Interchange of aequorin and obelin bioluminescence color is determined by substitution of one active site residue of each photoprotein

Stepanyuk, Galina A.; Gölz, Stefan; Markova, Svetlana V.; Frank, Ludmila A.; Lee, John; Vysotski, Eugene S.

doi:10.1016/j.febslet.2005.01.004

Cited by 68 publications

(89 citation statements)

References 48 publications

(62 reference statements)

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“…We found that the kinetics and sensitivity to Mg 21 of GFP-photoprotein fusions were similar to those of their cognate photoproteins. Indeed, values of these parameters for GA and GO responses were in the range of those reported for native (Hastings et al, 1969;Morin and Hastings, 1971a;Stephenson and Sutherland, 1981) and recombinant (Illarionov et al, 2000;Markova et al, 2002;Stepanyuk et al, 2005) aequorin and obelin. In the present study, we also found that kinetics and Mg 21 sensitivity of GA and GA N26D responses were similar to those of aequorin and aequorin N26D , respectively.…”

Section: Kinetic Properties Of Gfp-photoprotein Responses In Vitromentioning

confidence: 67%

“…Hence, our results show that kinetics of GFP-photoprotein fusions GA, GO, and GA N26D are markedly different, as expected from data obtained on photoproteins alone in previous studies. Indeed, onset and decay time constants of 10 msec and 833 msec, respectively, were obtained for aequorin (Hastings et al, 1969), whereas these values were 2.5-4 msec and 139-294 msec, respectively, for obelin depending on the Obelia species (Morin and Hastings, 1971a;Stephenson and Sutherland, 1981;Illarionov et al, 2000;Stepanyuk et al, 2005). Similarly, the aequorin N26D mutant reportedly exhibits slower decay kinetics than wild-type aequorin (Tricoire et al, 2006).…”

Section: Kinetics Of Calcium Sensor Responses In Vitromentioning

confidence: 83%

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Calcium imaging in single neurons from brain slices using bioluminescent reporters

Drobac

Tricoire

Chaffotte

et al. 2009

J of Neuroscience Research

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Responses of three bioluminescent Ca(2+) sensors were studied in vitro and in neurons from brain slices. These sensors consisted of tandem fusions of green fluorescent protein (GFP) with the photoproteins aequorin, obelin, or a mutant aequorin with high Ca(2+) sensitivity. Kinetics of GFP-obelin responses to a saturating Ca(2+) concentration were faster than those of GFP-aequorin at all Mg(2+) concentrations tested, whereas GFP-mutant aequorin responses were the slowest. GFP-photoproteins were efficiently expressed in pyramidal neurons following overnight incubation of acute neocortical slices with recombinant Sindbis viruses. Expression of GFP-photoproteins did not result in conspicuous modification of morphological or electrophysiological properties of layer V pyramidal cells. The three sensors allowed the detection of Ca(2+) transients associated with action potential discharge in single layer V pyramidal neurons. In these neurons, depolarizing steps of increasing amplitude elicited action potential discharge of increasing frequency. Bioluminescent responses of the three sensors were similar in several respects: detection thresholds, an exponential increase with stimulus intensity, photoprotein consumptions, and kinetic properties. These responses, which were markedly slower than kinetics measured in vitro, increased linearly during the action potential discharge and decayed exponentially at the end of the discharge. Onset slopes increased with stimulus intensity, whereas decay kinetics remained constant. Dendritic light emission contributed to whole-field responses, but the spatial resolution of bioluminescence imaging was limited to the soma and proximal apical dendrite. Nonetheless, the high signal-to-background ratio of GFP-photoproteins allowed the detection of Ca(2+) transients associated with 5 action potentials in single neurons upon whole-field bioluminescence recordings.

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Section: Kinetic Properties Of Gfp-photoprotein Responses In Vitromentioning

confidence: 67%

Section: Kinetics Of Calcium Sensor Responses In Vitromentioning

confidence: 83%

Calcium imaging in single neurons from brain slices using bioluminescent reporters

Drobac

Tricoire

Chaffotte

et al. 2009

J of Neuroscience Research

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“…Although there are differences in spectral properties among them, it has been already demonstrated that these are due to different interactions of the coelenteramide with the side chains of residues making up the binding cavity (32,36). Kinetic properties, Ca 2ϩ sensitivity, thermostability, and so on, are much more difficult to account for, in common with trying to account for enzymatic properties from structural information in general.…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of obelin after Ca²⁺-triggered bioluminescence suggests neutral coelenteramide as the primary excited state

Liu

Stepanyuk

Vysotski

et al. 2006

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

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104

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The crystal structure at 1.93-Å resolution is determined for the Ca 2؉ -discharged obelin containing three bound calcium ions as well as the product of the bioluminescence reaction, coelenteramide. This finding extends the series of available spatial structures of the ligand-dependent conformations of the protein to four, the obelin itself, and those after the bioluminescence reaction with or without bound Ca 2؉ and͞or coelenteramide. Among these structures, global conformational changes are small, typical of the class of ''calcium signal modulators'' within the EF-hand protein superfamily. Nevertheless, in the active site there are significant repositions of two residues. The His-175 imidazole ring flips becoming almost perpendicular to the original orientation corroborating the crucial importance of this residue for triggering bioluminescence. Tyr-138 hydrogen bonded to the coelenterazine N1-atom in unreacted obelin is moved away from the binding cavity after reaction. However, this Tyr is displaced by a water molecule from within the cavity, which now forms a hydrogen bond to the same atom, the amide N of coelenteramide. From this observation, a reaction scheme is proposed that would result in the neutral coelenteramide as the primary excited state product in photoprotein bioluminescence. From such a higher energy state it is now energetically feasible to account for the shorter wavelength bioluminescence spectra obtained from some photoprotein mutants or to populate the lower energy state of the phenolate anion to yield the blue bioluminescence ordinarily observed from native photoproteins.coelenterazine ͉ photoprotein ͉ EF hand ͉ luciferase ͉ aequorin

