Regeneration of the photoprotein aequorin

Shimomura, Osamu; Johnson, Frank H.

doi:10.1038/256236a0

Cited by 205 publications

(115 citation statements)

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“…The formation of aequorin is a slow process (2), whereas the luminescence reaction is very fast and proceeds to completion in the continuous presence of Ca 2ϩ . The aequorin response thus occurs as a flash that decays exponentially and whose onset rate does not depend on [Ca 2ϩ ] (15).…”

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confidence: 99%

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Calcium dependence of aequorin bioluminescence dissected by random mutagenesis

Tricoire¹,

Tsuzuki²,

Courjean³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Aequorin bioluminescence is emitted as a rapidly decaying flash upon calcium binding. Random mutagenesis and functional screening were used to isolate aequorin mutants showing slow decay rate of luminescence. Calcium sensitivity curves were shifted in all mutants, and an intrinsic link between calcium sensitivity and decay rate was suggested by the position of all mutations in or near EF-hand calcium-binding sites. From these results, a low calcium affinity was assigned to the N-terminal EF hand and a high affinity to the C-terminal EF-hand pair. In WT aequorin, the increase of the decay rate with calcium occurred at constant total photon yield and thus determined a corresponding increase of light intensity. Increase of the decay rate was underlain by variations of a fast and a slow component and required the contribution of all three EF hands. Conversely, analyses of double EF-hand mutants suggested that single EF hands are sufficient to trigger luminescence at a slow rate. Finally, a model postulating that proportions of a fast and a slow light-emitting state depend on calcium concentration adequately described the calcium dependence of aequorin bioluminescence. Our results suggest that variations of luminescence kinetics, which depend on three EF hands endowed with different calcium affinities, critically determine the amplitude of aequorin responses to biological calcium signals.EF hand ͉ kinetics ͉ luminescence ͉ photoprotein ͉ transduction T he photoprotein aequorin is a stable luciferase intermediate formed from the reaction of the protein apoaequorin (luciferase) and the substrate coelenterazine (luciferin), which emits light upon Ca 2ϩ binding (1-5). Aequorin contains three EF-hand Ca 2ϩ -binding sites (6-8) located close to its N (EF1) or C terminus (EF2,3 pair). Aequorin mutagenesis and crystal structure suggest that these three EF hands indeed bind Ca 2ϩ , but their individual contribution to luminescence is still a matter of debate (9-13).The steep increase of luminescence intensity with [Ca 2ϩ ] makes aequorin a useful reporter of intracellular calcium signals (14). The formation of aequorin is a slow process (2), whereas the luminescence reaction is very fast and proceeds to completion in the continuous presence of Ca 2ϩ . The aequorin response thus occurs as a flash that decays exponentially and whose onset rate does not depend on [Ca 2ϩ ] (15). It has been observed early that the decay rate of this response increases with [Ca 2ϩ ], whereas the total light emitted (light integral) remains relatively constant (15). This suggests that the increase of luminescence intensity with [Ca 2ϩ ] is determined by variations of the decay rate but not of the light integral. In other words, the shorter the duration of the flash (i.e., the faster the decay), the larger the amplitude of the response (i.e., light intensity). However, the relationships among the intensity, the decay rate, and the integral of bioluminescence and their links to EF-hand occupancy have not been clearly established.To analyze the co...

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confidence: 99%

“…T he photoprotein aequorin is a stable luciferase intermediate formed from the reaction of the protein apoaequorin (luciferase) and the substrate coelenterazine (luciferin), which emits light upon Ca 2ϩ binding (1)(2)(3)(4)(5). Aequorin contains three EF-hand Ca 2ϩ -binding sites (6-8) located close to its N (EF1) or C terminus (EF2,3 pair).…”

mentioning

confidence: 99%

Calcium dependence of aequorin bioluminescence dissected by random mutagenesis

Tricoire¹,

Tsuzuki²,

Courjean³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…For example, the luciferin of the sea pansy Renilla is coelenterazine [5], and the calcium-sensitive photoprotein aequorin from the jelly¢sh Aequorea also contains coelenterazine as its functional moiety [6,7].…”

Section: Introductionmentioning

confidence: 99%

Secretional luciferase of the luminous shrimpOplophorus gracilirostris: cDNA cloning of a novel imidazopyrazinone luciferase

Watanabe²,

et al. 2000

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The deep-sea shrimp Oplophorus gracilirostris secretes a luciferase that catalyzes the oxidation of coelenterazine to emit blue light. The luciferase (M r approx. 106 000) was found to be a complex composed of 35 kDa and 19 kDa proteins, and the cDNAs encoding these two proteins were cloned. The expression of the cDNAs in bacterial and mammalian cells indicated that the 19 kDa protein, not the 35 kDa protein, is capable of catalyzing the luminescent oxidation of coelenterazine. The primary sequence of the 35 kDa protein revealed a typical leucine-rich repeat sequence, whereas the catalytic 19 kDa protein shared no homology with any known luciferases including various imidazopyrazinone luciferases. ß

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“…Since active aequorin is ob-*Corresponding author. Fax: (1) (619) 534 7313. tained only when DTT or 2-mercaptoethanol is added to the incubation mixture [4,5], it is presumed that the reducing agent serves to reduce a disulfide bond. The aequorin molecule contains CYS'~~, Cysls2 and Cys"' [2,3], but it is not known whether these residues form a disulfide bond.…”

Section: Introductionmentioning

confidence: 99%

“…On binding Ca2', aequorin is converted to an enzyme (luciferase), which catalyzes the oxidation of coelenterazine by the bound oxygen, yielding as products light (&iX = 470 nm), CO, and coelenteramide (the oxidation product of coelenterazine). Apoaequorin may be regenerated into aequorin by incubation with coelenterazine, molecular oxygen, EDTA and 2-mercaptoethanol [4] or dithiothreitol (DTT) [5].…”

Section: Introductionmentioning

confidence: 99%