Response of Aequorin Bioluminescence to Rapid Changes in Calcium Concentration

Hastings, J. Woodland; Mitchell, George W.; Mattingly, Patrick H.; Blinks, John R.; Leeuwen, Menno van

doi:10.1038/2221047a0

Cited by 140 publications

(87 citation statements)

References 6 publications

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“…In WT, F was 615 Ϯ 21 ms at 1.24 mM Ca 2ϩ , close to the 833 ms reported assuming monoexponential decay (15). In contrast, other kinetic parameters appeared to depend on EF-hand domains.…”

Section: Bright and Slodk Mutationssupporting

confidence: 76%

“…Our data show that in WT aequorin, both the light intensity and the decay rate are greater at higher [Ca 2ϩ ], such that the total light (integral, ⌺ T ) is the same at different Ca 2ϩ concentrations. Although the [Ca 2ϩ ] dependence of the decay rate has been reported early (15), the present study reveals that it results from variations of a fast and a slow component. The fast and slow components coexisted across mutants and Ca 2ϩ concentrations and were even observed in double EF-hand mutants where only one EF hand is left unaffected.…”

Section: Discussioncontrasting

confidence: 58%

“…None of the reaction schemes and models of aequorin luminescence proposed so far (15,18) takes into account the variations of decay rate with [Ca 2ϩ ] as a key determinant of light intensity. Hence, we investigated whether a model based on these variations may capture the essential features of aequorin responses to Ca 2ϩ ( Fig.…”

Section: Bright and Slodk Mutationsmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

“…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.…”

mentioning

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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Section: Bright and Slodk Mutationssupporting

confidence: 76%

Section: Discussioncontrasting

confidence: 58%

Section: Bright and Slodk Mutationsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

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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Transient‐state kinetic analysis of complex formation between photoprotein clytin and GFP from jellyfish Clytia gregaria

Eremeeva

Berkel²,

Vysotski

2016

FEBS Letters

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Luminous organisms use different protein-mediated strategies to modulate light emission color. Here, we report the transient-state kinetic studies of the interaction between photoprotein clytin from Clytia gregaria and its antenna protein, cgreGFP. We propose that cgreGFP forms a transient complex with Ca 2+ -bound clytin before the excited singlet state of the coelenteramide product is formed. From the spectral distribution and donor-acceptor separation distance, we infer that clytin reaction intermediates may interact only with the middle side part of cgreGFP.

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