Structure of the Ca<sup>2+</sup>‐regulated photoprotein obelin at 1.7 Å resolution determined directly from its sulfur substructure

Liu, Zhijie; Vysotski, Eugene S.; Chen, Chun‐Jung; Rose, John P.; Lee, John; Wang, Bi-Cheng

doi:10.1110/ps.9.11.2085

Cited by 182 publications

(193 citation statements)

References 42 publications

Supporting

Mentioning

187

Contrasting

Unclassified

Order By: Relevance

“…5B). In obelin (also in aequorin), the Tyr-138 is hydrogen bonded to this same N of coelenterazine (15); however, in the Ca 2ϩ -discharged obelin here, it is moved out of the cavity with its side chain twisted Ͼ90°to become solvent exposed and hydrogen bonded to a surface Glu-55. In the structure of the discharged obelin without Ca 2ϩ (state IV), this Tyr-138 is also moved but remains in the binding cavity and water molecule W 2 does not move in to fill the vacant position.…”

Section: Discussionmentioning

confidence: 85%

See 1 more Smart Citation

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.

Self Cite

104

View full text Add to dashboard Cite

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

show abstract

Section: Discussionmentioning

confidence: 85%

“…The bioluminescence spectra are broad with maxima in the range 465-495 nm depending on the type of photoprotein (13). Spatial structures recently determined for aequorin and obelin confirm the presence of this 2-hydroperoxycoelenterazine and show that it is situated within a hydrophobic cavity and stabilized there by a hydrogen bond network (14,15).…”

mentioning

confidence: 88%

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.

Self Cite

104

View full text Add to dashboard Cite

show abstract

“…Interestingly, corresponding residues of the photoprotein obelin both interact with coelenterazine (A 46 and I 50 ; see supporting information and ref. 20). Finally, RHI and AHV mutants provide an example of mutations at the same positions that resulted in similar Ca 2ϩ EC 50 s but very different decay rates.…”

Section: Discussionmentioning

confidence: 99%

“…The structures of photoproteins suggest that each of the three EF-hand domains forms a functional unit (11,20). Indeed, results with double EF-hand mutants indicate that each individual EF hand is able to trigger aequorin bioluminescence.…”

Section: Discussionmentioning

confidence: 99%

Calcium dependence of aequorin bioluminescence dissected by random mutagenesis

Tricoire¹,

Tsuzuki²,

Courjean³

et al. 2006

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

View full text Add to dashboard Cite

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...

show abstract

“…However, over the last five years there have been several reports of successful application of sulphur SAD phasing. These experiments were carried out using Cu Kα radiation (1.54 Å) (Bond et al, 2001;de Graaff, et al, 2001;Yang et al, 2001;Lemke et al, 2002;, or synchrotron radiation at a wavelength of 1.54 Å to 1.77 Å (Dauter et al, 1999;Liu et al, 2000;Gordon et al, 2001;Brown et al, 2002;Li et al, 2002;Micossi et al, 2002;Ramagopal et al, 2003;Lartigue et al, 2004;Sekar et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

Structure determination of a novel protein by sulfur SAD using chromium radiation in combination with a new crystal-mounting method

2005

View full text Add to dashboard Cite

Synopsis A novel protein structure was solved by S-SAD using in-house chromium radiation. To reduce X-ray absorption of the cryo-buffer and cryoloop, a novel free crystal mounting technique was developed.Abstract A novel and easy crystal mounting technique was developed for the sulphur SAD method using chromium Kα radiation (2.29Å). Using this technique, the cryo-buffer and cryoloop around the protein crystal can be removed before data collection to eliminate their X-ray absorption. The superiority and the reproducibility of the datasets with our mounting technique were demonstrated using tetragonal hen egg-white lysozyme crystals. The structure of a novel protein, PH1109, from Pyrococcus horikoshii OT3 was solved using this technique. At the wavelength of chromium Kα radiation, anomalous signals, <|ΔF|>/<|F|> of PH1109 is expected to be 1.72%, as this protein of 144 residues includes 4 methionines and 2 cysteines. Sulphur SAD phasing was performed using SHELXD and SHELXE. In the case of the dataset obtained using our novel crystal mounting technique, 54.9 % of all residues were built with side-chains automatically by RESOLVE. On the other hand, only 16.0 % were built with side-chains for the dataset collected using the standard cryoloop. These results indicated that our crystal mounting technique was superior to the standard loop mounting method for measurement of small anomalous differences at longer wavelength, and yielded better results in sulphur substructure solution and initial phasing. The present study demonstrated that the sulphur SAD method with a chromium source becomes enhanced more practical for macromolecular structure determination using our crystal mounting technique.

show abstract

Structure of the Ca²⁺‐regulated photoprotein obelin at 1.7 Å resolution determined directly from its sulfur substructure

Cited by 182 publications

References 42 publications

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

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

Calcium dependence of aequorin bioluminescence dissected by random mutagenesis

Structure determination of a novel protein by sulfur SAD using chromium radiation in combination with a new crystal-mounting method

Contact Info

Product

Resources

About

Structure of the Ca2+‐regulated photoprotein obelin at 1.7 Å resolution determined directly from its sulfur substructure

Cited by 182 publications

References 42 publications

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

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

Calcium dependence of aequorin bioluminescence dissected by random mutagenesis

Structure determination of a novel protein by sulfur SAD using chromium radiation in combination with a new crystal-mounting method

Contact Info

Product

Resources

About

Structure of the Ca²⁺‐regulated photoprotein obelin at 1.7 Å resolution determined directly from its sulfur substructure

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

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