79 results on '"Branchini BR"'
Search Results
2. Synthesis and Bioluminescence of 'V'-Shaped Firefly Luciferin Analogues Based on A Novel Benzobisthiazole Core.
- Author
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Chang CH, Fontaine DM, Gómez S, Branchini BR, and Anderson JC
- Subjects
- Luciferases chemistry, Spectrum Analysis, Luminescent Measurements methods, Luciferases, Firefly, Firefly Luciferin chemistry, Light
- Abstract
The design of π-extended conjugation 'V'-shaped red shifted bioluminescent D-luciferin analogues based on a novel benzobisthiazole core is described. The divergent synthetic route allowed access to a range of amine donor substituents through an S
N Ar reaction. In spectroscopic studies, the 'V'-shaped luciferins exhibited narrower optical band gaps, more red-shifted absorption and emission spectra than D-luciferin. Their bioluminescence characteristics were recorded against four different luciferases (PpyLuc, FlucRed, CBR2 and PLR3). With native luciferase PpyLuc, the 'V'-shaped luciferins demonstrated more red-shifted emissions than D-luciferin (λbl =561 nm) by 60 to 80 nm. In addition, the benzobisthiazole luciferins showed a wide range of bioluminescence spectra from the visible light region (λbl =500 nm) to the nIR window (>650 nm). The computational results validate the design concept which can be used as a guide for further novel D-luciferin analogues based upon other 'V'-shaped heterocyclic cores., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)- Published
- 2023
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3. Structure elucidation of Keroplatus (Diptera:Keroplatidae) fungus gnat oxyluciferin.
- Author
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Kotlobay AA, Dubinnyi MA, Kovalchuk SI, Makhin AP, Miturich VS, Lyakhovich MS, Fontaine DM, Southworth TL, Shmygarev VI, Yatskin ON, Branchini BR, Yampolsky IV, and Kaskova ZM
- Abstract
Bioluminescence of insects is a well-known natural phenomenon in the focus of interest of scientific research. While the mechanisms of bioluminescence in Coleoptera have been extensively studied, there is a lack of information about the chemistry of light emission in Diptera species. Here we report the Keroplatus spp. oxyluciferin structure elucidation and identification as 3-hydroxykynurenic acid. Additionally, the present study provides the first direct evidence of the relationship between the bioluminescent systems of Orfelia and Keroplatus. However, the properties of the putative Orfelia oxyluciferin suggest that the light emission mechanisms are not identical., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2023 Elsevier Inc. All rights reserved.)
- Published
- 2023
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4. Bioluminescence, photophysical, computational and molecular docking studies of fully conformationally restricted enamine infraluciferin.
- Author
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Chang CH, Gómez S, Fontaine DM, Fikas P, Branchini BR, and Anderson JC
- Subjects
- Molecular Docking Simulation, Luciferases chemistry, Luminescent Measurements methods, Firefly Luciferin chemistry, Luciferins
- Abstract
A new rationally designed fully rotationally restricted luciferin has been synthesised. This synthetic luciferin, based upon the structure of infraluciferin, has two intramolecular H-bonds to reduce degrees of freedom, an amine group to enhance ICT process, and an alkenyl group to increase π-conjugation. In the spectroscopic measurements and computational calculations, enamine luciferin showed more red-shifted absorption and fluorescence emission than LH
2 and iLH2 . With PpyWT luciferase enamine luciferin gave bioluminescence at 564 nm which is similar to LH2 at 561 nm. Further investigation by docking studies revealed that the emission wavelength of enamine luciferin might be attributed to the unwanted twisted structure caused by Asp531 within the enzyme. With mutant luciferase FlucRed, the major emission peak was shifted to 606 nm, a distinct shoulder above 700 nm, and 21% of its spectrum located in the nIR range.- Published
- 2023
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5. Author Correction: A new brilliantly blue-emitting luciferin-luciferase system from Orfelia fultoni and Keroplatinae (Diptera).
- Author
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Viviani VR, Silva JR, Amaral DT, Bevilaqua VR, Abdalla FC, Branchini BR, and Johnson CH
- Published
- 2023
- Full Text
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6. A higher spectral range of beetle bioluminescence with infraluciferin.
- Author
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Jathoul AP, Branchini BR, Anderson JC, and Murray JAH
- Abstract
Coleopteran bioluminescence is unique in that beetle luciferases emit colors ranging between green (ca.550 nm) and red (ca.600 nm), including intermediate colors such as yellow and orange, allowing up to 3 simultaneous parameters to be resolved in vitro with natural luciferin ( D -LH
2 ). Here, we report a more than doubling of the maximum bioluminescence wavelength range using a single synthetic substrate, infraluciferin (iLH2 ). We report that different luciferases can emit colors ranging from visible green to near-infrared (nIR) with iLH2, including in human cells. iLH2 was designed for dual color far-red to nIR bioluminescence imaging (BLI) in small animals and has been utilized in different mouse models of cancer (including a metastatic hepatic model showing detailed hepatic morphology) and for robust dual parameter imaging in vivo (including in systemic hematological models). Here, we report the properties of different enzymes with iLH2 : Lampyrid wild-type (WT) Photinus pyralis ( Ppy ) firefly luciferase, Ppy -based derivatives previously engineered to be thermostable with D -LH2 , and also color-shifted Elaterid-based enzymes: blue-shifted Pyrearinus termitilluminans derivative Eluc (reported D-LH2 λmax = 538 nm) and red-shifted Pyrophorus plagiopthalamus derivative click beetle red (CBR) luciferase ( D -LH2 λmax = 618 nm). As purified enzyme, in bacteria or in human cells, Eluc emitted green light (λmax = 536 nm) with DL -iLH2 whereas Ppy Fluc (λmax = 689 nm), x2 Fluc (λmax = 704 nm), x5 Fluc (λmax = 694 nm), x11 Fluc (λmax = 694 nm) and CBR (λmax = 721 nm) produced far-red to nIR peak wavelengths. Therefore, with iLH2, enzyme λmaxes can be separated by ca.185nm, giving almost non-overlapping spectra. This is the first report of single-substrate bioluminescence color emission ranging from visible green to nIR in cells and may help shed light on the color tuning mechanism of beetle luciferases. We also report on the reason for the improvement in activity of x11 Fluc with iLH2 and engineer an improved infraluciferase (iluc) based on this mutant., Competing Interests: APJ was employed by Bioflares Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jathoul, Branchini, Anderson and Murray.)- Published
- 2022
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7. Systematic Comparison of Beetle Luciferase-Luciferin Pairs as Sources of Near-Infrared Light for In Vitro and In Vivo Applications.
- Author
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Branchini BR, Fontaine DM, Kohrt D, Huta BP, Racela AR, Fort BR, Southworth TL, and Roda A
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- Animals, Firefly Luciferin chemistry, HEK293 Cells, Humans, Infrared Rays, Luciferases chemistry, Luciferases genetics, Luminescent Measurements methods, Coleoptera, Luciferins
- Abstract
Luciferases catalyze light-emitting reactions that produce a rainbow of colors from their substrates (luciferins), molecular oxygen, and often additional cofactors. These bioluminescence (BL) systems have afforded an incredible variety of basic research and medical applications. Driven by the importance of BL-based non-invasive animal imaging (BLI) applications, especially in support of cancer research, new BL systems have been developed by engineering beetle luciferase (Luc) variants and synthetic substrate combinations to produce red to near-infrared (nIR) light to improve imaging sensitivity and resolution. To stimulate the application of BLI research and advance the development of improved reagents for BLI, we undertook a systematic comparison of the spectroscopic and BL properties of seven beetle Lucs with LH
2 and nine substrates, which included two new quinoline ring-containing analogs. The results of these experiments with purified Luc enzymes in vitro and in live HEK293T cells transfected with luc genes have enabled us to identify Luc/analog combinations with improved properties compared to those previously reported and to provide live cell BL data that may be relevant to in vivo imaging applications. Additionally, we found strong candidate enzyme/substrate pairs for in vitro biomarker applications requiring nIR sources with minimal visible light components. Notably, one of our new substrates paired with a previously developed Luc variant was demonstrated to be an excellent in vitro source of nIR and a potentially useful BL system for improved resolution in BLI.- Published
- 2022
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8. Dendritic cells maintain anti-tumor immunity by positioning CD8 skin-resident memory T cells.
- Author
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Vella JL, Molodtsov A, Angeles CV, Branchini BR, Turk MJ, and Huang YH
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- Animals, Biomarkers, CD8-Positive T-Lymphocytes metabolism, Dendritic Cells metabolism, Disease Models, Animal, Fluorescent Antibody Technique, Humans, Immunity, Immunophenotyping, Lymphocytes, Tumor-Infiltrating immunology, Lymphocytes, Tumor-Infiltrating metabolism, Lymphocytes, Tumor-Infiltrating pathology, Melanoma metabolism, Melanoma pathology, Memory T Cells metabolism, Mice, CD8-Positive T-Lymphocytes immunology, Dendritic Cells immunology, Melanoma immunology, Memory T Cells immunology
- Abstract
Tissue-resident memory (T
RM ) T cells are emerging as critical components of the immune response to cancer; yet, requirements for their ongoing function and maintenance remain unclear. APCs promote TRM cell differentiation and re-activation but have not been implicated in sustaining TRM cell responses. Here, we identified a novel role for dendritic cells in supporting TRM to melanoma. We showed that CD8 TRM cells remain in close proximity to dendritic cells in the skin. Depletion of CD11c+ cells results in rapid disaggregation and eventual loss of melanoma-specific TRM cells. In addition, we determined that TRM migration and/or persistence requires chemotaxis and adhesion mediated by the CXCR6/CXCL16 axis. The interaction between CXCR6-expressing TRM cells and CXCL16-expressing APCs was found to be critical for sustaining TRM cell-mediated tumor protection. These findings substantially expand our knowledge of APC functions in TRM T-cell homeostasis and longevity., (© 2021 Vella et al.)- Published
- 2021
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9. A New Ultrasensitive Bioluminescence-Based Method for Assaying Monoacylglycerol Lipase.
