Obelin from the Bioluminescent Marine Hydroid Obelia geniculata:  Cloning, Expression, and Comparison of Some Properties with Those of Other Ca²⁺-Regulated Photoproteins

Abstract

A cDNA encoding the Ca2+-regulated photoprotein of the bioluminescent marine hydroid Obelia geniculata was cloned and sequenced. The cDNA is a 774 bp fragment containing two overlapping open reading frames, one of which contained 585 bp encoding a 195 amino acid polypeptide which obviously has the primary structure of the apoprotein of a calcium-regulated photoprotein. Many of the residues are identical to those in other Ca2+-regulated photoproteins: 86% compared with that from Obelia longissima, 76% with that from Clytia (Phialidium), 64% with that from Aequorea, and 64% with that from Mitrocoma(Halistaura). The obelin from O. geniculata was overexpressed in Escherichia coli, refolded from inclusion bodies, and purified. The yield of highly purified recombinant protein was 55-80 mg/L of LB medium. O. geniculata obelin has absorption maxima at 280 and 460 nm and a shoulder at approximately 310 nm. The calcium-discharged protein loses visible absorption but exhibits a new absorption maximum at 343 nm. The bioluminescence of the obelin from O. geniculata is blue (lambda(max) = 495 nm). In contrast, the fluorescence of the calcium-discharged protein is yellow-green (lambda(max) = 520 nm; excitation at 340 nm). This is in sharp contrast to aequorin in which the bioluminescence and fluorescence emission spectra of the calcium-discharged protein are almost identical (lambda(max) = 465 nm). The Ca2+ concentration-effect curve for O. geniculata obelin is similar to those of many other photoproteins: at [Ca2+] below approximately 10(-8) M, calcium-independent luminescence is observed, and at [Ca2+] approximately 10(-3) M, the luminescence reaches a maximum. Between these extremes, the curve spans a vertical range of almost 8 log units with a maximum slope on a log-log plot of about 2.5. In the absence of Mg2+ the rate constant for the rise of bioluminescence determined by the stopped-flow technique is about 450 s(-1). The effects of Mg2+ on the kinetics of bioluminescence are complicated, but at all concentrations studied they are relatively small compared to the corresponding effects on aequorin luminescence. At least with respect to speed and sensitivity to Mg2+, the obelins from both O. longissima and O. geniculata would appear to be more suitable than aequorin for use as intracellular Ca2+ indicators.

CcbP, a calcium-binding protein from Anabaena sp. PCC 7120, provides evidence that calcium ions regulate heterocyst differentiation

“…This suggestion is also supported by the results of CcbP localization, which showed that it was present in vegetative cells but absent from heterocysts (Fig. 5) (30). Although the maximum fluorescence emission of recombinant obelin-Ca 2ϩ in vitro is at 517 nm (30), the peak of obelin-Ca 2ϩ fluorescence emission is at 460 nm in Anabaena cells (Fig.…”

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

“…For instance, in obelin (state II) the N 2 atoms of His-22 and His-24 (helix A) form hydrogen bonds with carbonyl oxygens of Trp-179 (helix H) and Gly-193 (C terminus); N 1 and N 2 atoms of Arg-21 (helix A) are hydrogen bonded with the carbonyl oxygen of Phe-178 (helix H), with the O␦ 1 oxygen of Asp-187 (C terminus), and with the oxygen of Pro-195, all situated in the C terminus. All these listed hydrogen bonds persist in the Ca 2ϩ -discharged obelin having bound coelenteramide and calcium (state III) and this strengthens the idea that the binding cavity remains inaccessible for solvent, thus providing the environment favoring the efficient fluorescence of the bound coelenteramide in the Ca 2ϩ -discharged photoproteins (8,24). Ca 2؉ -Binding Loops.…”

“…The best studied proteins using this luciferin are the Ca 2ϩ -regulated photoproteins such as those responsible for the bioluminescence of marine coelenterates (1), for example, aequorin from the jellyfish Aequorea (2, 3) and obelin from the hydroid Obelia (4,5). All of the photoproteins characterized to date consist of a single polypeptide chain with high sequence homology and contain three ''EF-hand'' Ca 2ϩ -binding consensus sequences (6)(7)(8) like some other Ca 2ϩ -binding proteins (9). Their bioluminescence property derives from the fact that they contain in their active site an ''activated'' coelenterazine, 2-hydroperoxycoelenterazine, tightly but noncovalently bound.…”

Calcium dependence of aequorin bioluminescence dissected by random mutagenesis

“…The fast kinetics of photoprotein luminescence provides a sensitive means of studying the early steps, which lead from calcium binding to activation of EF-hand-based calcium sensors. The structural homology among various photoproteins suggests that their [Ca 2ϩ ] depen-dence obeys the same rules, despite different maximum luminescence rates (22,23). Photoproteins contain a pseudo-EFhand motif, which does not bind Ca 2ϩ (6,13).…”