Halistaurin, phialidin and modified forms of aequorin as Ca2+ indicator in biological systems.
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ABSTRACT:
SUBMITTER: Shimomura O
PROVIDER: S-EPMC1145046 | biostudies-other | 1985 Jun
REPOSITORIES: biostudies-other
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Project description:Bioluminescence recording of Ca(2+) signals with the photoprotein aequorin does not require radiative energy input and can be measured with a low background and good temporal resolution. Shifting aequorin emission to longer wavelengths occurs naturally in the jellyfish Aequorea victoria by bioluminescence resonance energy transfer (BRET) to the green fluorescent protein (GFP). This process has been reproduced in the molecular fusions GFP-aequorin and monomeric red fluorescent protein (mRFP)-aequorin, but the latter showed limited transfer efficiency. Fusions with strong red emission would facilitate the simultaneous imaging of Ca(2+) in various cell compartments. In addition, they would also serve to monitor Ca(2+) in living organisms since red light is able to cross animal tissues with less scattering. In this study, aequorin was fused to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer Tomato-aequorin (tdTA) showed the highest BRET efficiency (largest energy transfer critical distance R(0)) and percentage of counts in the red band of all the fusions studied. In addition, red fluorophore maturation of tdTA within cells was faster than that of other fusions. Light output was sufficient to image ATP-induced Ca(2+) oscillations in single HeLa cells expressing tdTA. Ca(2+) rises caused by depolarization of mouse neuronal cells in primary culture were also recorded, and changes in fine neuronal projections were spatially resolved. Finally, it was also possible to visualize the Ca(2+) activity of HeLa cells injected subcutaneously into mice, and Ca(2+) signals after depositing recombinant tdTA in muscle or the peritoneal cavity. Here we report that tdTA is the brightest red bioluminescent Ca(2+) sensor reported to date and is, therefore, a promising probe to study Ca(2+) dynamics in whole organisms or tissues expressing the transgene.
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Project description:The photoprotein aequorin emits light by an intramolecular reaction when a trace of Ca2+ is added. The samples of aequorin that were purified by the conventional methods of column chromatography were separated by high-performance liquid chromatography into eight molecular forms (isoaequorins), which were designated aequorins A-H. Aequorins A, C and F were obtained in crystalline states. A wide range of properties were studied with aequorins A-F, which were essentially pure. These six isoaequorins showed relatively small differences in their spectroscopic properties, but their values of A0.1%/1 cm, 280 were found to be close to 3.0, about 10% more than the previously reported value of 2.70-2.71 that was obtained with the samples of conventionally purified aequorin. The Mr values ranged from 20,100 (aequorin F) to 22,800 (aequorin A), the luminescence activities ranged from 4.35 X 10(15) photons/mg (aequorin A) to 5.16 X 10(15) photons/mg (aequorin F), and the first-order reaction rate constants of luminescence ranged from 0.95 s-1 (aequorin A) to 1.33 s-1 (aequorin F). As regards sensitivity to Ca2+, aequorin D was the most sensitive, having a sensitivity about 0.4-0.5 pCa unit above that of the least sensitive kind (aequorin A).
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Project description:The Ca2+-binding photoprotein aequorin is a complex of apoAequorin (apoprotein) and (S)-2-peroxycoelenterazine. Aequorin can be regenerated by the incubation of apoAequorin with coelenterazine and molecular oxygen (O2). In this study, to investigate the molecular recognition of apoAequorin for coelenterazine using chemical probes, the chiral deaza-analogs of (S)- and (R)-deaza-CTZ (daCTZ) for coelenterazine and of (S)-2- and (R)-2-hydroxymethyl-deaza-CTZ (HM-daCTZ) for 2-peroxycoelenterazine were efficiently prepared by the improvement method. The chiral deaza-analogs of (S)-daCTZ and (S)-HM-daCTZ selectively inhibited the regeneration step to aequorin by binding the catalytic site of coelenterazine in the apoAequorin molecule. The crystal structures of the apoAequorin complexes with (S)-daCTZ and (S)-HM-daCTZ were determined, suggesting that the hydroxy moiety at the C6-hydroxyphenyl group and the carbonyl moiety of the imidazopyrazinone ring in coelenterazine are essential to bind the apoAequorin molecule through hydrogen bonding. Therefore, the chiral deaza-analogs of coelenterazine can be used as a probe to study the interaction between coelenterazine and the related proteins including photoprotein, luciferase, and coelenterazine-binding protein.
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