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Information on EC 1.13.12.24 - calcium-regulated photoprotein
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1.13.12.24 calcium-regulated photoproteinIUBMB Comments
Ca2+-regulated photoproteins are found in a variety of bioluminescent marine organisms, mostly coelenterates, and are responsible for their light emission. The best studied enzyme is from the jellyfish Aequorea victoria. The enzyme tightly binds the imidazolopyrazinone derivative coelenterazine, which is then peroxidized by oxygen. The hydroperoxide is stably bound until three Ca2+ ions bind to the protein, inducing a structural change that results in the formation of a 1,2-dioxetan-3-one ring, followed by decarboxylation and generation of a protein-bound coelenteramide in an excited state. The calcium-bound protein-product complex is known as a blue fluorescent protein. In vivo the energy is transferred to a green fluorescent protein (GFP) by Forster resonance energy transfer. In vitro, in the absence of GFP, coelenteramide emits a photon of blue light while returning to its ground state.
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Word Map
- 1.13.12.24
-
photoproteins
-
bioluminescence
- aequorin
-
luminescence
- obelia
- longissima
- coelenteramide
-
ctenophore
-
coelenterate
-
jellyfish
-
ef-hand
- 2-hydroperoxycoelenterazine
- mnemiopsis
- hydroid
- clytin
-
hydromedusan
- leidyi
- synthesis
- analysis
The expected taxonomic range for this enzyme is: Eumetazoa
Reaction Schemes
[apoaequorin]
+
+
+
3
=
[excited state blue fluorescent protein]
+
Synonyms
obelin, blue fluorescent protein, mnemiopsin, berovin, mitrocomin, mnemiopsin 2, ca2+-regulated photoprotein, calcium-regulated photoprotein, mnemiopsin 1, halistaurin, 
more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
aequorin
alcium-activated photoprotein
-
-
-
-
berovin
Ca2+-regulated photoprotein
-
-
-
-
clytin
halistaurin
-
-
-
-
mitrocomin
mnemiopsin
-
-
-
-
obelin
phialidin
-
-
-
-
aequorin
-
-
-
-
berovin
-
-
-
-
clytin
-
-
-
-
mitrocomin
-
-
-
-
obelin
-
-
-
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
[aequorin] + 3 Ca2+ = [aequorin] 1,2-dioxetan-3-one
(1c)
-
-
-
[aequorin] 1,2-dioxetan-3-one = [excited state blue fluorescent protein] + CO2
(1d)
-
-
-
[apoaequorin containing coelenterazine] + O2 = [aequorin]
(1b)
-
-
-
[apoaequorin] + coelenterazine + O2 + 3 Ca2+ = [excited state blue fluorescent protein] + CO2
overall reaction
-
-
-
[apoaequorin] + coelenterazine = [apoaequorin containing coelenterazine]
(1a)
-
-
-
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SYSTEMATIC NAME
IUBMB Comments
coelenterazine:oxygen 2-oxidoreductase (decarboxylating, calcium-dependent)
Ca2+-regulated photoproteins are found in a variety of bioluminescent marine organisms, mostly coelenterates, and are responsible for their light emission. The best studied enzyme is from the jellyfish Aequorea victoria. The enzyme tightly binds the imidazolopyrazinone derivative coelenterazine, which is then peroxidized by oxygen. The hydroperoxide is stably bound until three Ca2+ ions bind to the protein, inducing a structural change that results in the formation of a 1,2-dioxetan-3-one ring, followed by decarboxylation and generation of a protein-bound coelenteramide in an excited state. The calcium-bound protein-product complex is known as a blue fluorescent protein. In vivo the energy is transferred to a green fluorescent protein (GFP) by Forster resonance energy transfer. In vitro, in the absence of GFP, coelenteramide emits a photon of blue light while returning to its ground state.
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
[aequorin] 1,2-dioxetan-3-one
[aequorin] coelenteramide + CO2 + hnu
reaction in presence of Ca2+
-
-
?
[apoaequorin] + coelenterazine + O2
[aequorin] 1,2-dioxetan-3-one
-
-
-
?
[apoaequorin] + coelenterazine + O2
[apoaequorin] + coelenteramide + CO2 + hnu
-
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
[apoaequorin] + cp-coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
-
-
-
?