- Author
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Miceli M, Casati S, Allevi P, Berra S, Ottria R, Rota P, Branchini BR, and Ciuffreda P
- Subjects
- Anti-Anxiety Agents pharmacology, Enzyme Inhibitors pharmacology, Humans, Monoacylglycerol Lipases antagonists & inhibitors, Benzodioxoles pharmacology, Benzothiazoles metabolism, Biological Assay methods, Luciferases metabolism, Luminescence, Monoacylglycerol Lipases metabolism, Piperidines pharmacology
- Abstract
A novel bioluminescent Monoacylglycerol lipase (MAGL) substrate 6-O-arachidonoylluciferin, a D-luciferin derivative, was synthesized, physico-chemically characterized, and used as highly sensitive substrate for MAGL in an assay developed for this purpose. We present here a new method based on the enzymatic cleavage of arachidonic acid with luciferin release using human Monoacylglycerol lipase ( h MAGL) followed by its reaction with a chimeric luciferase, PLG2, to produce bioluminescence. Enzymatic cleavage of the new substrate by MAGL was demonstrated, and kinetic constants Km and Vmax were determined. 6-O-arachidonoylluciferin has proved to be a highly sensitive substrate for MAGL. The bioluminescence assay (LOD 90 pM, LOQ 300 pM) is much more sensitive and should suffer fewer biological interferences in cells lysate applications than typical fluorometric methods. The assay was validated for the identification and characterization of MAGL modulators using the well-known MAGL inhibitor JZL184. The use of PLG2 displaying distinct bioluminescence color and kinetics may offer a highly desirable opportunity to extend the range of applications to cell-based assays.
- Published
- 2021
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10. Control of B Cell Lymphoma by Gammaherpesvirus-Induced Memory CD8 T Cells.
- Author
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Preiss NK, Kang T, Usherwood YK, Huang YH, Branchini BR, and Usherwood EJ
- Subjects
- Animals, Epitopes, T-Lymphocyte genetics, Female, Lymphoma, B-Cell genetics, Lymphoma, B-Cell pathology, Mice, Mice, Transgenic, Murine hepatitis virus genetics, Neoplasm Proteins genetics, CD8-Positive T-Lymphocytes immunology, Epitopes, T-Lymphocyte immunology, Immunologic Memory, Lymphoma, B-Cell immunology, Murine hepatitis virus immunology, Neoplasm Proteins immunology
- Abstract
Persistent infection with gammaherpesviruses (γHV) can cause lymphomagenesis in immunocompromised patients. Murine γHV-68 (MHV-68) is an important tool for understanding immune factors contributing to γHV control; however, modeling control of γHV-associated lymphomagenesis has been challenging. Current model systems require very long incubation times or severe immune suppression, and tumor penetrance is low. In this report, we describe the generation of a B cell lymphoma on the C57BL/6 background, which is driven by the Myc oncogene and expresses an immunodominant CD8 T cell epitope from MHV-68. We determined MHV-68-specific CD8 T cells in latently infected mice use either IFN-γ or perforin/granzyme to control γHV-associated lymphoma, but perforin/granzyme is a more potent effector mechanism for lymphoma control than IFN-γ. Consistent with previous reports, CD4-depleted mice lost control of virus replication in persistently infected mice. However, control of lymphoma remained intact in the absence of CD4 T cells. Collectively, these data show the mechanisms of T cell control of B cell lymphoma in γHV-infected mice overlap with those necessary for control of virus replication, but there are also important differences. This study establishes a tool for further dissecting immune surveillance against, and optimizing adoptive T cell therapies for, γHV-associated lymphomas., (Copyright © 2020 by The American Association of Immunologists, Inc.)
- Published
- 2020
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11. A new brilliantly blue-emitting luciferin-luciferase system from Orfelia fultoni and Keroplatinae (Diptera).
- Author
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Viviani VR, Silva JR, Amaral DT, Bevilaqua VR, Abdalla FC, Branchini BR, and Johnson CH
- Subjects
- Animals, Chromatography, Ion Exchange, Diptera enzymology, Firefly Luciferin chemistry, Firefly Luciferin isolation & purification, Gene Expression Profiling, Luciferases chemistry, Luciferases isolation & purification, Luminescent Measurements, Mitochondria enzymology, Mitochondria metabolism, Spectrometry, Fluorescence, Diptera metabolism, Firefly Luciferin metabolism, Luciferases metabolism
- Abstract
Larvae of O. fultoni (Keroplatidae: Keroplatinae), which occur along river banks in the Appalachian Mountains in Eastern United States, produce the bluest bioluminescence among insects from translucent areas associated to black bodies, which are located mainly in the anterior and posterior parts of the body. Although closely related to Arachnocampa spp (Keroplatidae: Arachnocampininae), O.fultoni has a morphologically and biochemically distinct bioluminescent system which evolved independently, requiring a luciferase enzyme, a luciferin, a substrate binding fraction (SBF) that releases luciferin in the presence of mild reducing agents, molecular oxygen, and no additional cofactors. Similarly, the closely related Neoceroplatus spp, shares the same kind of luciferin-luciferase system of Orfelia fultoni. However, the molecular properties, identities and functions of luciferases, SBF and luciferin of Orfelia fultoni and other luminescent members of the Keroplatinae subfamily still remain to be fully elucidated. Using O. fultoni as a source of luciferase, and the recently discovered non-luminescent cave worm Neoditomiya sp as the main source of luciferin and SBF, we isolated and initially characterized these compounds. The luciferase of O. fultoni is a stable enzyme active as an apparent trimer (220 kDa) composed of ~70 kDa monomers, with an optimum pH of 7.8. The SBF, which is found in the black bodies in Orfelia fultoni and in smaller dark granules in Neoditomiya sp, consists of a high molecular weight complex of luciferin and proteins, apparently associated to mitochondria. The luciferin, partially purified from hot extracts by a combination of anion exchange chromatography and TLC, is a very polar and weakly fluorescent compound, whereas its oxidized product displays blue fluorescence with an emission spectrum matching the bioluminescence spectrum (~460 nm), indicating that it is oxyluciferin. The widespread occurrence of luciferin and SBF in both luminescent and non-luminescent Keroplatinae larvae indicate an additional important biological function for the substrate, and therefore the name keroplatin.
- Published
- 2020
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12. Mutagenesis and Structural Studies Reveal the Basis for the Activity and Stability Properties That Distinguish the Photinus Luciferases scintillans and pyralis .
- Author
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Branchini BR, Fontaine DM, Southworth TL, Huta BP, Racela A, Patel KD, and Gulick AM
- Subjects
- Amino Acids genetics, Animals, Catalytic Domain, Crystallization, Crystallography, X-Ray, Enzyme Stability drug effects, Enzyme Stability genetics, Escherichia coli genetics, Escherichia coli metabolism, Genetic Vectors, Guanidine pharmacology, Hot Temperature, Hydrogen-Ion Concentration, Ligands, Mutant Proteins chemistry, Protein Conformation, Spectrometry, X-Ray Emission, Fireflies enzymology, Luciferases, Firefly chemistry, Luciferases, Firefly genetics, Mutagenesis, Mutation
- Abstract
The dazzling yellow-green light emission of the common North American firefly Photinus pyralis and other bioluminescent organisms has provided a wide variety of prominent research applications like reporter gene assays and in vivo imaging methods. While the P. pyralis enzyme has been extensively studied, only recently has a second Photinus luciferase been cloned from the species scintillans . Even though the enzymes share very high sequence identity (89.8%), the color of the light they emit, their specific activity and their stability to heat, pH, and chemical denaturation are quite different with the scintillans luciferase being generally more resistant. Through the construction and evaluation of the properties of chimeric domain swapped, single point, and various combined variants, we have determined that only six amino acid changes are necessary to confer all of the properties of the scintillans enzyme to wild-type P. pyralis luciferase. Altered stability properties were attributed to four of the amino acid changes (T214N/S276T/H332N/E354N), and single mutations each predominantly changed emission color (Y255F) and specific activity (A222C). Results of a crystallographic study of the P. pyralis enzyme containing the six changes (Pps6) provide some insight into the structural basis for some of the documented property differences.
- Published
- 2019
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13. Near-infrared dual bioluminescence imaging in mouse models of cancer using infraluciferin.
