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This study elucidates functional artificial luciferases (ALucs) wholly synthesized for bioassays and molecular imaging. The ALucs bearing epitopes were newly created by amending the sequences of our previously reported ALucs in light of a multi-sequence alignment and hydrophobicity search. The synthesized ALucs are survived in live cells and stable in culture media for 25 days after secretion. The epitopes in ALucs are exposed during the secretion process and indeed valid for column purification and immunological assays. The ALucs exerted a 9400-times stronger optical intensity with a coelenterazine derivative (CTZ i), when compared with Renilla reniformis luciferase 8.6-535. A supersecondary structure of ALuc30 was predicted with respect to the X-ray crystallographic information of the coelenterazine-binding protein (CBP). The structure revealed that ALuc30 has a room for accommodating the iodide of CTZ i. This study guides on how to create functional artificial luciferases and predicts the structural details with the current bioinformatics technologies.
The different colors of light emitted by bioluminescent beetles that use an identical substrate and chemiexcitation reaction sequence to generate light remain a challenging and controversial mechanistic conundrum. The crystal structures of two beetle luciferases with red- and blue-shifted light relative to the green yellow light of the common firefly species provide direct insight into the molecular origin of the bioluminescence color. The structure of a blue-shifted green-emitting luciferase from the firefly Amydetes vivianii is monomeric with a structural fold similar to the previously reported firefly luciferases. The only known naturally red-emitting luciferase from the glow-worm Phrixothrix hirtus exists as tetramers and octamers. Structural and computational analyses reveal varying aperture between the two domains enclosing the active site. Mutagenesis analysis identified two conserved loops that contribute to the color of the emitted light. These results are expected to advance comparative computational studies into the conformational landscape of the luciferase reaction sequence.
Tumours are heterogeneous cell populations that undergo clonal evolution during tumour progression, metastasis and response to therapy. Short hairpin RNAs (shRNAs) generate stable loss-of-function phenotypes and are versatile experimental tools to explore the contribution of individual genetic alterations to clonal evolution. In these experiments tumour cells carrying shRNAs are commonly tracked with fluorescent reporters. While this works well for cell culture studies and leukaemia mouse models, fluorescent reporters are poorly suited for animals with solid tumours--the most common tumour types in cancer patients. Here we develop a toolkit that uses secreted luciferases to track the fate of two different shRNA-expressing tumour cell clones competitively, both in vitro and in vivo. We demonstrate that secreted luciferase activities can be measured robustly in the blood stream of tumour-bearing mice to accurately quantify, in a minimally invasive manner, the dynamic evolution of two genetically distinct tumour subclones in preclinical mouse models of tumour development, metastasis and therapy.
Both fluorescent and luminescent observation are widely used to examine real-time gene expression patterns in living organisms. Several fluuorescent and luminescent proteins with specific optical properties have been developed and applied for simultaneous, multi-color observation of more than two gene expression profiles. Compared to fluorescent proteins, however, the application of multi-color luminescent imaging in living organisms is still limited. In this study, we introduced two-color luciferases into the soil nematode C. elegans and performed simultaneous analysis of two gene expression profiles. Using a green-emitting luciferase Eluc (emerald luciferase) and red-emitting luciferase SLR (stable luciferase red), the expression patterns of two genes were simultaneously observed in single animals from embryonic to adult stages over its whole life span. In addition, dual gene activities were observed at the single embryo level, with the simultaneous observation of morphological changes. These are the first application of a two-color luciferase system into a whole animal and suggest that precise relationship of expression patterns of multiple genes of interest can be analyzed over the whole life of the animal, dependent on the changes in genetic and/or environmental conditions.
Firefly luciferases produce yellow-green light under physiological and alkaline conditions, however at acidic pH, higher temperatures or in the presence of heavy metals the color changes to red, a property called pH-sensitivity. Despite many decades of studies, the proton and metal binding sites responsible for pH-sensitivity remain enigmatic. Previously we suggested that the salt bridge E311/R337 keeps a closed conformation of the luciferin phenolate binding site. Here we further investigated the effect of this salt bridge and mutations of the neighbor residues H310 and E/N354, on metal and pH-sensitivity of firefly luciferases emitting distinct bioluminescence colors (Cratomorphus distinctus: 548 nm; Macrolampis sp2: 569 nm). The substitutions of H310 and E/N354 modulate metal sensitivity, whereas the carboxylate of E311 may work as the catalytic base essential for green bioluminescence and pH-sensitivity. Modeling studies showed that H310, E311 and E354 side-chains coordinate Zinc, constituting the metal binding site and the pH-sensor. Electrostatic potential and pKa calculations suggest that the external couple H310/E354 is affected by pH, whereas E311/R337 make a stabilized internal pair which retains excited oxyluciferin ejected proton near its phenolate group into a high energy state, promoting yellow-green bioluminescence. Protonation or metal binding weaken these electrostatic gates and their ability to retain the excited oxyluciferin released proton near its phenolate, promoting red light emission.