[apoaequorin] + f-coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
-
-
-
?
[apoaequorin] + h-coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
-
-
-
?
[apoaequorin] + hcp-coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
-
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
-
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
-
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
flash type light emission
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
the specific bioluminescence activities of five recombinant hydromedusan Ca2+-regulated photoproteins - aequorin, mitrocomin, clytin and obelins from Obelia longissima and Obelia geniculata is compared. Along with bioluminescence spectra, kinetics of light emission reactions and sensitivities to calcium, these photoproteins also differ in specific activities and consequently in quantum yields of biox02luminescent reactions. The highest specific activities are found for obelins and mitrocomin, whereas those of aequorin and clytin are lower
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
the interaction between C-terminal Tyr208 and Tyr13 of the berovin first alpha-helix is essential for the stabilization and proper orientation of the 2-hydroperoxy adduct of coelenterazine within the internal cavity as well as for supporting its closed conformation. In contrast to hydromedusan photoproteins, in berovin the interplay between Tyr residues is conditioned rather by the pi-pi interaction of their phenyl rings than by the formation of hydrogen bonds between OH-groups
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
-
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
the specific bioluminescence activities of five recombinant hydromedusan Ca2+-regulated photoproteins - aequorin, mitrocomin, clytin and obelins from Obelia longissima and Obelia geniculata is compared. Along with bioluminescence spectra, kinetics of light emission reactions and sensitivities to calcium, these photoproteins also differ in specific activities and consequently in quantum yields of biox02luminescent reactions. The highest specific activities are found for obelins and mitrocomin, whereas those of aequorin and clytin are lower
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
-
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
the specific bioluminescence activities of five recombinant hydromedusan Ca2+-regulated photoproteins - aequorin, mitrocomin, clytin and obelins from Obelia longissima and Obelia geniculata is compared. Along with bioluminescence spectra, kinetics of light emission reactions and sensitivities to calcium, these photoproteins also differ in specific activities and consequently in quantum yields of biox02luminescent reactions. The highest specific activities are found for obelins and mitrocomin, whereas those of aequorin and clytin are lower
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
-
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
the reaction mechanism of the bioluminescent protein mnemiopsin1 is revealed by X-ray crystallography and QM/MM simulations. A water molecule in the coelenteramide binding cavity is identified that forms a hydrogen bond with the amide nitrogen atom of coelenteramide, which, in turn, is hydrogen-bonded via another water molecule to Tyr-131. This observation supports the hypothesis that the function of the coelenteramide-bound water molecule is to catalyze the 2-hydroperoxycoelenterazine decarboxylation reaction by protonation of a dioxetanone anion, thereby triggering the bioluminescence reaction
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
-
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
the specific bioluminescence activities of five recombinant hydromedusan Ca2+-regulated photoproteins - aequorin, mitrocomin, clytin and obelins from Obelia longissima and Obelia geniculata is compared. Along with bioluminescence spectra, kinetics of light emission reactions and sensitivities to calcium, these photoproteins also differ in specific activities and consequently in quantum yields of biox02luminescent reactions. The highest specific activities are found for obelins and mitrocomin, whereas those of aequorin and clytin are lower
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
-
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
the specific bioluminescence activities of five recombinant hydromedusan Ca2+-regulated photoproteins - aequorin, mitrocomin, clytin and obelins from Obelia longissima and Obelia geniculata is compared. Along with bioluminescence spectra, kinetics of light emission reactions and sensitivities to calcium, these photoproteins also differ in specific activities and consequently in quantum yields of biox02luminescent reactions. The highest specific activities are found for obelins and mitrocomin, whereas those of aequorin and clytin are lower
-
-
?
additional information
?
-
aequorin exhibits an absorption near 454 nm. The native chromophore is Renilla luciferin or a nearly identical derivative. The binding of calcium ion to the photoprotein brings about a conformational change in the protein which results in the chromophore reacting with a protein side chain hydroperoxide to yield a protein bound luciferin hydroperoxide intermediate. This intermediate then decomposes to CO2 and an electronic excited state of the corresponding protein-oxyluciferin monoanion complex which leads to light emission
-
-
?
additional information
?
