Aequorin

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Sylvia Daunert - One of the best experts on this subject based on the ideXlab platform.

  • red shifted Aequorin variants incorporating non canonical amino acids applications in in vivo imaging
    PLOS ONE, 2016
    Co-Authors: Kristen Marie Grinstead, Laura Rowe, Emre Dikici, Charles Mark Ensor, Smita Joel, Pirouz Daftarian, Jean Marc Zingg, Sylvia Daunert
    Abstract:

    The increased importance of in vivo diagnostics has posed new demands for imaging technologies. In that regard, there is a need for imaging molecules capable of expanding the applications of current state-of-the-art imaging in vivo diagnostics. To that end, there is a desire for new reporter molecules capable of providing strong signals, are non-toxic, and can be tailored to diagnose or monitor the progression of a number of diseases. Aequorin is a non-toxic photoprotein that can be used as a sensitive marker for bioluminescence in vivo imaging. The sensitivity of Aequorin is due to the fact that bioluminescence is a rare phenomenon in nature and, therefore, it does not suffer from autofluorescence, which contributes to background emission. Emission of bioluminescence in the blue-region of the spectrum by Aequorin only occurs when calcium, and its luciferin coelenterazine, are bound to the protein and trigger a biochemical reaction that results in light generation. It is this reaction that endows Aequorin with unique characteristics, making it ideally suited for a number of applications in bioanalysis and imaging. Herein we report the site-specific incorporation of non-canonical or non-natural amino acids and several coelenterazine analogues, resulting in a catalog of 72 cysteine-free, Aequorin variants which expand the potential applications of these photoproteins by providing several red-shifted mutants better suited to use in vivo. In vivo studies in mouse models using the transparent tissue of the eye confirmed the activity of the Aequorin variants incorporating L-4-iodophehylalanine and L-4-methoxyphenylalanine after injection into the eye and topical addition of coelenterazine. The signal also remained localized within the eye. This is the first time that Aequorin variants incorporating non-canonical amino acids have shown to be active in vivo and useful as reporters in bioluminescence imaging.

  • Aequorin mutants with increased thermostability
    Analytical and Bioanalytical Chemistry, 2014
    Co-Authors: Xiaoge Qu, Laura Rowe, Emre Dikici, Mark Ensor, Sylvia Daunert
    Abstract:

    Bioluminescent labels can be especially useful for in vivo and live animal studies due to the negligible bioluminescence background in cells and most animals, and the non-toxicity of bioluminescent reporter systems. Significant thermal stability of bioluminescent labels is essential, however, due to the longitudinal nature and physiological temperature conditions of many bioluminescent-based studies. To improve the thermostability of the bioluminescent protein Aequorin, we employed random and rational mutagenesis strategies to create two thermostable double mutants, S32T/E156V and M36I/E146K, and a particularly thermostable quadruple mutant, S32T/E156V/Q168R/L170I. The double Aequorin mutants, S32T/E156V and M36I/E146K, retained 4 and 2.75 times more of their initial bioluminescence activity than wild-type Aequorin during thermostability studies at 37 °C. Moreover, the quadruple Aequorin mutant, S32T/E156V/Q168R/L170I, exhibited more thermostability at a variety of temperatures than either double mutant alone, producing the most thermostable Aequorin mutant identified thus far.

  • Aequorin variants with improved bioluminescence properties.
    Protein engineering design & selection : PEDS, 2009
    Co-Authors: Emre Dikici, Laura Rowe, Mark Ensor, Sapna K. Deo, L. Millner, C. Logue, Sylvia Daunert
    Abstract:

    The photoprotein Aequorin has been widely used as a bioluminescent label in immunoassays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. It is composed of apoAequorin (189 amino acid residues), the imidazopyrazine chromophore coelenterazine and molecular oxygen. The emission characteristics of Aequorin can be changed by rational design of the protein to introduce mutations in its structure, as well as by substituting different coelenterazine analogues to yield semi-synthetic Aequorins. Variants of Aequorin were created by mutating residues His16, Met19, Tyr82, Trp86, Trp108, Phe113 and Tyr132. Forty-two Aequorin mutants were prepared and combined with 10 different coelenterazine analogues in a search for proteins with different emission wavelengths, altered decay kinetics and improved stability. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants with significantly altered bioluminescent properties.

