Photoproteins as luminescent labels in binding assays (original) (raw)
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Journal of Photochemistry and Photobiology B-biology, 2017
Bioluminescence of a variety of marine coelenterates is determined by Ca 2+-regulated photoproteins. A strong interest in these proteins is for their wide analytical potential as intracellular calcium indicators and labels for in vitro binding assays. The presently known hydromedusan Ca 2+-regulated photoproteins contain three (aequorin and clytin) or five (obelin and mitrocomin) cysteine residues with one of them strictly conserved. We have constructed Cys-free aequorin and obelin by substitution of all cysteines to serine residues. Such mutants should be of interest for researchers by the possibility to avoid the incubation with dithiothreitol (or β-mercaptoethanol) required for producing an active photoprotein that is important for some prospective analytical assays in which the photoprotein is genetically fused with a target protein sensitive to the reducing agents. Cys-free mutants were expressed in Escherichia coli, purified, and characterized regarding the efficiency of photoprotein complex formation, functional activity, and conformational stability. The replacement of cysteine residues has been demonstrated to affect different properties of aequorin and obelin. Cys-free aequorin displays a twofold lower specific bioluminescence activity but preserves similar activation properties and light emission kinetics compared to the wild-type aequorin. In contrast, Cys-free obelin retains only~10% of the bioluminescence activity of wild-type obelin as well as binding coelenterazine and forming active photoprotein much less effectively. In addition, the substitution of Cys residues drastically changes the bioluminescence kinetics of obelin completely eliminating a "fast" component from the light signal decay curve. At the same time, the replacement of Cys residues increases conformational flexibility of both aequorin and obelin molecules, but again, the effect is more prominent in the case of obelin. The values of thermal midpoints of unfolding (T m) were determined to be 53.3 ± 0.2 and 44.6 ± 0.4°C for aequorin and Cys-free aequorin, and 49.1 ± 0.1 and 28.8 ± 0.3°C for obelin and Cys-free obelin, respectively. Thus, so far only Cys-free aequorin is suitable as a partner for fusing with a tag sensitive to reducing agents since the aequorin mutant preserves almost 50% of the bioluminescent activity and can be produced with a substantial yield.
2008
The systems named “preliminary charged” occupy a special place among numerous bioluminescence systems. The most well-known and studied representatives of such bioluminescence systems are Ca 2+ -regulated photoproteins, which are mainly responsible for the luminescence of marine coelenterates [1]. The photoprotein molecule is a stable enzyme–substrate complex composed of a monosubunit polypeptide and an oxygen- preactivated substrate, 2-hydroperoxycoelenterazine, which is stably but noncovalently bound to the protein. Bioluminescence is initiated by calcium ions and emerges due to oxidative decarboxylation of the substrate bound to the protein. This causes formation of the reaction product, coelenteramide (CLM), in an excited state, and ëé 2 . The transition of CLM from the excited to ground state is accompanied by light emission. The bioluminescence of photoproteins is independent of é 2 , because oxygen is already bound to the protein as a 2-hydroperoxy derivative of coelenterazine. The independence of oxygen is among the main distinctions of photoprotein reactions from the other bioluminescence reactions catalyzed by luciferases. The bioluminescence of photoproteins is observed in the range of 465–495 nm and depends on the particular organism from which the photoprotein is isolated. The photoproteins bound to 2-hydroperoxycoelenterazine do not fluoresce but display a bright fluorescence immediately after the reaction, when bound to CLM.
Ca 2+ -Regulated Photoproteins: Structural Insight into the Bioluminescence Mechanism
Accounts of Chemical Research, 2004
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 of what are called "Ca 2+ -regulated photoproteins", has been shown to have very attractive properties both in general applications and for basic structural biology investigations. This review will survey the new information into their molecular mechanism of bioluminescence action. † Russian Academy of Sciences, Siberian Branch.
FEBS Letters, 2005
The bioluminescence spectra from the Ca 2+-regulated photoproteins aequorin (k max = 469 nm) and obelin (k max = 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 wavelength with F88Y having k max = 453 nm. Removal of the H-bond by the substitution of Y82F in aequorin shifted its bioluminescence to k max = 501 nm. All mutants were stable with good activity and were expressible in mammalian cells, thereby demonstrating potential for monitoring multiple events in cells using multi-color detection.
Genetically Engineered Obelin as a Bioluminescent Label in an Assay for a Peptide
Analytical Biochemistry, 1999
The marine polyp Obelia longissima produces a protein, obelin, which emits light in a calcium-dependent manner. This photoprotein consists of a stable complex of its apoprotein, a chromophore, and oxygen. In the presence of calcium ions, the protein undergoes a change in conformation that allows it to catalyze the oxidation of the chromophore, coelenterazine, to coelenteramide with the release of light and CO 2. Photoproteins are attractive as labels in analytical applications because the bioluminescent signal that they produce is the result of a chemical reaction and, therefore, has virtually no background. Thus, bioluminescence allows for extremely sensitive detection. In that regard, the feasibility of using obelin as a label has been explored with the development of a competitive immunoassay for the determination of a small peptide analyte. To attach the obelin label in a controlled manner to the octapeptide, a fusion protein was produced using recombinant DNA techniques. The protein consisted of the C-terminus of the peptide fused to the N-terminus of obelin. The octapeptide-obelin fusion protein retained the bioluminescence properties of the native protein, and was subsequently used to generate dose-response curves for the free octapeptide.