-
no substrates: bisdeoxycoelenterazine, C6-methoxycoelenterazine, e-coelenterazine
-
-
?
additional information
?
-
poor substrates: n-coelenterazine, bis-coelenterazine, methoxy-coelenterazine, e-coelenterazine
-
-
?
additional information
?
-
the luminescence peak of aequorin is found at 472 nm. In presence of high concentrations of green fluorescent protein, the peak shifts to 509 nm
-
-
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
-
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
-
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
-
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
-
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
-
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
-
-
-
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
Mg2+
binding of Mg2+ to aequorin prevents the molecule from aggregating and stabilizes it in the monomeric form. Mg2+ binding induces conformational in the EF-hand loops. There are two Mg2+-binding sites, EF-hands I and III. EF-hand III binds to Mg2+ with higher affinity than EF-hand I, and only EF-hand III seems to be occupied by Mg2+ under physiological conditions
Ca2+
sequence displays three F-F hand structures that are characteristic for Ca2+-binding sites
Ca2+
the binding of calcium ion to the photoprotein brings about a conformational change in the protein which results in the chromophore reacting with a protein side chain hydroperoxide to yield a protein bound luciferin hydroperoxide intermediate. This intermediate then decomposes to CO2 and an electronic excited state of the corresponding protein-oxyluciferin monoanion complex which leads to light emission
Ca2+
when calcium binds at either or both EF hands I and IV the helices of these hands will change their relative orientations
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
EDTA
treatment with excess EDTA leads to a shift in the blue fluorescence to a greenish fluorescence due to a non-covalent complex of apoaequorin with coelenteramide (oxyluciferin) in a molar ratio of 1:1. By incubation with coelenterazine in the absence of reducing reagents, the greenish fluorescent protein is converted to aequorin at 25°C
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2-mercaptoethanol
reuired. About 1% residual activity in absence of 2-mercaptoethanol
dithiothreitol
in the presence of DTT, continuous luminescence is observed over 1 h. In the absence of DTT, the luminescence activity slowly decreases with a half period of 8.3 min
imidazole
luminescence activity of BFP-aq 1s stimulated 1.5- to 2fold by imidazole at concentrations of 30-300 mM. Imidazole has no effect on the binding affinity of coelenterazine and may act as a catalytic base
additional information
1H-pyrazole, 2-methylimidazole, 4-methylimidazole, histamine, L-histidine, urocanic acid, and 5-amino-4-imidazole carboxamide do not act as stimulators at concentrations of 30 and 150 mM
-
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DISEASE
TITLE OF PUBLICATION
LINK TO PUBMED
Colonic Neoplasms
Use of isogenic human cancer cells for high-throughput screening and drug discovery.
Muscular Dystrophy, Duchenne
Detection of unamplified target genes via CRISPR-Cas9 immobilized on a graphene field-effect transistor.
Paraplegia
Mouse models for hereditary spastic paraplegia uncover a role of PI4K2A in autophagic lysosome reformation.
Spastic Paraplegia, Hereditary
Mouse models for hereditary spastic paraplegia uncover a role of PI4K2A in autophagic lysosome reformation.
Stroke
Swing of the lever arm of a myosin motor at the isomerization and phosphate-release steps.
Uterine Cervical Neoplasms
Real-time monitoring of NADPH levels in living mammalian cells using fluorescence-enhancing protein bound to NADPHs.