- Author
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Stowe CL, Burley TA, Allan H, Vinci M, Kramer-Marek G, Ciobota DM, Parkinson GN, Southworth TL, Agliardi G, Hotblack A, Lythgoe MF, Branchini BR, Kalber TL, Anderson JC, and Pule MA
- Subjects
- Animals, Crystallography, X-Ray, Disease Models, Animal, Luminescent Proteins analysis, Luminescent Proteins chemistry, Luminescent Proteins genetics, Male, Mice, Neoplasm Transplantation, Protein Conformation, Recombinant Fusion Proteins analysis, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Luminescent Measurements methods, Neoplasms diagnostic imaging, Neoplasms pathology, Optical Imaging methods
- Abstract
Bioluminescence imaging (BLI) is ubiquitous in scientific research for the sensitive tracking of biological processes in small animal models. However, due to the attenuation of visible light by tissue, and the limited set of near-infrared bioluminescent enzymes, BLI is largely restricted to monitoring single processes in vivo. Here we show, that by combining stabilised colour mutants of firefly luciferase (FLuc) with the luciferin (LH
2 ) analogue infraluciferin (iLH2 ), near-infrared dual BLI can be achieved in vivo. The X-ray crystal structure of FLuc with a high-energy intermediate analogue, 5'-O-[N-(dehydroinfraluciferyl)sulfamoyl] adenosine (iDLSA) provides insight into the FLuc-iLH2 reaction leading to near-infrared light emission. The spectral characterisation and unmixing validation studies reported here established that iLH2 is superior to LH2 for the spectral unmixing of bioluminescent signals in vivo; which led to this novel near-infrared dual BLI system being applied to monitor both tumour burden and CAR T cell therapy within a systemically induced mouse tumour model., Competing Interests: CS, TB, HA, MV, GK, DC, GP, TS, GA, AH, ML, BB, TK, JA, MP No competing interests declared, (© 2019, Stowe et al.)- Published
- 2019
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14. Bioluminescence chemistry of fireworm Odontosyllis .
- Author
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Kotlobay AA, Dubinnyi MA, Purtov KV, Guglya EB, Rodionova NS, Petushkov VN, Bolt YV, Kublitski VS, Kaskova ZM, Ziganshin RH, Nelyubina YV, Dorovatovskii PV, Eliseev IE, Branchini BR, Bourenkov G, Ivanov IA, Oba Y, Yampolsky IV, and Tsarkova AS
- Subjects
- Animals, Biosynthetic Pathways, Color, Indoles chemistry, Indoles metabolism, Luminescent Agents metabolism, Luminescent Measurements, Luminescent Proteins metabolism, Molecular Structure, Oxidation-Reduction, Polychaeta metabolism, Pyrazines chemistry, Pyrazines metabolism, Luminescent Agents chemistry, Polychaeta chemistry
- Abstract
Marine polychaetes Odontosyllis undecimdonta , commonly known as fireworms, emit bright blue-green bioluminescence. Until the recent identification of the Odontosyllis luciferase enzyme, little progress had been made toward characterizing the key components of this bioluminescence system. Here we present the biomolecular mechanisms of enzymatic (leading to light emission) and nonenzymatic (dark) oxidation pathways of newly described O. undecimdonta luciferin. Spectral studies, including 1D and 2D NMR spectroscopy, mass spectrometry, and X-ray diffraction, of isolated substances allowed us to characterize the luciferin as an unusual tricyclic sulfur-containing heterocycle. Odontosyllis luciferin does not share structural similarity with any other known luciferins. The structures of the Odontosyllis bioluminescent system's low molecular weight components have enabled us to propose chemical transformation pathways for the enzymatic and nonspecific oxidation of luciferin., Competing Interests: Conflict of interest statement: I.V.Y. is shareholder of Planta LLC. Planta LLC filed patent applications related to the use of Odontosyllis luciferin., (Copyright © 2019 the Author(s). Published by PNAS.)
- Published
- 2019
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15. Prêt-à-porter nanoYESα and nanoYESβ bioluminescent cell biosensors for ultrarapid and sensitive screening of endocrine-disrupting chemicals.
- Author
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Lopreside A, Calabretta MM, Montali L, Ferri M, Tassoni A, Branchini BR, Southworth T, D'Elia M, Roda A, and Michelini E
- Subjects
- Genes, Reporter, Limit of Detection, Luciferases genetics, Medicago sativa chemistry, Plant Extracts chemistry, Reproducibility of Results, Saccharomyces cerevisiae genetics, Glycine max chemistry, Water Pollutants, Chemical analysis, Biosensing Techniques, Endocrine Disruptors analysis, Luminescent Measurements methods, Nanotechnology, Saccharomyces cerevisiae metabolism, Smartphone
- Abstract
Cell-based assays utilizing reporter gene technology have been widely exploited for biosensing, as they provide useful information about the bioavailability and cell toxicity of target analytes. The long assay time due to gene transcription and translation is one of the main drawbacks of cell biosensors. We report the development of two yeast biosensors stably expressing human estrogen receptors α and β and employing NanoLuc as the reporter protein to upgrade the widely used yeast estrogen screening (YES) assays. A viability control strain was also developed based on a chimeric green-emitting luciferase, PLG2, expressed for the first time in Saccharomycescerevisiae. Thanks to their brightness, NanoLuc and PLG2 provided excellent sensitivity, enabling the implementation of these biosensors into low-cost smartphone-based devices. The developed biosensors had a rapid (1 h) response and reported on (anti)estrogenic activity via human estrogen receptors α and β as well as general sample toxicity. Under optimized conditions, we obtained LODs of 7.1 ± 0.4 nM and 0.38 ± 0.08 nM for E2 with nanoYESα and nanoYESβ, respectively. As a proof of concept, we analyzed real samples from plants showing significant estrogenic activity or known to contain significant amounts of phytoestrogens. Graphical abstract.
- Published
- 2019
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16. Smartphone-based multicolor bioluminescent 3D spheroid biosensors for monitoring inflammatory activity.
- Author
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Michelini E, Calabretta MM, Cevenini L, Lopreside A, Southworth T, Fontaine DM, Simoni P, Branchini BR, and Roda A
- Subjects
- HEK293 Cells, Humans, Inflammation genetics, NF-kappa B genetics, Signal Transduction genetics, Smartphone, Spheroids, Cellular, Tumor Necrosis Factor-alpha genetics, Biosensing Techniques, Inflammation diagnosis, Luminescent Measurements, Tumor Necrosis Factor-alpha isolation & purification
- Abstract
Whole-cell biosensors present many advantages, including being able to monitor the toxicity and bioavailability of chemicals; cells grown in traditional 2D cultures, however, do not reproduce the complexity of in vivo physiology. In the last years, 3D cell-culture models have garnered great attention due to their capability to better mimic in vivo cellular responses to external stimuli, providing excellent model living organisms. In order to obtain a predictive, sensitive, and robust yet low-cost 3D cell biosensor, we developed a smartphone-based bioluminescent 3D cell biosensor platform for effect-based analysis. We exploited the Nuclear Factor-kappa B (NF-kB) signal transduction pathway, which is induced by several types of stressors and is involved in the regulation of cell-cycle/growth, inflammation, apoptosis, and immunity. The smartphone-based biosensor relies on immobilized HEK293 spheroids genetically engineered with powerful red- and green-emitting luciferases utilized as inflammation and viability reporters. It provides a limit of detection for Tumor Necrosis Factor (TNFα) of 0.15 ± 0.05 ng/mL and could be a useful tool to initially screen environmental samples or other compounds on-site, especially for additional more accurate chemical analyses., (Copyright © 2018 Elsevier B.V. All rights reserved.)
- Published
- 2019
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17. A Firefly Luciferase Dual Color Bioluminescence Reporter Assay Using Two Substrates To Simultaneously Monitor Two Gene Expression Events.
- Author
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Branchini BR, Southworth TL, Fontaine DM, Kohrt D, Florentine CM, and Grossel MJ
- Subjects
- HEK293 Cells, Humans, Luciferases, Firefly analysis, Luciferases, Firefly genetics, Luminescent Agents analysis, Promoter Regions, Genetic, Protein Engineering, Substrate Specificity, Transcriptional Activation, Transfection, Firefly Luciferin metabolism, Gene Expression, Genes, Reporter, Luciferases, Firefly metabolism, Luminescent Agents metabolism, Luminescent Measurements methods
- Abstract
Effective methods for monitoring eukaryotic gene expression and regulation based on bioluminescence - the emission of light by living organisms - are well established. Typically, the expression of a gene of interest is reported on with high sensitivity and over a wide dynamic range by the emission of light from a variety of engineered luciferase genes from beetles and marine organisms. The luciferase reporter genes are expressed downstream of the target gene or promoter and detected after exogenous addition of luciferin substrates. We describe a novel bioluminescence reporter method for the simultaneous monitoring of two genes expressing engineered firefly luciferase variants that emit readily distinguishable green and red light signals. The key feature is the selectivity of the enzymes for two luciferin substrates that determine each emission color. To validate our method, we performed a complex promoter transactivation experiment side-by-side with the Dual-Luciferase Reporter protocol and obtained essentially identical results. Additional comparative experiments demonstrated that our assay system provided improvements in background, cell normalization, and detectability compared to representative available methods. With access to a luminometer equipped with two optical filters, this method is an excellent choice for genetic reporter assays that can be performed with a single reagent solution.