  • Handbook of Biosensors and Biochips - Recombinant Aequorin‐Based Systems for Biomarker Analysis
    Handbook of Biosensors and Biochips, 2008
    Co-Authors: Laura Rowe, Emre Dikici, Mark Ensor, Sapna K. Deo, Krystal Teasley, Sylvia Daunert
    Abstract:

    Detection methods utilized in the development of biosensors and biochips often require labeling strategies. Commonly employed labels include radioisotopes, enzymes, and fluorohphores. Bioluminescent proteins offer an alternative labeling option and are becoming increasingly popular because of their versatility, sensitivity, and nontoxicity. Bioluminescent labels emit light of varying colors as a method of releasing the energy that is produced by their specific chemical reactions. Aequorin is an example of one such bioluminescent protein which has been studied extensively and used in a variety of bioanalytical techniques. In this chapter, we describe the qualities of Aequorin and the potential that it has as a generic label. This chapter also details several Aequorin-based immunoassays that have been developed for the monitoring of biomarkers important to both hormonal disorders and cardiovascular disease. Lastly, we discuss the progress and the pitfalls associated with developing multianalyte detection schemes and robust sensing technologies based on bioluminescent, Aequorin labeling. Keywords: Aequorin; label; reporter; calcium detection; bioluminescence; photoprotein; immunoassay; lab-on-a-CD; multianalyte detection

  • Surface properties of "jellyfish": Langmuir monolayer and Langmuir-Blodgett film studies of recombinant Aequorin.
    Langmuir, 2007
    Co-Authors: Chengshan Wang, Mark Ensor, Sylvia Daunert, Miodrag Micic, Roger M. Leblanc
    Abstract:

    : In this paper, we studied the surface properties of recombinant Aequorin at the air-water interface. Using the Langmuir monolayer technique, the surface properties of Aequorin were studied, including the surface pressure and surface potential-area isotherms, compression-decompression cycles, and stability on Trizma Base (Tris/HCl) buffer at pH 7.6. The results showed that Aequorin formed a stable Langmuir monolayer and the surface pressure-area isotherms were dependent on both pH and ionic strength. At a pH higher or lower than 7.6, the limiting molecular area decreased. The circular dichroism (CD) spectra of Aequorin in aqueous solutions explained this result: when the pH was higher than 7.6, the alpha-helix conformation changed to unordered structures, whereas at a pH lower than 7.6, the alpha-helix conformation changed to beta-sheet. The addition of calcium chloride to the Tris/HCl buffer subphase (pH 7.6) caused an increase of the limiting molecular area of the Aequorin Langmuir monolayer. The fluorescence spectra of a Langmuir-Blodgett (LB) film of Aequorin in the presence of calcium chloride indicated that the Aequorin transformed to the apoAequorin.

Sapna K. Deo - One of the best experts on this subject based on the ideXlab platform.

  • Aequorin variants with improved bioluminescence properties.
    Protein engineering design & selection : PEDS, 2009
    Co-Authors: Emre Dikici, Laura Rowe, Mark Ensor, Sapna K. Deo, L. Millner, C. Logue, Sylvia Daunert
    Abstract:

    The photoprotein Aequorin has been widely used as a bioluminescent label in immunoassays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. It is composed of apoAequorin (189 amino acid residues), the imidazopyrazine chromophore coelenterazine and molecular oxygen. The emission characteristics of Aequorin can be changed by rational design of the protein to introduce mutations in its structure, as well as by substituting different coelenterazine analogues to yield semi-synthetic Aequorins. Variants of Aequorin were created by mutating residues His16, Met19, Tyr82, Trp86, Trp108, Phe113 and Tyr132. Forty-two Aequorin mutants were prepared and combined with 10 different coelenterazine analogues in a search for proteins with different emission wavelengths, altered decay kinetics and improved stability. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants with significantly altered bioluminescent properties.