Networks of protein-protein interactions play key roles in numerous important biological processes in living subjects. An effective methodology to assess protein-protein interactions in living cells of interest is protein-fragment complement assay (PCA). Particularly the assays using fluorescent proteins are powerful techniques, but they do not directly track interactions because of its irreversibility or the time for chromophore formation. By contrast, PCAs using bioluminescent proteins can overcome these drawbacks. We herein describe an imaging method for real-time analysis of protein-protein interactions using multicolor luciferases with different spectral characteristics. The sensitivity and signal-to-background ratio were improved considerably by developing a carboxy-terminal fragment engineered from a click beetle luciferase. We demonstrate its utility in spatiotemporal characterization of Smad1-Smad4 and Smad2-Smad4 interactions in early developing stages of a single living Xenopus laevis embryo. We also describe the value of this method by application of specific protein-protein interactions in cell cultures and living mice. This technique supports quantitative analyses and imaging of versatile protein-protein interactions with a selective luminescence wavelength in opaque or strongly auto-fluorescent living subjects.
Luciferases from copepods Metridia longa and Gaussia princeps are successfully used as bioluminescent reporters for in vivo and in vitro assays. Here, we report the minimal sequence of copepod luciferases required for bioluminescence activity that was revealed by gradual deletions of sequence encoding the smallest MLuc7 isoform of M. longa luciferase. The single catalytic domain is shown to reside within the G32-A149 MLuc7 sequence and to be formed by both non-identical repeats, including 10 conserved Cys residues. Because this part of MLuc7 displays high homology with those of other copepod luciferases, our suggestion is that the determined boundaries of the catalytic domain are the same for all known copepod luciferases. The involvement of the flexible C-terminus in the retention of the bioluminescent reaction product in the substrate-binding cavity was confirmed by structural modeling and kinetics study. We also demonstrate that the ML7-N10 mutant (15.4 kDa) with deletion of ten amino acid residues at the N-terminus can be successfully used as a miniature bioluminescent reporter in living cells. Application of a shortened reporter may surely reduce the metabolic load on the host cells and decrease steric and functional interference at its use as a part of hybrid proteins.
Here we present a study of the thermal inactivation and the refolding of the proteins in Gram positive Bacillus subtilis. To enable use of bacterial luciferases as the models for protein thermal inactivation and refolding in B. subtilis cells, we developed a variety of bright luminescent B. subtilis strains which express luxAB genes encoding luciferases of differing thermolability. The kinetics of the thermal inactivation and the refolding of luciferases from Photorhabdus luminescens and Photobacterium leiognathi were compared in Gram negative and Gram positive bacteria. In B. subtilis cells, these luciferases are substantially more thermostable than in Escherichia coli. Thermal inactivation of the thermostable luciferase P. luminescens in B. subtilis at 48.5°С behaves as a first-order reaction. In E.coli, the first order rate constant (Kt) of the thermal inactivation of luciferase in E. coli exceeds that observed in B. subtilis cells 2.9 times. Incubation time dependence curves for the thermal inactivation of the thermolabile luciferase of P. leiognathi luciferase in the cells of E. coli and B. subtilis may be described by first and third order kinetics, respectively. Here we shown that the levels and the rates of refolding of thermally inactivated luciferases in B. subtilis cells are substantially lower that that observed in E. coli. In dnaK-negative strains of B. subtilis, both the rates of thermal inactivation and the efficiency of refolding are similar to that observed in wild-type strains. These experiments point that the role that DnaKJE plays in thermostability of luciferases may be limited to bacterial species resembling E. coli.