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
UniProt
brenda
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
physiological function
protein-protein energy transfer between green fluoresecent protein and aequorin, possibly involving a Forster-type mechanism in vivo as well as in vitro
physiological function
when calcium binds at either or both EF hands I and IV the helices of these hands will change their relative orientations. Displacement of the helices flanking the C-terminal loop would disrupt the hydrogen-bonding network of the loop, resulting in a relocation of the side chain of Tyr 184. This would disrupt the hydrogen bonds to His 169 and the peroxide. The peroxide would be free to attack the adjacent carbonylic C3 to initiate the light-emitting reaction. The C-terminal tail could become partly or completely uncoupled from the helices, opening up the ligand-binding site to permit the egress of one or both reaction products
physiological function
the enzyme is responsible for the bioluminescence of the ctenophore Beroe abyssicola
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UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). AEQ1_AEQVI
196
0
22514
Swiss-Prot
Secretory Pathway (Reliability: 2)
CLYT_CLYGR
198
0
22541
Swiss-Prot
other Location (Reliability: 2)
OBL_OBEGE
195
0
22163
Swiss-Prot
Chloroplast (Reliability: 4)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method at 20°C, crystal structures of the apoAequorin complexes with the chiral deaza-analogs (S)-daCTZ and (S)-HM-daCTZ are determined, suggesting that the hydroxy moiety at the C6-hydroxyphenyl group and the carbonyl moiety of the imidazopyrazinone ring in coelenterazine are essential to bind the apoAequorin molecule through hydrogen bonding
to 1.7 A resolution. A calcium ion is observed in each of the three EF-hand loops that have the canonical calcium-binding sequence, and each is coordinated in the characteristic pentagonal bipyramidal configuration. The calcium-loaded apo-protein retains the same compact scaffold and overall fold as the unreacted photoprotein containing the bound substrate, 2-hydroperoxycoelenterazine
to 2.3 A resolution. Aequorin is a globular molecule containing a hydrophobic core cavity that accommodates the ligand coelenterazine-2-hydroperoxide
recombinantly expressed in Escherichia coli with Hexa-His-tag tail at N-terminal
sitting drop vapor diffusion method, crystal structure of Cd2+-loaded apo-mnemiopsin1 at 2.15 A resolution. The structure reveals that mnemiopsin1 has a two-domain fold with four alpha-helices in each domain
to 1.7 A resolution. A calcium ion is observed in each of the three EF-hand loops that have the canonical calcium-binding sequence, and each is coordinated in the characteristic pentagonal bipyramidal configuration. The calcium-loaded apo-protein retains the same compact scaffold and overall fold as the unreacted photoprotein containing the bound substrate, 2-hydroperoxycoelenterazine and also the same as the Ca2+-discharged obelin bound with the product, coelenteramide
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C145S/C152S/C180S
21% of wild-type activity. Contrary to wild-type, 49% residual activity in absence of 2-mercaptoethanol. The regeneration of the triple mutant aequorin is sharply inhibited by 2-mercaptoethanol
C145S/C180S
68% of wild-type activity. Contrary to wild-type, 28% residual activity in absence of 2-mercaptoethanol
C152S/C180S
17% of wild-type activity. Contrary to wild-type, 14% residual activity in absence of 2-mercaptoethanol
D119A
-
mutant is an active photoprotein, Ca2+ affinity is reduced by a factor of 20 compared to the wild type
D153G
shift of maximum luminescence to shorter wavelengths. t1/2 is decreased with respect to the half-life time of the native protein (0.7 s). Mutation results in a significant decrease in stability at 65°C
F149
residue participates in stabilization of the coelenterazine peroxide and the triggering of photon emission by linking the third EF-hand to Trp-129 and His-169 coelenterazine binding residues
L170I
16fold increase of wild-type activity, an increase of the photoprotein lifetime at 37°C and increased thermostability
Q168
residue participates in stabilization of the coelenterazine peroxide and the triggering of photon emission by linking the third EF-hand to Trp-129 and His-169 coelenterazine binding residues
Q168R
11fold increase of wild-type activity, increase of the photoprotein lifetime at 37°C and increased thermostablility
W86F
shift of maximum luminescence to shorter wavelengths. The single mutant reaches the highest stability against thermal shock
W86F/D153G
decreased t1/2-value. Maximum emission spectrum at 401 nm. Mutation results in a significant decrease in stability at 65°C
Y82F
Y82F/D153G
decreased t1/2-value. Maximum emission spectrum at 478 nm
Y82F/W86F
maximum emission spectrum at 400 nm. Y82F mutation results in shift of emission to longer wavelength, while the W86F mutation shifts the emission to shorter wavelengths. Compared to wild type aequorin, the Y82F/W86F variant displays a 2fold increase of light half-life. Mutation results in a significant decrease in stability at 65°C