  • Handbook of Biosensors and Biochips - Recombinant Aequorin‐Based Systems for Biomarker Analysis
    Handbook of Biosensors and Biochips, 2008
    Co-Authors: Laura Rowe, Emre Dikici, Mark Ensor, Sapna K. Deo, Krystal Teasley, Sylvia Daunert
    Abstract:

    Detection methods utilized in the development of biosensors and biochips often require labeling strategies. Commonly employed labels include radioisotopes, enzymes, and fluorohphores. Bioluminescent proteins offer an alternative labeling option and are becoming increasingly popular because of their versatility, sensitivity, and nontoxicity. Bioluminescent labels emit light of varying colors as a method of releasing the energy that is produced by their specific chemical reactions. Aequorin is an example of one such bioluminescent protein which has been studied extensively and used in a variety of bioanalytical techniques. In this chapter, we describe the qualities of Aequorin and the potential that it has as a generic label. This chapter also details several Aequorin-based immunoassays that have been developed for the monitoring of biomarkers important to both hormonal disorders and cardiovascular disease. Lastly, we discuss the progress and the pitfalls associated with developing multianalyte detection schemes and robust sensing technologies based on bioluminescent, Aequorin labeling. Keywords: Aequorin; label; reporter; calcium detection; bioluminescence; photoprotein; immunoassay; lab-on-a-CD; multianalyte detection

  • Bioluminescence resonance energy transfer from Aequorin to a fluorophore: an artificial jellyfish for applications in multianalyte detection
    Analytical and Bioanalytical Chemistry, 2005
    Co-Authors: Sapna K. Deo, Mara Mirasoli, Sylvia Daunert
    Abstract:

    In nature, the green light emission observed in the jellyfish Aequorea victoria is a result of a non-radiative energy transfer from the excited-state Aequorin to the green fluorescent protein. In this work, we have modified the photoprotein Aequorin by attaching selected fluorophores at a unique site on the protein. This will allow for in vitro transfer of bioluminescent energy from Aequorin to the fluorophore thus creating an “artificial jellyfish”. The fluorophores are selected such that the excitation spectrum of the fluorophore overlaps with the emission spectrum of Aequorin. By modifying Aequorin with different fluorophores, bioluminescent labels with different emission maxima are produced, which will allow for the simultaneous detection of multiple analytes. By examining the X-ray crystal structure of the protein, four different sites for introduction of the unique cysteine residue were evaluated. Two fluorophores with differing emission maxima were attached individually to the mutants through the sulfhydryl group of the cysteine molecule. Two of the fluorophore-labeled mutants showed a peak corresponding to fluorophore emission thus indicating resonance energy transfer from Aequorin to the fluorophore.

  • An immunoassay for Leu-enkephalin based on a C-terminal Aequorin-peptide fusion.
    Analytical chemistry, 2001
    Co-Authors: Sapna K. Deo, Sylvia Daunert
    Abstract:

    Recently we demonstrated that the fusion of an octapeptide to the C-terminus of a cysteine-free mutant of Aequorin showed no inhibitory effect on the luminescence activity of the photoprotein. This observation is of particular importance when the use of Aequorin as a label in the development of immunoassays for peptides whose activity lies in their C-terminal region or the epitope for antibody recognition is at their C-terminus is desired. In the case of opioid peptides, antibodies are directed toward their C-terminus as they differ from each other at this terminus. The goal of this study was to develop an immunoassay for Leu-enkephalin, a mammalian opioid peptide, using a C-terminal Aequorin−peptide fusion protein. For that, the N-terminus of Leu-enkephalin was genetically fused to the C-terminus of a cysteine-free mutant of Aequorin. It was observed that the C-terminal conjugated Aequorin maintained its luminescence activity. An immunoassay for Leu-enkephalin was then developed using the Aequorin−Leu-en...