Detecting the folding/unfolding pathways of biological macromolecules is one of the urgent problems of molecular biophysics. The unfolding of bacterial luciferase from Vibrio harveyi is well-studied, unlike that of Photobacterium leiognathi, despite the fact that both of them are actively used as a reporter system. The aim of this study was to compare the conformational transitions of these luciferases from two different protein subfamilies during equilibrium unfolding with urea. Intrinsic steady-state and time-resolved fluorescence spectra and circular dichroism spectra were used to determine the stages of the protein unfolding. Molecular dynamics methods were applied to find the differences in the surroundings of tryptophans in both luciferases. We found that the unfolding pathway is the same for the studied luciferases. However, the results obtained indicate more stable tertiary and secondary structures of P. leiognathi luciferase as compared to enzyme from V. harveyi during the last stage of denaturation, including the unfolding of individual subunits. The distinctions in fluorescence of the two proteins are associated with differences in the structure of the C-terminal domain of α-subunits, which causes different quenching of tryptophan emissions. The time-resolved fluorescence technique proved to be a more effective method for studying protein unfolding than steady-state methods.
In vivo bioluminescence imaging (BLI) has been an invaluable noninvasive method to visualize molecular and cellular behaviors in laboratory animals. Bioluminescent reporter mice harboring luciferases for general use have been limited to a classical luciferase, Luc2, from Photinus pyralis, and have been extremely powerful for various in vivo studies. However, applicability of reporter mice for in vivo BLI could be further accelerated by increasing light intensity through the use of other luciferases and/or by improving the biodistribution of their substrates in the animal body. Here we created two Cre-dependent reporter mice incorporating luciferases oFluc derived from Pyrocoeli matsumurai and Akaluc, both of which had been reported previously to be brighter than Luc2 when using appropriate substrates; we then tested their bioluminescence in neural tissues and other organs in living mice. When expressed throughout the body, both luciferases emitted an intense yellow (oFluc) or far-red (Akaluc) light easily visible to the naked eye. oFluc and Akaluc were similarly bright in the pancreas for in vivo BLI; however, Akaluc was superior to oFluc for brain imaging, because its substrate, AkaLumine-HCl, was distributed to the brain more efficiently than the oFluc substrate, D-luciferin. We also demonstrated that the lights produced by oFluc and Akaluc were sufficiently spectrally distinct from each other for dual-color imaging in a single living mouse. Taken together, these novel bioluminescent reporter mice are an ideal source of cells with bright bioluminescence and may facilitate in vivo BLI of various tissues/organs for preclinical and biomedical research in combination with a wide variety of Cre-driver mice.
A library of 367 protein kinase inhibitors, the GSK Published Kinase Inhibitor Set (PKIS), which has been annotated for protein kinase family activity and is available for public screening efforts, was assayed against the commonly used luciferase reporter enzymes from the firefly, Photinus pyralis (FLuc) and marine sea pansy, Renilla reniformis (RLuc). A total of 22 compounds (∼6% of the library) were found to inhibit FLuc with 10 compounds showing potencies ≤1 µM. Only two compounds were found to inhibit RLuc, and these showed relatively weak potency values (∼10 µM). An inhibitor series of the VEGFR2/TIE2 protein kinase family containing either an aryl oxazole or benzimidazole-urea core illustrate the different structure activity relationship profiles FLuc inhibitors can display for kinase inhibitor chemotypes. Several FLuc inhibitors were broadly active toward the tyrosine kinase and CDK families. These data should aid in interpreting the results derived from screens employing the GSK PKIS in cell-based assays using the FLuc reporter. The study also underscores the general need for strategies such as the use of orthogonal reporters to identify kinase or non-kinase mediated cellular responses.
Currently, a variety of red and green beetle luciferase variants are available for bioluminescence imaging (BLI). In addition, new luciferin analogues providing longer wavelength luminescence have been developed that show promise for improved deep tissue imaging. However, a detailed assessment of these analogues (e.g., Akalumine-HCl, CycLuc1, and amino naphthyl luciferin (NH2-NpLH2)) combined with state of the art luciferases has not been performed. The aim of this study was to evaluate for the first time the in vivo brightness and spectral characteristics of firefly (Luc2), click beetle green (CBG99), click beetle red 2 (CBR2), and Akaluc luciferases when paired with different D-luciferin (D-LH2) analogues in vivo.
