Preparation and preliminary study of crystals of the recombinant calcium-regulated photoprotein obelin from the bioluminescent hydroid <i>Obelia longissima</i>

Vysotski, Eugene S.; Liu, Zhijie; Rose, John P.; Wang, B. C.; Lee, John

doi:10.1107/s0907444999011828

Cited by 7 publications

(5 citation statements)

References 16 publications

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“…The final product was homogeneous according to LC-electrospray ionization mass spectrometry. The molecular weight of the W92F obelin (22,040 Da) determined with this method is in an excellent agreement with that calculated from the amino acid sequence excluding Met1 (22 040 Da), as was also observed for WT obelin (24). For investigation of coelenterazine binding kinetics, the apoWT and apoW92F were purified according to the procedure described in (22).…”

Section: Methodssupporting

confidence: 80%

Violet Bioluminescence and Fast Kinetics from W92F Obelin: Structure-Based Proposals for the Bioluminescence Triggering and the Identification of the Emitting Species

et al. 2003

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Obelin from the hydroid Obelia longissima and aequorin are members of a subfamily of Ca(2+)-regulated photoproteins that is a part of the larger EF-hand calcium binding protein family. On the addition of Ca(2+), obelin generates a blue bioluminescence emission (lambda(max) = 485 nm) as the result of the oxidative decarboxylation of the bound substrate, coelenterazine. The W92F obelin mutant is noteworthy because of the unusually high speed with which it responds to sudden changes of [Ca(2+)] and because it emits violet light rather than blue due to a prominent band with lambda(max) = 405 nm. Increase of pH in the range from 5.5 to 8.5 and using D(2)O both diminish the contribution of the 405 nm band, indicating that excited state proton transfer is involved. Fluorescence model studies have suggested the origin of the 485 nm emission as the excited state of an anion of coelenteramide, the bioluminescence reaction product, and 405 nm from the excited neutral state. Assuming that the dimensions of the substrate binding cavity do not change during the excited state formation, a His22 residue within hydrogen bonding distance to the 6-(p-hydroxy)-phenyl group of the excited coelenteramide is a likely candidate for accepting the phenol proton to produce an ion-pair excited state, in support of recent suggestions for the bioluminescence emitting state. The proton transfer could be impeded by removal of the Trp92 H-bond, resulting in strong enhancement of a 405 nm band giving the violet color of bioluminescence. Comparative analysis of 3D structures of the wild-type (WT) and W92F obelins reveals that there are structural displacements of certain key Ca(2+)-ligating residues in the loops of the two C-terminal EF hands as well as clear differences in hydrogen bond networks in W92F. For instance, the hydrogen bond between the side-chain oxygen atom of Asp169 and the main-chain nitrogen of Arg112 binds together the incoming alpha-helix of loop III and the exiting alpha-helix of loop IV in WT, providing probably concerted changes in these EF hands on calcium binding. But this linkage is not found in W92F obelin. These differences apparently do not change the overall affinity to calcium of W92F obelin but may account for the kinetic differences between the WT and mutant obelins. From analysis of the hydrogen bond network in the coelenterazine binding cavity, it is proposed that the trigger for bioluminescence reaction in these Ca(2+)-regulated photoproteins may be a shift of the hydrogen bond donor-acceptor separations around the coelenterazine-2-hydroperoxy substrate, initiated by small spatial adjustment of the exiting alpha-helix of loop IV.

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Section: Methodssupporting

confidence: 80%

Violet Bioluminescence and Fast Kinetics from W92F Obelin: Structure-Based Proposals for the Bioluminescence Triggering and the Identification of the Emitting Species

et al. 2003

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“…The purified protein was concentrated to approximately 8–10 mg/ml, desalted on a BioGel P2 column equilibrated with 1 mM EDTA, 10 mM Na/K phosphate buffer, pH 7.3, and concentrated again to approximately the same concentration. Protein was homogeneous according to LC‐Electrospray Ionization Mass Spectrometry and the mass was in excellent agreement with that calculated from sequence excluding Met1, as also observed for WT obelin [10]. Protein concentration was determined with the Bio‐Rad DC Protein Assay Kit with bovine serum albumin as a protein standard.…”