  • C-Terminal and N-Terminal Fusions of Aequorin with Small Peptides in Immunoassay Development
    Bioconjugate chemistry, 2001
    Co-Authors: Sapna K. Deo, Jennifer C. Lewis, Sylvia Daunert
    Abstract:

    Aequorin fusion proteins have been used extensively in intracellular Ca2+ measurements and in the development of binding assays. Gene fusions to Aequorin for production of fusion proteins have been...

Satoshi Inouye - One of the best experts on this subject based on the ideXlab platform.

  • Slow luminescence kinetics of semi-synthetic Aequorin: expression, purification and structure determination of cf3-Aequorin
    Journal of biochemistry, 2018
    Co-Authors: Satoshi Inouye, Yuri Tomabechi, Takamitsu Hosoya, Shun-ichi Sekine, Mikako Shirouzu
    Abstract:

    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 A 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.

  • Purification of histidine-tagged Aequorin with a reactive cysteine residue for chemical conjugations and its application for bioluminescent sandwich immunoassays
    Protein Expression and Purification, 2012
    Co-Authors: Satoshi Inouye, Jun'ichi Sato
    Abstract:

    Abstract 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 40.3 mg of His-Cys4-Aequorin from 2 L of cultured cells with over 95% purity. The chemical conjugates of His-Cys4-Aequorin with maleimide-acitivated 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.

  • Recombinant Aequorin with a reactive cysteine residue for conjugation with maleimide-activated antibody
    Analytical biochemistry, 2008
    Co-Authors: Satoshi Inouye, Jun'ichi Sato
    Abstract:

    Abstract The mutated recombinant Aequorin with a reactive cysteine residue (Cys–Aequorin) was highly purified and then conjugated with a maleimide-activated antibody without significant loss of luminescence activity. The conjugate ratio of Cys–Aequorin to heavy chain of immunoglobulin G (IgG) was estimated to be 1:1. To test the bioluminescent immunoassay with Aequorin-labeled antibody, α-fetoprotein (AFP), a serological marker of liver cancer, was used as a model analyte. The measurable range of AFP was 0.02 to 200 ng/ml with the coefficient of variation between 2.1 and 4.5%.

  • blue fluorescent protein from the calcium sensitive photoprotein Aequorin catalytic properties for the oxidation of coelenterazine as an oxygenase
    FEBS Letters, 2006
    Co-Authors: Satoshi Inouye, Satoko Sasaki
    Abstract:

    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.

  • The crystal structures of semi-synthetic Aequorins
    Protein science : a publication of the Protein Society, 2005
    Co-Authors: Sachiko Toma, Satoshi Inouye, Khoon Tee Chong, Atsushi Nakagawa, Katsunori Teranishi, Osamu Shimomura
    Abstract:

    The photoprotein Aequorin emits light by an intramolecular reaction in the presence of a trace amount of Ca2+. Semi-synthetic Aequorins, produced by replacing the coelenterazine moiety in Aequorin with the analogues of coelenterazine, show widely different sensitivities to Ca2+. To understand the structural basis of the Ca2+-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 Ca2+, and the loop of EF-hand IV is clearly deformed. It is most likely that the binding of Ca2+ 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 Ca2+ 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 Ca2+-sensitivity and reaction rate are explained by the capability of the involved groups and structures to undergo conformational changes in response to the Ca2+-binding.

Osamu Shimomura - One of the best experts on this subject based on the ideXlab platform.

  • The crystal structures of semi-synthetic Aequorins
    Protein science : a publication of the Protein Society, 2005
    Co-Authors: Sachiko Toma, Satoshi Inouye, Khoon Tee Chong, Atsushi Nakagawa, Katsunori Teranishi, Osamu Shimomura
    Abstract:

    The photoprotein Aequorin emits light by an intramolecular reaction in the presence of a trace amount of Ca2+. Semi-synthetic Aequorins, produced by replacing the coelenterazine moiety in Aequorin with the analogues of coelenterazine, show widely different sensitivities to Ca2+. To understand the structural basis of the Ca2+-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 Ca2+, and the loop of EF-hand IV is clearly deformed. It is most likely that the binding of Ca2+ 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 Ca2+ 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 Ca2+-sensitivity and reaction rate are explained by the capability of the involved groups and structures to undergo conformational changes in response to the Ca2+-binding.