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“…In contrast, the Ca 2þ -discharged obelins show fluorescence maxima about 25 nm to longer wavelength than their bioluminescence. Furthermore, the obelin bioluminescence spectrum is bimodal, with a minor higher energy band having a maximum around 400 nm, not evident in aequorin bioluminescence (9,10). From spectral studies of model compounds, this high energy band is identified as from the excited level of coelenteramide in its neutral state (11).…”

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confidence: 99%

Picosecond Fluorescence Relaxation Spectroscopy of the Calcium-Discharged Photoproteins Aequorin and Obelin

et al. 2009

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Addition of calcium ions to the Ca 2þ -regulated photoproteins, such as aequorin and obelin, produces a blue bioluminescence originating from a fluorescence transition of the protein-bound product, coelenteramide. The kinetics of several transient fluorescent species of the bound coelenteramide is resolved after picosecond-laser excitation and streak camera detection. The initially formed spectral distributions at picosecond-times are broad, evidently comprised of two contributions, one at higher energy (∼25 000 cm -1 ) assigned as from the Ca 2þ -discharged photoprotein-bound coelenteramide in its neutral state. This component decays much more rapidly (t 1/2 ∼ 2 ps) in the case of the Ca 2þ -discharged obelin than aequorin (t 1/2 ∼ 30 ps). The second component at lower energy shows several intermediates in the 150-500 ps times, with a final species having spectral maxima 19 400 cm -1 , bound to Ca 2þ -discharged obelin, and 21 300 cm -1 , bound to Ca 2þ -discharged aequorin, and both have a fluorescence decay lifetime of 4 ns. It is proposed that the rapid kinetics of these fluorescence transients on the picosecond time scale, correspond to times for relaxation of the protein structural environment of the binding cavity.Bioluminescent animals are found in a variety of types occurring both terrestrially and in the ocean. More often than not, the chemistry of their light emission processes and the proteins involved are found quite unrelated. Well-studied cases, for example, are the bioluminescence of the firefly, which involves ATP and a substrate firefly luciferin, a benzthiozole derivative, and that of the photoprotein aequorin from the bioluminescent jellyfish Aequorea, which is triggered for light emission by Ca 2þ . Coelenterazine, an imidazopyrazinone derivative (Figure 1), is the luciferin (a generic term for the substrate) involved in many marine bioluminescent systems, including the ones subject to this present study, the Ca 2þ -regulated photoproteins, aequorin and obelin from the hydrazoan Obelia (1-3).A significant advance in uncovering the mechanism of bioluminescence from aequorin and obelin resulted from the determination of the high-resolution spatial structure of the two photoproteins (4-6). The coelenterazine was revealed residing in an internal cavity substituted with a peroxy group ( Figure 1B), as long suspected from earlier indirect evidence (7). Such a compound would be very unstable in free solution, but in the protein it appears to be frozen in place via a H-bond network to amino acid residues comprising the binding cavity. Model chemiluminescence studies with coelenterazine analogues had shown such a peroxide to be an intermediate, closing to a dioxetanone in the reaction pathway (8). The free energy produced by decarboxylation of this dioxetanone around 70 kcal/mol is sufficient to account for the energy of the photons of blue bioluminescence.Aequorin and obelin are EF-hand proteins belonging to the large family of Ca 2þ -binding proteins. They each contain three Ca 2þ -ligati...

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Interchange of aequorin and obelin bioluminescence color is determined by substitution of one active site residue of each photoprotein

Cited by 68 publications

References 48 publications

Calcium imaging in single neurons from brain slices using bioluminescent reporters

Calcium imaging in single neurons from brain slices using bioluminescent reporters

Crystal structure of obelin after Ca²⁺-triggered bioluminescence suggests neutral coelenteramide as the primary excited state

Picosecond Fluorescence Relaxation Spectroscopy of the Calcium-Discharged Photoproteins Aequorin and Obelin

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Interchange of aequorin and obelin bioluminescence color is determined by substitution of one active site residue of each photoprotein

Cited by 68 publications

References 48 publications

Calcium imaging in single neurons from brain slices using bioluminescent reporters

Calcium imaging in single neurons from brain slices using bioluminescent reporters

Crystal structure of obelin after Ca2+-triggered bioluminescence suggests neutral coelenteramide as the primary excited state

Picosecond Fluorescence Relaxation Spectroscopy of the Calcium-Discharged Photoproteins Aequorin and Obelin

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Crystal structure of obelin after Ca²⁺-triggered bioluminescence suggests neutral coelenteramide as the primary excited state