- Published
- 2018
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18. Red-emitting chimeric firefly luciferase for in vivo imaging in low ATP cellular environments.
- Author
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Branchini BR, Southworth TL, Fontaine DM, Kohrt D, Welcome FS, Florentine CM, Henricks ER, DeBartolo DB, Michelini E, Cevenini L, Roda A, and Grossel MJ
- Subjects
- Animals, Fireflies, HEK293 Cells, HeLa Cells, Humans, Luciferases, Firefly metabolism, Adenosine Triphosphate analysis, Luciferases, Firefly chemistry, Luminescent Measurements
- Abstract
Beetle luciferases have been adapted for live cell imaging where bioluminescence is dependent on the cellular availability of ATP, O
2 , and added luciferin. Previous Photinus pyralis red-emitting variants with high Km values for ATP have performed disappointingly in live cells despite having much higher relative specific activities than enzymes like Click Beetle Red (CBR). We engineered a luciferase variant PLR3 having a Km value for ATP similar to CBR and ∼2.6-fold higher specific activity. The red-emitting PLR3 was ∼2.5-fold brighter than CBR in living HEK293T and HeLa cells, an improvement consistent with the importance of the Km value in low ATP environments., (Copyright © 2017 Elsevier Inc. All rights reserved.)- Published
- 2017
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19. Cloning of the Orange Light-Producing Luciferase from Photinus scintillans-A New Proposal on how Bioluminescence Color is Determined.
- Author
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Branchini BR, Southworth TL, Fontaine DM, Murtiashaw MH, McGurk A, Talukder MH, Qureshi R, Yetil D, Sundlov JA, and Gulick AM
- Subjects
- Amino Acid Substitution, Animals, Cloning, Molecular, Crystallography, X-Ray, Fireflies, Hydrogen Bonding, Luciferases, Firefly chemistry, Luciferases, Firefly genetics, Mutagenesis, Site-Directed, Protein Conformation, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Color, Luciferases, Firefly metabolism, Luminescence
- Abstract
Unlike the enchanting yellow-green flashes of light produced on warm summer evenings by Photinus pyralis, the most common firefly species in North America, the orange lights of Photinus scintillans are infrequently observed. These Photinus species, and likely all bioluminescent beetles, use the same substrates beetle luciferin, ATP and oxygen to produce light. It is the structure of the particular luciferase enzyme that is the key to determining the color of the emitted light. We report here the molecular cloning of the P. scintillans luc gene and the expression and characterization of the corresponding novel recombinant luciferase enzyme. A comparison of the amino acid sequence with that of the highly similar P. pyralis enzyme and subsequent mutagenesis studies revealed that the single conservative amino acid change tyrosine to phenylalanine at position 255 accounted for the entire emission color difference. Additional mutagenesis and crystallographic studies were performed on a H-bond network, which includes the position 255 residue and five other stringently conserved beetle luciferase residues, that is proximal to the substrate/emitter binding site. The results are interpreted in the context of a speculative proposal that this network is key to the understanding of bioluminescence color determination., (© 2016 The American Society of Photobiology.)
- Published
- 2017
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20. Bioluminescence Imaging of Spheroids for High-throughput Longitudinal Studies on 3D Cell Culture Models.
- Author
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Cevenini L, Calabretta MM, Lopreside A, Branchini BR, Southworth TL, Michelini E, and Roda A
- Subjects
- Cell Culture Techniques, Extracellular Matrix metabolism, HEK293 Cells, Humans, Inflammation metabolism, NF-kappa B metabolism, Tumor Cells, Cultured, Luminescence, Models, Biological, Spheroids, Cellular
- Abstract
Bioluminescent (BL) cell-based assays based on two-dimensional (2D) monolayer cell cultures represent well-established bioanalytical tools for preclinical screening of drugs. However, cells in 2D cultures do not often reflect the morphology and functionality of living organisms, thus limiting the predictive value of 2D cell-based assays. Conversely, 3D cell models have the capability to generate the extracellular matrix and restore cell-to-cell communications; thus, they are the most suitable model to mimic in vivo physiology. In this work, we developed a nondestructive real-time BL imaging assay of spheroids for longitudinal studies on 3D cell models. A high-throughput BL 3D cell-based assay in micropatterned 96-well plate format is reported. The assay performance was assessed using the transcriptional regulation of nuclear factor K beta response element in human embryonic kidney (HEK293) cells. We compared concentration-response curves for tumor necrosis factor-α with those obtained using conventional 2D cell cultures. One of the main advantages of this approach is the nonlysing nature of the assay, which allows for repetitive measurements on the same sample. The assay can be implemented in any laboratory equipped with basic cell culture facilities and paves the way to the development of new 3D bioluminescent cell-based assays., (© 2017 The American Society of Photobiology.)
- Published
- 2017
- Full Text
- View/download PDF
21. Cloning of the Blue Ghost (Phausis reticulata) Luciferase Reveals a Glowing Source of Green Light.
- Author
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Branchini BR, Southworth TL, Salituro LJ, Fontaine DM, and Oba Y
- Subjects
- Amino Acid Sequence, Animals, Appalachian Region, Base Pairing, Cloning, Molecular, Escherichia coli genetics, Fireflies classification, Luciferases, Firefly chemistry, Luciferases, Firefly metabolism, Male, Open Reading Frames, Phylogeny, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Reverse Transcriptase Polymerase Chain Reaction, Luciferases, Firefly genetics, Luminescence
- Abstract
In the southern Appalachian area of the United States, the Phausis reticulata firefly, commonly known as the "Blue Ghost," performs a unique display of bioluminescence. Adult male organisms are observed darting rapidly along paths and riverbeds in dark forests producing long-lasting and mesmerizing bluish-white luminous streaks. Starting with eighteen adult male firefly lanterns, we used a reverse transcriptase and rapid amplification of cDNA ends (RACE) approach to clone the 1635 base pair open reading frame of the P. reticulata luc gene corresponding to a 545 residue protein. Expression of the recombinant luciferase protein in Escherichia coli and characterization studies revealed the true color of the light emission to be green (λ
max = 552 nm), strongly suggesting that the field observations result from a Purkinje shift. While the P. reticulata luciferase amino acid sequence is 74.3% identical to the North American Photinus pyralis luciferase, we were surprised to find that it was 88.4% and 87.7% identical to luciferases from C. ruficollis and D. axillaris both native to mainland Japan. Phylogenetic analysis confirmed the close relationship of the three enzymes that is surprising given the great distance between their natural habitats and the inability of the Japanese fireflies to produce bright bioluminescence., (© 2016 The American Society of Photobiology.)- Published
- 2017
- Full Text
- View/download PDF
22. A Highly Sensitive Biosensor for ATP Using a Chimeric Firefly Luciferase.
- Author
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Branchini BR and Southworth TL
- Subjects
- Adenosine Triphosphate metabolism, Animals, Fireflies enzymology, Fireflies genetics, HEK293 Cells, Humans, Luciferases, Firefly genetics, Luciferases, Firefly metabolism, Luminescent Agents metabolism, Protein Engineering methods, Adenosine Triphosphate analysis, Biosensing Techniques methods, Luciferases, Firefly analysis, Luminescent Agents analysis, Luminescent Measurements methods
- Abstract
Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications based on the detection of the enzymes or using the proteins to measure ATP levels. Based on studies of chimeric luciferases, we engineered a novel luciferase called PLG2 that has enhanced specific activity, and thermal and pH stability compared to the commonly used Photinus pyralis luciferase. We present here protocols for preparing a single assay mixture containing PLG2 that can be used to readily detect femtomole levels of ATP. Our methodology can be used with a variety of samples, including human and bacterial cells, where measurements of ATP can be used as a biosensor for the detection of viable cells., (© 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
23. Probing Bioluminescence Resonance Energy Transfer in Quantum Rod-Luciferase Nanoconjugates.
- Author
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Alam R, Karam LM, Doane TL, Coopersmith K, Fontaine DM, Branchini BR, and Maye MM
- Subjects
- Luciferases metabolism, Nanoconjugates ultrastructure, Quantum Dots ultrastructure, Bioluminescence Resonance Energy Transfer Techniques methods, Luciferases chemistry, Nanoconjugates chemistry, Quantum Dots chemistry
- Abstract
We describe the necessary design criteria to create highly efficient energy transfer conjugates containing luciferase enzymes derived from Photinus pyralis (Ppy) and semiconductor quantum rods (QRs) with rod-in-rod (r/r) microstructure. By fine-tuning the synthetic conditions, CdSe/CdS r/r-QRs were prepared with two different emission colors and three different aspect ratios (l/w) each. These were hybridized with blue, green, and red emitting Ppy, leading to a number of new BRET nanoconjugates. Measurements of the emission BRET ratio (BR) indicate that the resulting energy transfer is highly dependent on QR energy accepting properties, which include absorption, quantum yield, and optical anisotropy, as well as its morphological and topological properties, such as aspect ratio and defect concentration. The highest BR was found using r/r-QRs with lower l/w that were conjugated with red Ppy, which may be activating one of the anisotropic CdSe core energy levels. The role QR surface defects play on Ppy binding, and energy transfer was studied by growth of gold nanoparticles at the defects, which indicated that each QR set has different sites. The Ppy binding at those sites is suggested by the observed BRET red-shift as a function of Ppy-to-QR loading (L), where the lowest L results in highest efficiency and furthest shift.