  • The discovery of Aequorin and green fluorescent protein
    Journal of Microscopy, 2005
    Co-Authors: Osamu Shimomura
    Abstract:

    We discovered Aequorin and green fluorescent protein (GFP) in 1961 from the same species of jellyfish (Shimomura et al ., 1962). Our target was a luminescent substance, Aequorin, and GFP was isolated as a by-product of Aequorin owing to its bright conspicuous fluorescence. Both are unusual proteins but they had no particular importance when we first reported them. Their importance became apparent in the course of studies, and now, 40 years after their discovery, they are well known and widely used, Aequorin as a calcium probe and GFP as a marker protein. In the characterization of these proteins, information obtained from the bioluminescence of the ostracod Cypridina played an extremely important role. Without the information gained on Cypridina luminescence, the characterization of Aequorin would not have been possible. Because I had studied the bioluminescence of Cypridina before I worked on Aequorin, I would like to begin my story with my encounter with Cypridina .

  • The crystal structure of the photoprotein Aequorin at 2.3 Å resolution
    Nature, 2000
    Co-Authors: James F. Head, Satoshi Inouye, Katsunori Teranishi, Osamu Shimomura
    Abstract:

    Aequorin is a calcium-sensitive photoprotein originally obtained from the jellyfish Aequorea aequorea. Because it has a high sensitivity to calcium ions and is biologically harmless, Aequorin is widely used as a probe to monitor intracellular levels of free calcium. The Aequorin molecule contains four helix-loop-helix 'EF-hand' domains, of which three can bind calcium. The molecule also contains coelenterazine as its chromophoric ligand. When calcium is added, the protein complex decomposes into apoAequorin, coelenteramide and CO2, accompanied by the emission of light. ApoAequorin can be regenerated into active Aequorin in the absence of calcium by incubation with coelenterazine, oxygen and a thiol agent. Cloning and expression of the complementary DNA for Aequorin were first reported in 1985 (refs 2, 6), and growth of crystals of the recombinant protein has been described; however, techniques have only recently been developed to prepare recombinant Aequorin of the highest purity, permitting a full crystallographic study. Here we report the structure of recombinant Aequorin determined by X-ray crystallography. Aequorin is found to be a globular molecule containing a hydrophobic core cavity that accommodates the ligand coelenterazine-2-hydroperoxide. The structure shows protein components stabilizing the peroxide and suggests a mechanism by which calcium activation may occur.

  • Thein SituRegeneration and Extraction of Recombinant Aequorin fromEscherichia coliCells and the Purification of Extracted Aequorin
    Protein expression and purification, 1999
    Co-Authors: Osamu Shimomura, Satoshi Inouye
    Abstract:

    Abstract Recombinant apoAequorin expressed in the periplasmic space of Escherichia coli cells was regenerated into Aequorin and extracted from the cells, simultaneously, using a buffer that contained coelenterazine. Due to the mild extraction conditions, the impurities in the extract were minimal. Thus, the purification of extracted Aequorin could be accomplished in only two steps, anion-exchange chromatography and hydrophobic interaction chromatography, simply by adsorption and elution in both steps. The purified recombinant Aequorin was pure, based on various data, including HPLC analysis and light-emitting activity. The yield of purified Aequorin was 25–35 mg from 600 ml of culture, which was over 75% of the total amount of apoAequorin expressed in E. coli cells.