Section: Methodssupporting

confidence: 62%

Structural basis for the emission of violet bioluminescence from a W92F obelin mutant

et al. 2001

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Mutation of the Trp92 that is known to lie within the active site of the photoprotein obelin from Obelia longissima, results in a shift of the bioluminescence color from blue (V V max = 485 nm) to violet. The corrected spectrum shows a new band with V V max = 410 nm now contributing equally to the one at longer wavelength. The crystal structure of this W92F obelin determined at 1.72 A î resolution shows that there is no significant change in the dimensions of the active site between WT obelin (recombinant Ca 2+ -regulated photoprotein from Obelia longissima) and the mutant. It is proposed that the bioluminescence spectral shift results from removal of a hydrogen bond from the indole of W92 nearby a hydroxyl belonging to the 6-phenyl substituent of the substrate coelenterazine. Propagation of this change through a conjugated bond system in the excited state of the product coelenteramide affects the coupling of the N1-position and the hydrogen-bonded Y138. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

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“…Induction was initiated with 1 mM IPTG when the culture reached an OD 590 of 0.6-0.8. After addition of IPTG, cultivation of the cells was continued for 3 h. The WT obelin and its mutant were purified and activated with coelenterazine (Prolume, Pinetop, USA) as described elsewhere (Vysotski et al, 1999(Vysotski et al, , 2001Illarionov et al, 2000). The purified photoproteins were homogeneous according to SDS-PAGE.…”

Section: Preparation Of the Photoprotein Samplesmentioning

confidence: 99%

Structures of the Ca²⁺-regulated photoprotein obelin Y138F mutant before and after bioluminescence support the catalytic function of a water molecule in the reaction

Natashin

Ding

Eremeeva

et al. 2014

Acta Cryst D Biol Crystallogr

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Ca(2+)-regulated photoproteins, which are responsible for light emission in a variety of marine coelenterates, are a highly valuable tool for measuring Ca(2+) inside living cells. All of the photoproteins are a single-chain polypeptide to which a 2-hydroperoxycoelenterazine molecule is tightly but noncovalently bound. Bioluminescence results from the oxidative decarboxylation of 2-hydroperoxycoelenterazine, generating protein-bound coelenteramide in an excited state. Here, the crystal structures of the Y138F obelin mutant before and after bioluminescence are reported at 1.72 and 1.30 Å resolution, respectively. The comparison of the spatial structures of the conformational states of Y138F obelin with those of wild-type obelin gives clear evidence that the substitution of Tyr by Phe does not affect the overall structure of both Y138F obelin and its product following Ca(2+) discharge compared with the corresponding conformational states of wild-type obelin. Despite the similarity of the overall structures and internal cavities of Y138F and wild-type obelins, there is a substantial difference: in the cavity of Y138F obelin a water molecule corresponding to W2 in wild-type obelin is not found. However, in Ca(2+)-discharged Y138F obelin this water molecule now appears in the same location. This finding, together with the observed much slower kinetics of Y138F obelin, clearly supports the hypothesis that the function of a water molecule in this location is to catalyze the 2-hydroperoxycoelenterazine decarboxylation reaction by protonation of a dioxetanone anion before its decomposition into the excited-state product. Although obelin differs from other hydromedusan Ca(2+)-regulated photoproteins in some of its properties, they are believed to share a common mechanism.

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Preparation and preliminary study of crystals of the recombinant calcium-regulated photoprotein obelin from the bioluminescent hydroid Obelia longissima

Cited by 7 publications

References 16 publications

Violet Bioluminescence and Fast Kinetics from W92F Obelin: Structure-Based Proposals for the Bioluminescence Triggering and the Identification of the Emitting Species

Violet Bioluminescence and Fast Kinetics from W92F Obelin: Structure-Based Proposals for the Bioluminescence Triggering and the Identification of the Emitting Species

Structural basis for the emission of violet bioluminescence from a W92F obelin mutant

Structures of the Ca²⁺-regulated photoprotein obelin Y138F mutant before and after bioluminescence support the catalytic function of a water molecule in the reaction

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Preparation and preliminary study of crystals of the recombinant calcium-regulated photoprotein obelin from the bioluminescent hydroid Obelia longissima

Cited by 7 publications

References 16 publications

Violet Bioluminescence and Fast Kinetics from W92F Obelin: Structure-Based Proposals for the Bioluminescence Triggering and the Identification of the Emitting Species

Violet Bioluminescence and Fast Kinetics from W92F Obelin: Structure-Based Proposals for the Bioluminescence Triggering and the Identification of the Emitting Species

Structural basis for the emission of violet bioluminescence from a W92F obelin mutant

Structures of the Ca2+-regulated photoprotein obelin Y138F mutant before and after bioluminescence support the catalytic function of a water molecule in the reaction

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Structures of the Ca²⁺-regulated photoprotein obelin Y138F mutant before and after bioluminescence support the catalytic function of a water molecule in the reaction