- Published
- 2016
- Full Text
- View/download PDF
24. Detection of nitric oxide production in cell cultures by luciferin-luciferase chemiluminescence.
- Author
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Woldman YY, Eubank TD, Mock AJ, Stevens NC, Varadharaj S, Turco J, Gavrilin MA, Branchini BR, and Khramtsov VV
- Subjects
- Animals, Aorta cytology, Aorta drug effects, Aorta metabolism, Bradykinin pharmacology, Cattle, Cell Line, Cyclic GMP metabolism, Diphosphates chemistry, Diphosphates metabolism, Endothelial Cells cytology, Endothelial Cells drug effects, Firefly Luciferin metabolism, Guanosine Triphosphate metabolism, Guanylate Cyclase metabolism, Lipopolysaccharides pharmacology, Luciferases metabolism, Luminescence, Macrophages cytology, Macrophages drug effects, Mice, Nitric Oxide biosynthesis, Nitric Oxide Donors metabolism, Nitric Oxide Donors pharmacology, Nitrites chemistry, Nitrites metabolism, Nitroso Compounds metabolism, Nitroso Compounds pharmacology, Receptors, Cytoplasmic and Nuclear metabolism, Sensitivity and Specificity, Soluble Guanylyl Cyclase, Sulfate Adenylyltransferase metabolism, Biological Assay, Endothelial Cells metabolism, Firefly Luciferin chemistry, Luciferases chemistry, Luminescent Measurements standards, Macrophages metabolism, Nitric Oxide analysis
- Abstract
A chemiluminescent method is proposed for quantitation of NO generation in cell cultures. The method is based on activation of soluble guanylyl cyclase by NO. The product of the guanylyl cyclase reaction, pyrophosphate, is converted to ATP by ATP sulfurylase and ATP is detected in a luciferin-luciferase system. The method has been applied to the measurement of NO generated by activated murine macrophages (RAW 264.7) and bovine aortic endothelial cells. For macrophages activated by lipopolysaccharide and γ-interferon, the rate of NO production is about 100 amol/(cell·min). The rate was confirmed by the measurements of nitrite, the product of NO oxidation. For endothelial cells, the basal rate of NO generation is 5 amol/(cell·min); the rate approximately doubles upon activation by bradykinin, Ca(2+) ionophore A23187 or mechanical stress. For both types of cells the measured rate of NO generation is strongly affected by inhibitors of NO synthase. The sensitivity of the method is about 50 pM/min, allowing the registration of NO generated by 10(2)-10(4) cells. The enzyme-linked chemiluminescent method is two orders of magnitude more sensitive than fluorescent detection using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM)., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
25. An enhanced chimeric firefly luciferase-inspired enzyme for ATP detection and bioluminescence reporter and imaging applications.
- Author
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Branchini BR, Southworth TL, Fontaine DM, Kohrt D, Talukder M, Michelini E, Cevenini L, Roda A, and Grossel MJ
- Subjects
- Animals, Enzyme Stability, Fireflies enzymology, HEK293 Cells, Humans, Hydrogen-Ion Concentration, Luminescent Measurements, Protein Engineering, Temperature, Adenosine Triphosphate metabolism, Genes, Reporter genetics, Luciferases, Firefly genetics, Molecular Imaging methods, Recombinant Fusion Proteins genetics
- Abstract
Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. We previously reported the approximately 2-fold enhanced activity and 1.4-fold greater bioluminescence quantum yield properties of a chimeric enzyme that contains the N-domain of Photinus pyralis luciferase joined to the C-domain of Luciola italica luciferase. Subsequently, we identified 5 amino acid changes based on L. italica that are the main determinants of the improved bioluminescence properties. Further engineering to enhance thermal and pH stability produced a novel luciferase called PLG2. We present here a systematic comparison of the spectral and physical properties of the new protein with P. pyralis luciferase and demonstrate the potential of PLG2 for use in assays based on the detection of femtomole levels of ATP. In addition, we compared the performance of a mammalian codon-optimized version of the cDNA for PLG2 with the luc2 gene in HEK293T cells. Using an optimized low-cost assay system, PLG2 activity can be monitored in mammalian cell lysates and living cells with 4.4-fold and approximately 3.0-fold greater sensitivity, respectively. PLG2 could be an improved alternative to Promega's luc2 for reporter and imaging applications., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
26. Experimental Support for a Single Electron-Transfer Oxidation Mechanism in Firefly Bioluminescence.
- Author
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Branchini BR, Behney CE, Southworth TL, Fontaine DM, Gulick AM, Vinyard DJ, and Brudvig GW
- Subjects
- Animals, Electron Transport, Electrons, Fireflies chemistry, Fireflies metabolism, Firefly Luciferin chemistry, Firefly Luciferin metabolism, Luciferases, Firefly chemistry, Luminescence, Models, Molecular, Oxidation-Reduction, Superoxides chemistry, Superoxides metabolism, Fireflies enzymology, Luciferases, Firefly metabolism
- Abstract
Firefly luciferase produces light by converting substrate beetle luciferin into the corresponding adenylate that it subsequently oxidizes to oxyluciferin, the emitter of bioluminescence. We have confirmed the generally held notions that the oxidation step is initiated by formation of a carbanion intermediate and that a hydroperoxide (anion) is involved. Additionally, structural evidence is presented that accounts for the delivery of oxygen to the substrate reaction site. Herein, we report key convincing spectroscopic evidence of the participation of superoxide anion in a related chemical model reaction that supports a single electron-transfer pathway for the critical oxidative process. This mechanism may be a common feature of bioluminescence processes in which light is produced by an enzyme in the absence of cofactors.
- Published
- 2015
- Full Text
- View/download PDF
27. Near infrared bioluminescence resonance energy transfer from firefly luciferase--quantum dot bionanoconjugates.
- Author
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Alam R, Karam LM, Doane TL, Zylstra J, Fontaine DM, Branchini BR, and Maye MM
- Subjects
- Animals, Diagnostic Imaging methods, Fireflies, Luminescent Measurements, Quantum Dots ultrastructure, Fluorescence Resonance Energy Transfer methods, Luciferases, Firefly chemistry, Luminescence, Quantum Dots chemistry
- Abstract
The bioluminescence resonance energy transfer (BRET) between firefly luciferase enzymes and semiconductive quantum dots (QDs) with near infrared emission is described. The QD were phase transferred to aqueous buffers using a histidine mediated phase transfer route, and incubated with a hexahistidine tagged, green emitting variant of firefly luciferase from Photinus pyralis (PPyGRTS). The PPyGRTS were bound to the QD interface via the hexahistidine tag, which effectively displaces the histidine layer and binds directly to the QD interfaces, allowing for short donor-acceptor distances (∼5.5 nm). Due to this, high BRET efficiency ratios of ∼5 were obtained. These PPyGRTS-QD bio-nano conjugates were characterized by transmission electron microscopy, thermal gravimetric analysis, Fourier transform infrared spectroscopy and BRET emission studies. The final optimized conjugate was easily observable by night vision imaging, demonstrating the potential of these materials in imaging and signaling/sensing applications.
- Published
- 2014
- Full Text
- View/download PDF
28. A Photinus pyralis and Luciola italica chimeric firefly luciferase produces enhanced bioluminescence.
- Author
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Branchini BR, Southworth TL, Fontaine DM, Davis AL, Behney CE, and Murtiashaw MH
- Subjects
- Adenosine Triphosphate chemistry, Animals, Enzyme Assays, Firefly Luciferin chemistry, Luciferases, Firefly biosynthesis, Luciferases, Firefly genetics, Luminescent Measurements, Protein Engineering, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins genetics, Fireflies enzymology, Luciferases, Firefly chemistry, Recombinant Fusion Proteins chemistry
- Abstract
We report the enhanced bioluminescence properties of a chimeric enzyme (PpyLit) that contains the N-domain of recombinant Photinus pyralis luciferase joined to the C-domain of recombinant Luciola italica luciferase. Compared to the P. pyralis enzyme, the novel PpyLit chimera exhibited 1.8-fold enhanced flash-height specific activity, 2.0-fold enhanced integration-based specific activity, 2.9-fold enhanced catalytic efficiency (kcat/Km), and a 1.4-fold greater bioluminescence quantum yield. The results of this study provide an underlying basis of this unusual example of a chimeric enzyme with enhanced catalytic properties that are not simply the sum of the contributions of the two luciferases.
- Published
- 2014
- Full Text
- View/download PDF
29. Multicolor bioluminescence boosts malaria research: quantitative dual-color assay and single-cell imaging in Plasmodium falciparum parasites.