  • Light-emitting properties of recombinant semisynthetic Aequorins and recombinant fluorescein-conjugated Aequorin for measuring cellular calcium
    Cell calcium, 1993
    Co-Authors: Osamu Shimomura, Yoshito Kishi, B Musicki, S Inouye
    Abstract:

    15 kinds of recombinant semi-synthetic Aequorins and a recombinant fluorescein-conjugated Aequorin were prepared and their properties in Ca2+-triggered luminescence were studied. The semi-synthetic Aequorins showed a wide range of Ca2+-sensitivity. The luminescence intensity of a high-sensitivity type (hcp-Aequorin) was greater than 104-times that of a low-sensitivity type (n-Aequorin) at pCa 6.0–6.5. The fluorescein-conjugated Aequorin exhibited fluorescence in addition to the Ca2+-triggered luminescence, thus it can be used to visualize the diffusion and distribution of Aequorin in cells. The data obtained, particularly the Ca2+-sensitivity curves, are useful in selecting a suitable semi-synthetic Aequorin for an experiment.

O. Shimomura - One of the best experts on this subject based on the ideXlab platform.

  • Cause of spectral variation in the luminescence of semisynthetic Aequorins.
    Biochemical Journal, 1995
    Co-Authors: O. Shimomura
    Abstract:

    Aequorin emits light in the presence of Ca2+, decomposing into apoAequorin, coelenteramide and CO2. Semisynthetic Aequorins, produced by replacing the coelenterazine moiety in Aequorin with analogues of coelenterazine, showed widely different sensitivities to Ca2+ as well as certain spectral variations. A group of semisynthetic Aequorins, e-type Aequorins, showed bimodal luminescence, with peaks at 400-405 nm and 440-475 nm in various intensity ratios, whereas all other Aequorins luminesced with only one peak, in the range 440-475 nm. The cause of the spectral variation was studied by various experiments including: (1) comparison with the fluorescence of the spent solution and the luminescence of the spent solution produced by added coelenterazine; (2) luminescence in 2H2O; (3) the rate of conformational change of apoAequorin; (4) the rates of regeneration in the presence and absence of O2. The results suggested that the spectrum of Ca(2+)-triggered luminescence is strongly affected by the ionic charge on the amide N atom of the coelenteramide that is bound to apoAequorin. When the amide N atom is negatively charged, light is emitted with a 440-475 nm peak. In the case of e-type Aequorins, the negative charge on the amide N atom is less because of the structure of e-coelenterazine involved, resulting in the emission of a 400-405 nm peak from the uncharged form of coelenteramide; the intensity ratio of 400-405 nm peak to 440-475 nm peak is determined by the amount of negative charge resting on the amide N atom of e-coelenteramide at the time of light emission. Most of the spectral variations in luminescence and fluorescence can be explained on the basis of ionic and hydrophobic interaction between a coelenteramide and apoAequorin.

  • The relative rate of Aequorin regeneration from apoAequorin and coelenterazine analogues.
    The Biochemical journal, 1993
    Co-Authors: O. Shimomura, Y Kishi, S Inouye
    Abstract:

    The regeneration of an active semi-synthetic Aequorin, from apoAequorin produced in cells and a coelenterazine analogue, is a key step in measuring Ca2+ in the cells. The relative rates of the regeneration of semi-synthetic Aequorins from apoAequorin and 28 synthetic coelenterazine analogues were compared. The results indicated that the rate is strongly influenced by the analogues used. The regeneration of ordinary Aequorin with normal coelenterazine was relatively fast (50% regeneration in 22 min), whereas the rates of regenerating semi-synthetic Aequorins with coelenterazine analogues varied widely, and all were slower than that of regenerating ordinary Aequorin, except for e-type coelenterazines (containing an extra ethano group). The regeneration with e-type coelenterazines was significantly faster, indicating the possible superiority of e-type analogues in the intracellular regeneration of Aequorin, especially when an increased sensitivity to Ca2+ is needed.

  • Preparation and handling of Aequorin solutions for the measurement of cellular Ca2
    Cell calcium, 1991
    Co-Authors: O. Shimomura
    Abstract:

    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.