E50G
the luminescence activity of the variant is about 17times greater than that of wild-type photoprotein. The activity of E50G variant increases as a result of more flexibility that is brought about by Gly essential for adopting the correct conformation for functional activity. In comparison with wild-type protein, the variant shows higher optimum temperature and calcium sensitivity as well as slower rate of luminesx02cence decay
E50G/D47N
mutation in loop I of mnemiopsin 2 leads to some conformational alterations in the secondary and tertiary structures which affect both the interaction of the photoprotein with the substrate and Ca2+ coordination
E50G/E53T
mutation in loop I of mnemiopsin 2 leads to some conformational alterations in the secondary and tertiary structures which affect both the interaction of the photoprotein with the substrate and Ca2+ coordination
F88H
mutation shifts the bioluminescence maximum from 482 nm for wild-type to 459 nm. 105% of wild-type bioluminescence yield
F88R
mutation shifts the bioluminescence maximum from 482 nm for wild-type to 474 nm. 99% of wild-type bioluminescence yield
F88W
mutation shifts the bioluminescence maximum from 482 nm for wild-type to 477 nm. 100% of wild-type bioluminescence yield
F88Y
mutation shifts the bioluminescence maximum from 482 nm for wild-type to 453 nm. 100% of wild-type bioluminescence yield
additional information
Y82F
mutation leads to removal of a H-bond from Tyr82 to the bound coelenteramide, and shifts its bioluminescence from 469 nm for wild-type to 501 nm. Mutant is stable with good activity and expressible in mammalian cells
Y82F
mutation shifts the peak towards longer wavelengths. Increased half time of initial light by 3.6 s. Mutation results in a significant decrease in stability at 65°C
additional information
mutation of all 3 Cys resiudes to Ser. Cys-free aequorin displays a two-fold lower specific bioluminescence activity but preserves similar activation properties and light emission kinetics compared to the wild-type aequorin. Mutant aequorin shows increased conformational flexibility
additional information
mutations with increased luminescence intensity neighbor the His-16 or His-169 coelenterazine binding residues or are located in the first EF-hand
additional information
replacement of the loop sequence of EF-hand I with other known loop sequences of Ca2+-binding proteins. Mutants replaced with the EF-hand I and EF-hand III from photoproteins show sufficient luminescence activity, but mutants replaced by EF-hands from Renilla luciferin-binding protein and calmodulin do not. The Ca2+-binding affinity to aequorin is reduced by a positive charge at the second position of the canonical EF-hand motif sequence
additional information
the C-terminal proline residue of aequorin is essential for the long-term stability of the bound coelenterazine. Aequorin lacking the proline residue has only 1% of the specific activity of the wild-type after 2 h, and is virtually inactive after 18 h. Replacement of the C-terminal proline residue with histidine or glutamic acid decreases the specific activity to 10 and 190% of that of the wild-type respectively, these variants show half-lives of 2.4 h and 2.3 h, respectively
additional information
mutation of all 5 Cys resiudes to Ser. Cys-free obelin retains only about 10% of the bioluminescence activity of wild-type obelin and binds coelenterazine and forms active photoprotein much less effectively. The mutation drastically changes the bioluminescence kinetics of obelin completely eliminating a fast component from the light signal decay curve. Replacement of Cys residues increases conformational flexibility
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
44
wild-type, 50% inactivation of aequorin bioluminescence by a 30-min heat shock
48
mutant L170I, 50% inactivation of aequorin bioluminescence by a 30-min heat shock
50
53
mutant Q168R, 50% inactivation of aequorin bioluminescence by a 30-min heat shock
57
mutant Q168R/L170I, 50% inactivation of aequorin bioluminescence by a 30-min heat shock
80
95
3 min, complete loss of activity. Fluorescence recovers to over 93% after incubating for a further 20 min at 24°C
50
the inactivation rate constants (kinact) at 50°C is 0.001/min for apo-mnemiopsin
50
the inactivation rate constants (kinact) at 50°C is 0.001/min for apo-mnemiopsin
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
aequorin is extremely sensitive to presence of Ca2+, the solutions usually faintly luminescing spontaneously, resulting in a gradual loss of capacity to luminesce. Spontaneous luminescence, with the accompanying decrease in activity, can be eliminated by saturating the aequorin plus EDTA solutions with (NH4)2SO4 and keeping at -20°
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
upon treatment with urea and 2-mercaptoethanol, a blue fluorescent compound (AF-350), and a protein (apoaequorin-SH) are obtained
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CLONED (Commentary)
ORGANISM
UNIPROT <