that the aequorin luminescence varies with the [Ca2 +] in vivo as it does in vitro, i.e., to the second power [50], a 7-fold rise in luminescence represents a ~ 2.6-fold increase in [Ca2 +]. As the resting level of [Ca2 +] in the cytosol of zebrafish embryonic cells has been reported to be ~ 60 nM [19], we thus estimate the peak Ca2 + rise during E-YSL wave propagation to be ~ 156 nM.
Preferential loading of the complementary bioluminescent (f-aequorin) and fluorescent (Calcium Green-1 dextran) Ca^2 + reporters into the yolk syncytial layer (YSL) of zebrafish embryos, revealed the generation of stochastic patterns of fast, short-range, and slow, long-range Ca^2 + waves that propagate exclusively through the external YSL (E-YSL). Starting abruptly just after doming (~ 4.5 h post-fertilization: hpf), and ending at the shield stage (~ 6.0 hpf) these distinct classes of waves propagated at mean velocities of ~ 50 and ~ 4 μm/s, respectively. Although the number and pattern of these waves varied between embryos, their initiation site and arcs of propagation displayed a distinct dorsal bias, suggesting an association with the formation and maintenance of the nascent dorsal-ventral axis. Wave initiation coincided with a characteristic clustering of YSL nuclei (YSN), and their associated perinuclear ER, in the E-YSL. Furthermore, the inter-YSN distance (IND) appeared to be critical such that Ca^2 + wave propagation occurred only when this was <~ 8 μm; an IND >~ 8 μm was coincidental with wave termination at shield stage. Treatment with the IP₃R antagonist, 2-APB, the Ca^2 + buffer, 5,5′-dibromo BAPTA, and the SERCA-pump inhibitor, thapsigargin, resulted in a significant disruption of the E-YSL Ca^2 + waves, whereas exposure to the RyR antagonists, ryanodine and dantrolene, had no significant effect. These findings led us to propose that the E-YSL Ca^2 + waves are generated mainly via Ca^2 + release from IP₃Rs located in the perinuclear ER, and that the clustering of the YSN is an essential step in providing a CICR pathway required for wave propagation. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.
Purification and characterization of a novel thermostable luciferase from Benthosema pterotum
2013, Journal of Photochemistry and Photobiology B: Biology
A novel luciferase from Benthosema pterotum, collected from Port of Jask, close to Persian Gulf, was purified for the first time, using Q-Sepharose anion exchange chromatography. The molecular mass of the novel enzyme, measured by SDS–PAGE technique, was about 27 kDa and its K_m value is 0.4 μM; both values are similar to those of other coelenterazine luciferases. B. pterotum (BP) luciferase showed maximum intensity of emitted light at 40 °C, in 20 mM Tris buffer, pH 9 and 20 mM magnesium concentration. Experimental measurements indicated that BP luciferase is a relatively thermostable enzyme; furthermore it shows a high residual activity at extreme pH values. Its biological activity is strongly inhibited by 1 mM Cu²⁺, Zn²⁺ and Ni²⁺, while calcium and mainly magnesium ions strongly increase BP luciferase activity. The B. pterotum luciferase generated blue light with a maximum emission wavelength at 475 nm and showed some similarity with other luciferases, while other parameters appeared quite different, in this way, confirming that a novel protein has been purified.
Aequorin-based genetic approaches to visualize Ca <sup>2+</sup> signaling in developing animal systems
2012, Biochimica et Biophysica Acta - General Subjects
In recent years, as our understanding of the various roles played by Ca^2 + signaling in development and differentiation has expanded, the challenge of imaging Ca^2 + dynamics within living cells, tissues, and whole animal systems has been extended to include specific signaling activity in organelles and non-membrane bound sub-cellular domains.
In this review we outline how recent advances in genetics and molecular biology have contributed to improving and developing current bioluminescence-based Ca^2 + imaging techniques. Reporters can now be targeted to specific cell types, or indeed organelles or domains within a particular cell.
These advances have contributed to our current understanding of the specificity and heterogeneity of developmental Ca^2 + signaling. The improvement in the spatial resolution that results from specifically targeting a Ca^2 + reporter has helped to reveal how a ubiquitous signaling messenger like Ca^2 + can regulate coincidental but different signaling events within an individual cell; a Ca^2 + signaling paradox that until now has been hard to explain.
Techniques used to target specific reporters via genetic means will have applications beyond those of the Ca^2 + signaling field, and these will, therefore, make a significant contribution in extending our understanding of the signaling networks that regulate animal development. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signalling.

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Regular ArticleLuminescence of Aequorin Is Triggered by the Binding of Two Calcium Ions

Abstract

Regular Article
Luminescence of Aequorin Is Triggered by the Binding of Two Calcium Ions