- Author
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Cevenini L, Camarda G, Michelini E, Siciliano G, Calabretta MM, Bona R, Kumar TR, Cara A, Branchini BR, Fidock DA, Roda A, and Alano P
- Subjects
- Antimalarials pharmacology, Cell Line, Fluorescent Antibody Technique, Humans, Luciferases genetics, Luciferases metabolism, Plasmids genetics, Plasmids metabolism, Plasmodium falciparum drug effects, Plasmodium falciparum metabolism, Promoter Regions, Genetic, Protozoan Proteins genetics, Single-Cell Analysis, Luminescent Measurements, Microscopy, Video, Parasitology methods, Plasmodium falciparum isolation & purification
- Abstract
New reliable and cost-effective antimalarial drug screening assays are urgently needed to identify drugs acting on different stages of the parasite Plasmodium falciparum, and particularly those responsible for human-to-mosquito transmission, that is, the P. falciparum gametocytes. Low Z' factors, narrow dynamic ranges, and/or extended assay times are commonly reported in current gametocyte assays measuring gametocyte-expressed fluorescent or luciferase reporters, endogenous ATP levels, activity of gametocyte enzymes, or redox-dependent dye fluorescence. We hereby report on a dual-luciferase gametocyte assay with immature and mature P. falciparum gametocyte stages expressing red and green-emitting luciferases from Pyrophorus plagiophthalamus under the control of the parasite sexual stage-specific pfs16 gene promoter. The assay was validated with reference antimalarial drugs and allowed to quantitatively and simultaneously measure stage-specific drug effects on parasites at different developmental stages. The optimized assay, requiring only 48 h incubation with drugs and using a cost-effective luminogenic substrate, significantly reduces assay cost and time in comparison to state-of-the-art analogous assays. The assay had a Z' factor of 0.71 ± 0.03, and it is suitable for implementation in 96- and 384-well microplate formats. Moreover, the use of a nonlysing D-luciferin substrate significantly improved the reliability of the assay and allowed one to perform, for the first time, P. falciparum bioluminescence imaging at single-cell level.
- Published
- 2014
- Full Text
- View/download PDF
30. Chemical Analysis of the Luminous Slime Secreted by the Marine Worm Chaetopterus (Annelida, Polychaeta).
- Author
-
Branchini BR, Behney CE, Southworth TL, Rawat R, and Deheyn DD
- Subjects
- Animals, Luminescence, Spectrometry, Mass, Electrospray Ionization, Mucus chemistry, Polychaeta chemistry, Polychaeta metabolism, Riboflavin chemistry
- Abstract
The marine annelid Chaetopterus variopedatus produces bioluminescence by an unknown and potentially novel mechanism. We have advanced the study of this fascinating phenomenon, which has not been investigated for nearly 60 years after initial studies were first reported for this species. Here, we show that the luminous slime produced by the worm exhibits blue fluorescence that matches the bioluminescence emission. This result suggests that the oxyluciferin emitter is present. However, while the blue fluorescence decays over time green fluorescence is increasingly revealed that is likely associated with products of the luminescence reaction. LC/MS and fluorescence analysis of harvested luminescent material revealed riboflavin as the major green fluorescent component. Riboflavin is usually associated with the mechanism of light production in bacteria, yet luminous bacteria were not found in the worm mucus, and accordingly were not reported to be directly responsible for the light emission, which is under nervous control in the worm. We therefore propose a hypothesis in which riboflavin or a structurally related derivative serves as the emitter in the worm's light producing reaction., (© 2013 The American Society of Photobiology.)
- Published
- 2014
- Full Text
- View/download PDF
31. Novel multistep BRET-FRET energy transfer using nanoconjugates of firefly proteins, quantum dots, and red fluorescent proteins.
- Author
-
Alam R, Zylstra J, Fontaine DM, Branchini BR, and Maye MM
- Subjects
- Animals, Fireflies enzymology, Fluorescence Resonance Energy Transfer, Red Fluorescent Protein, Luciferases, Firefly chemistry, Luminescent Proteins chemistry, Quantum Dots
- Abstract
Sequential bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) from firefly luciferase to red fluorescent proteins using quantum dot or rod acceptor/donor linkers is described. The effect of morphology and tuned optical properties on the efficiency of this unique BRET-FRET system was evaluated.
- Published
- 2013
- Full Text
- View/download PDF
32. Novel heterocyclic analogues of firefly luciferin.
- Author
-
Woodroofe CC, Meisenheimer PL, Klaubert DH, Kovic Y, Rosenberg JC, Behney CE, Southworth TL, and Branchini BR
- Subjects
- Luminescence, Spectrophotometry, Ultraviolet, Firefly Luciferin chemistry, Heterocyclic Compounds chemistry
- Abstract
Five novel firefly luciferin analogues in which the benzothiazole ring system of the natural substrate was replaced with benzimidazole, benzofuran, benzothiophene, benzoxazole, and indole were synthesized. The fluorescence, bioluminescence, and kinetic properties of the compounds were evaluated with recombinant Photinus pyralis wild type luciferase. With the exception of indole, all of the substrates containing heterocycle substitutions produced readily measurable flashes of light with luciferase. Compared to that of luciferin, the intensities ranged from 0.3 to 4.4% in reactions with varying pH optima and times to reach maximal intensity. The heteroatom changes influenced both the fluorescence and bioluminescence emission spectra, which displayed maxima of 479-528 and 518-574 nm, respectively. While there were some interesting trends in the spectroscopic and bioluminescence properties of this group of structurally similar substrate analogues, the most significant findings were associated with the benzothiophene-containing compound. This synthetic substrate produced slow decay glow kinetics that increased the total light-based specific activity of luciferase more than 4-fold versus the luciferin value. Moreover, over the pH range of 6.2-9.4, the emission maximum is 523 nm, an unusual 37 nm blue shift compared to that of the natural substrate. The extraordinary bioluminescence properties of the benzothiophene luciferin should translate into greater sensitivity for analyte detection in a wide variety of luciferase-based applications.
- Published
- 2012
- Full Text
- View/download PDF
33. Crystal structure of firefly luciferase in a second catalytic conformation supports a domain alternation mechanism.
- Author
-
Sundlov JA, Fontaine DM, Southworth TL, Branchini BR, and Gulick AM
- Subjects
- Adenosine Monophosphate metabolism, Binding Sites, Catalysis, Coenzyme A Ligases chemistry, Coenzyme A Ligases metabolism, Crystallography, X-Ray, Luciferases, Firefly metabolism, Models, Molecular, Protein Conformation, Protein Structure, Tertiary, Luciferases, Firefly chemistry
- Abstract
Beetle luciferases catalyze a two-step reaction that includes the initial adenylation of the luciferin substrate, followed by an oxidative decarboxylation that ultimately produces light. Evidence for homologous acyl-CoA synthetases supports a domain alternation catalytic mechanism in which these enzymes' C-terminal domain rotates by ~140° to adopt two conformations that are used to catalyze the two partial reactions. While many structures exist of acyl-CoA synthetases in both conformations, to date only biochemical evidence supports domain alternation with luciferase. We have determined the structure of a cross-linked luciferase enzyme that is trapped in the second conformation. This new structure supports the role of the second catalytic conformation and provides insights into the biochemical mechanism of the luciferase oxidative step.
- Published
- 2012
- Full Text
- View/download PDF
34. Designing quantum rods for optimized energy transfer with firefly luciferase enzymes.
- Author
-
Alam R, Fontaine DM, Branchini BR, and Maye MM
- Subjects
- Energy Transfer, Fluorescence Resonance Energy Transfer methods, Luciferases, Firefly chemistry, Quantum Dots
- Abstract
The bioluminescence resonance energy transfer (BRET) between firefly luciferase from Photinus pyralis (Ppy) with core/shell semiconductive quantum rods (QRs) has been studied as a function of QR aspect ratio and internal microstructure. The QRs were found to be ideal energy acceptors, and Ppy-to-core distances were optimized using rod-in-rod microstructures that were achieved by the synthetic control of rod morphology, surface chemistry, and Ppy:QR loading. The BRET ratios of >44 measured are the highest efficiencies to date.
- Published
- 2012
- Full Text
- View/download PDF
35. Bioluminescence is produced from a trapped firefly luciferase conformation predicted by the domain alternation mechanism.
- Author
-
Branchini BR, Rosenberg JC, Fontaine DM, Southworth TL, Behney CE, and Uzasci L
- Subjects
- Amino Acid Sequence, Animals, Fireflies genetics, Fireflies metabolism, Firefly Luciferin metabolism, Luciferases, Firefly genetics, Luminescence, Molecular Sequence Data, Mutation, Protein Conformation, Protein Structure, Tertiary, Fireflies enzymology, Luciferases, Firefly chemistry, Luciferases, Firefly metabolism
- Abstract
According to the domain alternation mechanism and crystal structure evidence, the acyl-CoA synthetases, one of three subgroups of a superfamily of adenylating enzymes, catalyze adenylate- and thioester-forming half-reactions in two different conformations. The enzymes accomplish this by presenting two active sites through an ~140° rotation of the C-domain. The second half-reaction catalyzed by another subgroup, the beetle luciferases, is a mechanistically dissimilar oxidative process that produces bioluminescence. We have demonstrated that a firefly luciferase variant containing cysteine residues at positions 108 and 447 can be intramolecularly cross-linked by 1,2-bis(maleimido)ethane, trapping the enzyme in a C-domain-rotated conformation previously undocumented in the available luciferase crystal structures. The cross-linked luciferase cannot adenylate luciferin but is nearly fully capable of bioluminescence with synthetic luciferyl adenylate because it retains the ability to carry out the oxidative half-reaction. The cross-linked luciferase is apparently trapped in a conformation similar to those adopted by acyl-CoA synthetases as they convert acyl adenylates into the corresponding CoA thioesters.