We recently reported the cDNA sequences of 11 copepod luciferases from the superfamily Augaptiloidea in the order Calanoida. They were classified into two groups, Metridinidae and Heterorhabdidae/Lucicutiidae families, by phylogenetic analyses. To elucidate the evolutionary processes, we have now further isolated 12 copepod luciferases from Augaptiloidea species (Metridia asymmetrica, Metridia curticauda, Pleuromamma scutullata, Pleuromamma xiphias, Lucicutia ovaliformis and Heterorhabdus tanneri). Codon-based synonymous/nonsynonymous tests of positive selection for 25 identified copepod luciferases suggested that positive Darwinian selection operated in the evolution of Heterorhabdidae luciferases, whereas two types of Metridinidae luciferases had diversified via neutral mechanism. By in silico analysis of the decoded amino acid sequences of 25 copepod luciferases, we inferred two protein sequences as ancestral copepod luciferases. They were expressed in HEK293 cells where they exhibited notable luciferase activity both in intracellular lysates and cultured media, indicating that the luciferase activity was established before evolutionary diversification of these copepod species.
Bioluminescence relies on the oxidation of a luciferin substrate catalysed by a luciferase enzyme. Luciferins and luciferases are generic terms used to describe a large variety of substrates and enzymes. Whereas luciferins can be shared by phylogenetically distant organisms which feed on organisms producing them, luciferases have been thought to be lineage-specific enzymes. Numerous light emission systems would then have co-emerged independently along the tree of life resulting in a plethora of non-homologous luciferases. Here, we identify for the first time a candidate luciferase of a luminous echinoderm, the ophiuroid Amphiura filiformis Phylogenomic analyses identified the brittle star predicted luciferase as homologous to the luciferase of the sea pansy Renilla (Cnidaria), contradicting with the traditional viewpoint according to which luciferases would generally be of convergent origins. The similarity between the Renilla and Amphiura luciferases allowed us to detect the latter using anti-Renilla luciferase antibodies. Luciferase expression was specifically localized in the spines which were demonstrated to be the bioluminescent organs in vivo However, enzymes homologous to the Renilla luciferase but unable to trigger light emission were also identified in non-luminous echinoderms and metazoans. Our findings strongly indicate that those enzymes, belonging to the haloalkane dehalogenase family, might then have been convergently co-opted into luciferases in cnidarians and echinoderms. In these two benthic suspension-feeding species, similar ecological pressures would constitute strong selective forces for the functional shift of these enzymes and the emergence of bioluminescence.
A unique combinatorial bioluminescence (BL) imaging system was developed for determining molecular events in mammalian cells with various colors and BL intensity patterns. This imaging system consists of one or multiple reporter luciferases and a series of novel coelenterazine (CTZ) analogues named "S-series". For this study, ten kinds of novel S-series CTZ analogues were synthesized and characterized concerning the BL intensities, spectra, colors, and specificity of various marine luciferases. The characterization revealed that the S-series CTZ analogues luminesce with blue-to-orange-colored BL spectra with marine luciferases, where the most red-shifted BL spectrum peaked at 583 nm. The colors completed a visible light color palette with those of our precedent C-series CTZ analogues. The synthesized substrates S1, S5, S6, and S7 were found to have a unique specificity with marine luciferases, such as R86SG, NanoLuc (shortly, NLuc), and ALuc16. They collectively showed unique BL intensity patterns to identify the marine luciferases together with colors. The marine luciferases, R86SG, NLuc, and ALuc16, were multiplexed into multi-reporter systems, the signals of which were quantitatively unmixed with the specific substrates. When the utility was applied to a single-chain molecular strain probe, the imaging system simultaneously reported three different optical indexes for a ligand, i.e., unique BL intensity and color patterns for identifying the reporters, together with the ligand-specific fold intensities in mammalian cells. This study directs a new combinatorial BL imaging system to specific image molecular events in mammalian cells with multiple optical indexes.