The theoretical studies of light emitters in bioluminescence of Ca-regulated photoprothin obelin
2008
ATP is the major source for metabolic energy in the cell and affects numerous cellular processes. Generally ATP is measured by luminescence assay using firefly luciferin-luciferase reaction as total ATP content in a mass of cells. We developed a luminescence microscope to measure ATP at the single cell level and applied it to monitor cytosolic ATP level throughout the apoptotic process in a HeLa cell transfected luciferase gene. As a result, apoptotic stimuli by four medicines (staurosporine, carbonyl cyanide p-trifluoro-methoxyphenylhydrazone, cycloheximide and actinomycin D) induced elevation of the ATP level after 1-2 h. This result confirmed previous data reported by luminometric assay. However, we observed a phenomenon just before cell death that some cells emit flash luminescence after 4-8 h stimulus. It is uncertain whether or not the flash luminescence is due to increased cytosolic ATP; such a result can not be obtained by conventional luminometric assay. The bioluminescence imaging assay at the cellular level would make a new research field, not only in ATP measurement but also in intracellular signal transduction, regulation of gene expression and other areas.
Calcium-regulated photoproteins of marine coelenterates
Molecular Biology, 2006
SPATIAL STRUCTURE AND MECHANISM OF THE BIOLUMINESCENT REACTION To date the spatial structure has been resolved for luciferases of bacteria [21], firefly [22], and dinoflagellate [23]. As for Ca 2+-regulated photoproteins, structures are known for their complexes with the substrate or the product [20, 24, 25], and with Ca 2+ initiating the bioluminescent reaction [26, 27]. These achievements are due to the high stability of photopro
Photochemistry and Photobiology, 2018
Site-directed mutagenesis is a powerful tool to investigate the structure-function relationship of proteins and a function of certain amino acid residues in catalytic conversion of substrates during enzymatic reactions. Hence, it is not surprising that this approach was repeatedly applied to elucidate the role of certain amino acid residues in various aspects of photoprotein bioluminescence, mostly for aequorin and obelin, and to design mutant photoproteins with altered properties (modified calcium affinity, faster or slower bioluminescence kinetics, different emission color) which would either allow the development of novel bioluminescent assays or improvement of characteristics of the already existing ones. This information, however, is scattered over different articles. In this review, we systematize the findings that were made using site-directed mutagenesis studies regarding the impact of various amino acid residues on bioluminescence of hydromedusan Ca 2+-regulated photoproteins. All key residues that have been identified are pinpointed, and their influence on different aspects of photoprotein functioning such as active photoprotein complex formation, bioluminescence reaction, calcium response and light emitter formation is discussed.
Journal of Proteomics, 2011
We present here a new analytical strategy for identification and characterisation of fluorescent proteins from marine organisms. By applying basic proteomics tools it is possible to screen large sample collections for fluorescent proteins of desired characteristics prior to gene cloning. Our methodology which includes isolation, spectral characterisation, stability testing, gel-based separation and mass spectrometric identification was optimised on samples collected during the Danish Galathea 3 expedition. Four corals of the Fungia, Sarcophyton and Acropora species emitting green fluorescence were tested. Each of the fluorescent extracts behaves differently under denaturing conditions but complete fluorescence loss was not observed. Optimised electrophoretic conditions yielded effective separation of active fluorescent proteins in both 1DE and 2DE. Mass spectrometric analysis of the proteins in the fluorescent spots excised directly from unstained 2DE gels provides sequence information that might be sufficient to design degenerate primers for gene cloning. Identified fluorescent proteins are in agreement with the coral species determined by visual examination of the samples. The presented methodology is a viable alternative to direct gene cloning for the discovery of novel fluorescent proteins and will be further validated on other samples collected during the Galathea 3 expedition. Novel fluorescent proteins Marine organisms 2DE Mass spectrometry De novo sequencing J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 1 ) 4 4 -5 5
Photochemistry and Photobiology
Site-directed mutagenesis is a powerful tool to investigate the structure-function relationship of proteins and a function of certain amino acid residues in catalytic conversion of substrates during enzymatic reactions. Hence, it is not surprising that this approach was repeatedly applied to elucidate the role of certain amino acid residues in various aspects of photoprotein bioluminescence, mostly for aequorin and obelin, and to design mutant photoproteins with altered properties (modified calcium affinity, faster or slower bioluminescence kinetics, different emission color) which would either allow the development of novel bioluminescent assays or improvement of characteristics of the already existing ones. This information, however, is scattered over different articles. In this review, we systematize the findings that were made using site-directed mutagenesis studies regarding the impact of various amino acid residues on bioluminescence of hydromedusan Ca 2+-regulated photoproteins. All key residues that have been identified are pinpointed, and their influence on different aspects of photoprotein functioning such as active photoprotein complex formation, bioluminescence reaction, calcium response and light emitter formation is discussed.