- Published
- 2011
- Full Text
- View/download PDF
36. Sequential bioluminescence resonance energy transfer-fluorescence resonance energy transfer-based ratiometric protease assays with fusion proteins of firefly luciferase and red fluorescent protein.
- Author
-
Branchini BR, Rosenberg JC, Ablamsky DM, Taylor KP, Southworth TL, and Linder SJ
- Subjects
- Caspase 3 metabolism, Factor Xa metabolism, Luciferases, Firefly genetics, Luminescent Proteins genetics, Recombinant Fusion Proteins analysis, Spectroscopy, Near-Infrared, Substrate Specificity, Thrombin metabolism, Red Fluorescent Protein, Enzyme Assays methods, Fluorescence Resonance Energy Transfer methods, Fluorescent Dyes analysis, Luciferases, Firefly analysis, Luminescent Proteins analysis, Peptide Hydrolases metabolism
- Abstract
We report here the preparation of ratiometric luminescent probes that contain two well-separated emission peaks produced by a sequential bioluminescence resonance energy transfer (BRET)-fluorescence resonance energy transfer (FRET) process. The probes are single soluble fusion proteins consisting of a thermostable firefly luciferase variant that catalyze yellow-green (560nm maximum) bioluminescence and a red fluorescent protein covalently labeled with a near-infrared fluorescent dye. The two proteins are connected by a decapeptide containing a protease recognition site specific for factor Xa, thrombin, or caspase 3. The rates of protease cleavage of the fusion protein substrates were monitored by recording emission spectra and plotting the change in peak ratios over time. Detection limits of 0.41nM for caspase 3, 1.0nM for thrombin, and 58nM for factor Xa were realized with a scanning fluorometer. Our results demonstrate for the first time that an efficient sequential BRET-FRET energy transfer process based on firefly luciferase bioluminescence can be employed to assay physiologically important protease activities., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
37. A portable bioluminescence engineered cell-based biosensor for on-site applications.
- Author
-
Roda A, Cevenini L, Michelini E, and Branchini BR
- Subjects
- Androgens analysis, Estrogens analysis, Biosensing Techniques instrumentation, Genetic Engineering, Luminescent Measurements instrumentation
- Abstract
We have developed a portable biosensing device based on genetically engineered bioluminescent (BL) cells. Cells were immobilized on a 4 × 3 multiwell cartridge using a new biocompatible matrix that preserved their vitality. Using a fiber optic taper, the cartridge was placed in direct contact with a cooled CCD sensor to image and quantify the BL signals. Yeast and bacterial cells were engineered to express recognition elements, whose interaction with the analyte led to luciferase expression, via reporter gene technology. Three different biosensors were developed. The first detects androgenic compounds using yeast cells carrying a green-emitting P. pyralis luciferase regulated by the human androgen receptor and a red mutant of the same species as internal vitality control. The second biosensor detects two classes of compounds (androgens and estrogens) using yeast strains engineered to express green-or red-emitting mutant firefly luciferases in response to androgens or estrogens, respectively. The third biosensor detects lactose analogue isopropyl β-d-1-thiogalactopyranoside using two E. coli strains. One strain exploits the lac operon as recognition element for the expression of P. pyralis luciferase. The other strain serves as a vitality control expressing Gaussia princeps luciferase, which requires a different luciferin substrate. The immobilized cells were stable for up to 1 month. The analytes could be detected at nanomolar levels with good precision and accuracy when the specific signal was corrected using the internal vitality control. This portable device can be used for on-site multiplexed bioassays for different compound classes., (Copyright © 2011 Elsevier B.V. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
38. Chemically modified firefly luciferase is an efficient source of near-infrared light.
- Author
-
Branchini BR, Ablamsky DM, and Rosenberg JC
- Subjects
- Fluorescence Resonance Energy Transfer, Fluorescent Dyes chemistry, Luciferases, Firefly metabolism, Maleimides chemistry, Models, Molecular, Infrared Rays, Luciferases, Firefly chemistry, Luminescence
- Abstract
Bioluminescence and bioluminescence resonance energy transfer (BRET) are two naturally occurring light emission phenomena that have been adapted to a wide variety of important research applications including in vivo imaging and enzyme assays. The luciferase enzyme from the North American firefly, which produces yellow-green light, is a key component of many of these applications. Recognizing the heightened interest in the potential of near-infrared (nIR) light to improve these technologies, we have demonstrated that spectral emissions with maxima of 705 and 783 nm can be efficiently produced by a firefly luciferase variant covalently labeled with nIR fluorescent dyes. In one case, an outstanding BRET ratio of 34.0 was achieved emphasizing the importance of selective labeling with fluorescent dyes and the efficiency provided by the intramolecular BRET process. Additionally, we constructed a biotinylated fusion protein that similarly produced nIR light. This novel material was immobilized on solid supports containing streptavidin, demonstrating, in principle, that it may be used for receptor-based imaging. Also, the matrix-bound labeled fusion protein was used to measure factor Xa activity at physiological concentrations in blood. We believe this to be the first report of bright nIR light sources produced by chemical modification of a beetle luciferase.
- Published
- 2010
- Full Text
- View/download PDF
39. Red-emitting luciferases for bioluminescence reporter and imaging applications.
- Author
-
Branchini BR, Ablamsky DM, Davis AL, Southworth TL, Butler B, Fan F, Jathoul AP, and Pule MA
- Subjects
- Animals, Cell Line, Humans, Kinetics, Luciferases, Firefly genetics, Mutagenesis, Site-Directed, Recombinant Proteins genetics, Recombinant Proteins metabolism, Temperature, Genes, Reporter, Luciferases, Firefly metabolism, Luminescent Agents metabolism, Luminescent Measurements methods
- Abstract
North American firefly Photinus pyralis luciferase, which emits yellow-green light (557nm), has been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. Luciferase variants with red-shifted bioluminescence and high specific activity can be paired with green-emitting counterparts for use in dual-color reporter assays or can be used alone for in vivo imaging. Beginning with a previously reported red-emitting thermostable mutant and using mutagenesis techniques, we engineered two luciferases with redder emission maxima while maintaining satisfactory specific activities and thermostability. The novel enzymes were expressed in HEK293 cells, where they performed similarly to Promega's codon-optimized click beetle red luciferase in model reporter assays. When the firefly luciferase variants were codon-optimized and retested using optimized substrate concentrations, they provided 50- to 100-fold greater integrated light intensities than the click beetle enzyme. These results suggest that the novel enzymes should provide superior performance in dual-color reporter and in vivo imaging applications, and they illustrate the importance of codon optimization for assays in mammalian cells.
- Published
- 2010
- Full Text
- View/download PDF
40. In vivo bioluminescence imaging for the study of intestinal colonization by Escherichia coli in mice.
- Author
-
Foucault ML, Thomas L, Goussard S, Branchini BR, and Grillot-Courvalin C
- Subjects
- Animals, Colony Count, Microbial, Escherichia coli genetics, Luciferases genetics, Luciferases, Bacterial genetics, Mice, Mice, Inbred BALB C, Plasmids, Recombinant Proteins genetics, Recombinant Proteins metabolism, Staining and Labeling methods, Escherichia coli growth & development, Escherichia coli Infections microbiology, Gastrointestinal Tract microbiology, Luciferases metabolism, Luciferases, Bacterial metabolism, Luminescence, Whole Body Imaging methods
- Abstract
Bioluminescence imaging (BLI) is emerging as a powerful tool for real-time monitoring of infections in living animals. However, since luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. Strains of Escherichia coli harboring the genes for either the bacterial luciferase from Photorhabdus luminescens or the PpyRE-TS and PpyGR-TS firefly luciferase mutants of Photinus pyralis (red and green thermostable P. pyralis luciferase mutants, respectively) have been engineered and used to monitor intestinal colonization in the streptomycin-treated mouse model. There was excellent correlation between the bioluminescence signal measured in the feces (R2=0.98) or transcutaneously in the abdominal region of whole animals (R2=0.99) and the CFU counts in the feces of bacteria harboring the luxABCDE operon. Stability in vivo of the bioluminescence signal was achieved by constructing plasmid pAT881(pGB2OmegaPamiluxABCDE), which allowed long-term monitoring of intestinal colonization without the need for antibiotic selection for plasmid maintenance. Levels of intestinal colonization by various strains of E. coli could be compared directly by simple recording of the bioluminescence signal in living animals. The difference in spectra of light emission of the PpyRE-TS and PpyGR-TS firefly luciferase mutants and dual bioluminescence detection allowed direct in vitro and in vivo quantification of two bacterial populations by measurement of red and green emitted signals and thus monitoring of the two populations simultaneously. This system offers a simple and direct method to study in vitro and in vivo competition between mutants and the parental strain. BLI is a useful tool to study intestinal colonization.
- Published
- 2010
- Full Text
- View/download PDF
41. The effects of elevated osmotic concentration on control of germination in the gemmules of freshwater sponges Eunapius fragilis and Anheteromeyania ryderi.