Bioluminescent proteins (BLPs) are a class of proteins with various mechanisms of light emission such as bioluminescence and fluorescence from luminous organisms. While valuable for commercial and medical applications, identification of BLPs, including luciferases and fluorescent proteins (FPs), is rather challenging, owing to their high variety of protein sequences. Moreover, characterization of BLPs facilitates mutagenesis analysis to enhance bioluminescence and fluorescence. Therefore, this study proposes a novel methodological approach to estimating the propensity scores of 400 dipeptides and 20 amino acids in order to design two prediction methods and characterize BLPs based on a scoring card method (SCM). The SCMBLP method for predicting BLPs achieves an accuracy of 90.83% for 10-fold cross-validation higher than existing support vector machine based methods and a test accuracy of 82.85%. A dataset consisting of 269 luciferases and 216 FPs is also established to design the SCMLFP prediction method, which achieves training and test accuracies of 97.10% and 96.28%, respectively. Additionally, four informative physicochemical properties of 20 amino acids are identified using the estimated propensity scores to characterize BLPs as follows: 1) high transfer free energy from inside to the protein surface, 2) high occurrence frequency of residues in the transmembrane regions of the protein, 3) large hydrophobicity scale from the native protein structure, and 4) high correlation coefficient (R = 0.921) between the amino acid compositions of BLPs and integral membrane proteins. Further analyzing BLPs reveals that luciferases have a larger value of R (0.937) than FPs (0.635), suggesting that luciferases tend to locate near the cell membrane location rather than FPs for convenient receipt of extracellular ions. Importantly, the propensity scores of dipeptides and amino acids and the identified properties facilitate efforts to predict, characterize, and apply BLPs, including luciferases, photoproteins, and FPs. The web server is available at http://iclab.life.nctu.edu.tw/SCMBLP/index.html.
Secreted luciferases are highly useful bioluminescent reporters for cell-based assays and drug discovery. A variety of secreted luciferases from marine organisms have been described that harbor an N-terminal signal peptide for release along the classical secretory pathway. Here, we have characterized the secretion of Gaussia luciferase in more detail.
The present study introduces a unique BL signature imaging system with novel CTZ analogues named "C-series." Nine kinds of C-series CTZ analogues were first synthesized, and BL intensity patterns and spectra were then examined according to the marine luciferases. The results show that the four CTZ analogues named C3, C4, C6, and C7, individually or collectively luminesce with completely distinctive BL spectral signatures and intensity patterns according to the luciferases: Renilla luciferase (RLuc), NanoLuc, and artificial luciferase (ALuc). The signatural reporters were multiplexed into a multi-reporter system comprising RLuc8.6-535SG and ALuc16. The usefulness of the signatural reporters was further determined with a multi-probe system that consists of two single-chain probes embedding RLuc8 and ALuc23. This study is a great addition to the study of conventional bioassays with a unique methodology, and for the specification of each signal in a single- or multi-reporter system using unique BL signatures and patterns of reporter luciferases.
Bioluminescence (BL) is unique cold body radiation of light, generated by luciferin-luciferase reactions and commonly used in various bioassays and molecular imaging. However, most of the peak emissions of BL populate the blue-yellow region and have broad spectral bandwidths and thus superimpose each other, causing optical cross-leakages in multiplex assays. This study synthesized a new series of coelenterazine (CTZ) analogues, named K-series, that selectively illuminates marine luciferases with unique, blue-shifted spectral properties. The optical property and specificity of the K-series CTZ analogues were characterized by marine luciferases, with K2 and K5 found to specifically luminesce with ALuc- and RLuc-series marine luciferases, respectively. The results confirmed that the luciferase specificity and color variation of the CTZ analogues minimize the cross-leakages of BL signals and enable high-throughput screening of specific ligands in the mixture. The specificity and color variation of the substrates were further tailored to marine luciferases (or single-chain bioluminescent probes) to create a multiplex quadruple assay system with four integrated, single-chain bioluminescent probes, with each probe designed to selectively luminesce only with its specific ligand (first authentication) and a specific CTZ analogue (second authentication). This unique multiplex quadruple bioluminescent assay system is an efficient optical platform for specific and high-throughput imaging of multiple optical markers in bioassays without optical cross-leakages.
Multicolor bioluminescence imaging using near-infrared emitting luciferases is an attractive application to detect two cell populations within one animal model. Herein, we describe how to distinguish dual-color bioluminescent signals co-localized in the same compartment. We tested CBG2 click beetle (λ = 660 nm) and CBR2 click beetle (λ = 730 nm) luciferases paired with NH2-NpLH2 luciferin. Following a spectral unmixing algorithm, single spectral contributions can be resolved and quantified, enabling the visualization of multiple cell types in deep tissue by injection of a single substrate. For complete details on the use and execution of this protocol, please refer to Zambito et al. (2020).
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