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Metridia longa is a marine copepod from which a blue bioluminescence originates as a secretion from epidermal glands in response to various stimuli. We demonstrate that Metridia luciferase is specific for coelenterazine to produce blue light (lambda(max) = 480 nm). Using an expression cDNA library and functional screening, we cloned and sequenced the cDNA(More)
Two kinds of Ca(2+)-regulated photoprotein obelin with altered color of bioluminescence were obtained by active-center amino acid substitution. The mutant W92F-H22E emits violet light (lambda(max) = 390 nm) and the mutant Y139F emits greenish light (lambda(max) = 498 nm), with small spectral overlap, both display high activity and stability and thus may be(More)
The crystal structures of calcium-loaded apo-aequorin and apo-obelin have been determined at resolutions 1.7A and 2.2 A, respectively. A calcium ion is observed in each of the three EF-hand loops that have the canonical calcium-binding sequence, and each is coordinated in the characteristic pentagonal bipyramidal configuration. The calcium-loaded(More)
Ca2+-regulated photoproteins are members of the EF-hand calcium-binding protein family. The addition of Ca2+ produces a blue bioluminescence by triggering a decarboxylation reaction of protein-bound hydroperoxycoelenterazine to form the product, coelenteramide, in an excited state. Based on the spatial structures of aequorin and several obelins, we have(More)
The bioluminescent jellyfish has contributed two famous proteins to modern science: green fluorescent protein or GFP, which finds wide use as a probe in cell biology studies, and aequorin, which has been used for intracellular calcium measurement for more than 30 years. More recently, obelin, a protein from the bioluminescent hydroid and also in the family(More)
The Ca(2+)-regulated photoprotein obelin was substituted at Trp92 by His, Lys, Glu, and Arg. All mutants fold into stable conformations and produce bimodal bioluminescence spectra with enhanced contribution from a violet emission. The W92R mutant has an almost monomodal bioluminescence (lambdamax=390 nm) and monomodal fluorescence (lambdamax=425 nm) of the(More)
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,(More)
The bioluminescence spectra from the Ca2+-regulated photoproteins aequorin (lambdamax=469 nm) and obelin (lambdamax=482 nm) differ because aequorin has an H-bond from its Tyr82 to the bound coelenteramide, not present in obelin at the corresponding Phe88. Substitutions of this Phe88 by Tyr, Trp, or His shifted the obelin bioluminescence to shorter(More)
The crystal structure at 1.93-A resolution is determined for the Ca2+-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(More)