- Author
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Loomis SH, Bettridge A, and Branchini BR
- Subjects
- Animals, Cell Division physiology, Fresh Water, Inositol metabolism, Osmotic Pressure, Oxygen Consumption physiology, Porifera metabolism, Sorbitol metabolism, Porifera physiology
- Abstract
Freshwater sponges produce gemmules during the fall as an adaptation to survive cold winters. Most gemmules are produced in a state of diapause and must undergo a vernalization period before diapause is broken and they enter the quiescence state. Quiescent gemmules will germinate if placed at room temperature. We examined the mechanism of germination in two species of freshwater sponges, Eunapius fragilis and Anheteromeyania ryderi. Germination, cell division, and oxygen consumption are all inhibited when the osmotic concentration of the gemmules of either species is maintained at or above 50 mOsm by placing them in a solution of impermeable osmolytes. The internal osmotic concentration of cells of quiescent gemmules is maintained above 100 mOsm by the presence of sorbitol (in E. fragilis) and myoinositol (in A. ryderi). During the early stages of germination, levels of sorbitol and myoinositol decline to less than 50 mM by 20 h after the initiation of germination. The onset of cell division and beginning of germination correlate with the drop in osmolyte levels below 50 mOsm. Thus, an early trigger initiating germination is most likely the catabolism of sorbitol or myoinositol leading to a drop in the osmotic concentration of the cells.
- Published
- 2009
- Full Text
- View/download PDF
42. Combining intracellular and secreted bioluminescent reporter proteins for multicolor cell-based assays.
- Author
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Michelini E, Cevenini L, Mezzanotte L, Ablamsky D, Southworth T, Branchini BR, and Roda A
- Subjects
- Cell Line, Humans, Luminescence, Color, Luciferases chemistry
- Abstract
Bioluminescent (BL) proteins are a promising tool for diverse applications based on reporter gene technology thanks to their high sensitivity and range of linear response. Due to their widespread use in the environmental, medical and agro-food fields, there is a great need for new BL reporter proteins with improved characteristics to provide researches a wide range of suitable reporters. Few efforts have been made in this direction and further improvement of BL reporter features (e.g., thermostability, narrower emission bandwidth, emission at different wavelengths) tailored for specific applications would be a remarkable progress toward the development of ultrasensitive multiplexed assays either in vitro or in vivo. The suitability of using red- and green-emitting thermostable mutants of Photinus pyralis firefly luciferase and two click beetle luciferases in combination with a secreted luciferase from Gaussia princeps was evaluated to develop a triple-color mammalian assay. Two triple-reporter model mammalian systems were developed in a human hepatoblastoma cell line to monitor the transcriptional regulation of cholesterol 7-alpha hydroxylase (cyp7a1), the enzyme that catalyzes the first and rate-limiting step of the main pathway responsible for cholesterol degradation in humans. These model systems allowed us to evaluate the feasibility of using two intracellular BL reporters and a secreted one in the same cell-based assay. The selection of reporter proteins characterized by similar expression levels was identified as a critical point for the development of a multicolor assay.
- Published
- 2008
- Full Text
- View/download PDF
43. Synergistic mutations produce blue-shifted bioluminescence in firefly luciferase.
- Author
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Branchini BR, Ablamsky DM, Rosenman JM, Uzasci L, Southworth TL, and Zimmer M
- Subjects
- Base Sequence, Chromatography, High Pressure Liquid, DNA Primers, Kinetics, Luciferases, Firefly genetics, Luminescence, Models, Molecular, Mutagenesis, Site-Directed, Spectrometry, Mass, Electrospray Ionization, Luciferases, Firefly metabolism
- Abstract
Light emission from the North American firefly Photinus pyralis, which emits yellow-green (557 nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. In a previous study designed to produce luciferases for simultaneously monitoring two gene expression events, we identified a very promising blue-shifted emitter (548 nm) that contained the mutations Val241Ile, Gly246Ala, and Phe250Ser [Branchini, B. R., Southworth, T. L., Khattak, N. F., Michelini, E., and Roda, A. (2005) Red- and green-emitting firefly luciferase mutants for bioluminescent reporter applications, Anal. Biochem. 345, 140-148]. To establish the basis of the unusual blue-shifted emission, we determined that a simple additive effect of the three individual mutations did not account for the spectral properties of the triple mutant. Instead, the bioluminescence emission spectra of two double mutants containing Phe250Ser and either Val241Ile or Gly246Ala very closely resembled that of the triple mutant. Additional mutagenesis results confirmed that the blue-shifted emission of the double mutants was determined by the synergistic behavior of active site residues. Molecular modeling studies of the Gly246Ala and Phe250Ser double mutant supported the notion that the blue-shifted emission was due to localized changes that increased the hydrophobicity at the emitter site as a result of the addition of a single methyl group at position 246. Moreover, the modeling data suggested that the Ala246 side chain remained close to the emitter through an additional H-bond between Ala246 and the hydroxyl group of Phe250, providing a possible structural basis for the synergistic behavior.
- Published
- 2007
- Full Text
- View/download PDF
44. Thermostable red and green light-producing firefly luciferase mutants for bioluminescent reporter applications.
- Author
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Branchini BR, Ablamsky DM, Murtiashaw MH, Uzasci L, Fraga H, and Southworth TL
- Subjects
- Animals, Enzyme Stability genetics, Fireflies enzymology, Hot Temperature, Hydrogen-Ion Concentration, Light, Luciferases, Firefly genetics, Mutagenesis, Site-Directed, Mutation, Biological Assay methods, Luciferases, Firefly analysis, Luciferases, Firefly chemistry, Luminescent Measurements
- Abstract
Light emission from the North American firefly Photinus pyralis, which emits yellow-green (557-nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. We present studies on the production of a set of thermostable red- and green-emitting luciferase mutants with bioluminescent properties suitable for dual-color reporter assays, biosensor measurements with internal controls, and imaging techniques. Starting with the luciferase variant Ser284Thr, we introduced the mutations Thr214Ala, Ala215Leu, Ile232Ala, Phe295Leu, and Glu354Lys to produce a new red-emitting enzyme with a bioluminescence maximum of 610 nm, narrow emission bandwidth, favorable kinetic properties, and excellent thermostability at 37 degrees C. By adding the same five changes to luciferase mutant Val241Ile/Gly246Ala/Phe250Ser, we produced a protein with an emission maximum of 546 nm, providing a set of thermostable enzymes whose bioluminescence maxima were separated by 64 nm. Model studies established that the luciferases could be detected at the attomole level and six orders of magnitude higher. In microplate luminometer format, mixtures containing 1.0 fmol total luciferase were quantified from measurements of simultaneously emitted red and green light. The results presented here provide evidence that it is feasible to monitor two distinct activities at 37 degrees C with these novel thermostable proteins.
- Published
- 2007
- Full Text
- View/download PDF
45. Luciferase from the Italian firefly Luciola italica: molecular cloning and expression.
- Author
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Branchini BR, Southworth TL, DeAngelis JP, Roda A, and Michelini E
- Subjects
- Amino Acid Sequence, Animals, Cloning, Molecular, DNA, Complementary metabolism, Gene Expression, Luciferases, Firefly isolation & purification, Luciferases, Firefly metabolism, Molecular Sequence Data, Phylogeny, Sequence Alignment, Time Factors, Fireflies enzymology, Luciferases, Firefly genetics
- Abstract
The cDNA encoding the luciferase from the Italian firefly Luciola italica was cloned using reverse transcriptase-PCR and a gene-specific primer set based on the DNA sequence of Luciola mingrelica. The cDNA sequence of L. italica luciferase was determined to be 1647 base pairs in length with an open reading frame of 548 amino acids. Phylogenetic analysis of the protein sequence demonstrated that this luciferase is closely related to that of other fireflies of the Lampyridae family, particularly within the Luciolinae subfamily, showing 96% homology to luciferases from the fireflies Hotaria unmunsana and Hotaria parvula. The specific activity of the L. italica luciferase was 78% of the North American enzyme, after correction for emission color differences. The bioluminescence emission of the Italian firefly is pH sensitive with maxima at 566 nm and 614 nm at pH 7.8 and 6.0, respectively. Interestingly, the total bioluminescence output was approximately 2-fold greater than that of P. pyralis luciferase due to differences in turnover characteristics evidenced by extended light emission decay kinetics. We expect that this newly discovered luciferase will be suitable for a wide range of bioluminescence applications including in vivo imaging and multiplex assays.
- Published
- 2006
- Full Text
- View/download PDF
46. Red- and green-emitting firefly luciferase mutants for bioluminescent reporter applications.
- Author
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Branchini BR, Southworth TL, Khattak NF, Michelini E, and Roda A
- Subjects
- Animals, Fireflies genetics, Firefly Luciferin chemistry, Genes, Reporter, Luciferases, Firefly genetics, Amino Acid Substitution, Fireflies enzymology, Luciferases, Firefly chemistry, Luminescence, Luminescent Measurements methods, Point Mutation
- Abstract
Light emission from the North American firefly Photinus pyralis, which emits yellow-green (557-nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. Here, we present studies leading to the production of a set of red- and green-emitting luciferase mutants with bioluminescent properties suitable for expanding the use of the P. pyralis system to dual-color reporter assays, biosensor measurements with internal controls, and imaging techniques. Using a combination of mutagenesis methods, we determined that the Ser284Thr mutation was sufficient to create an excellent red-emitting luciferase with a bioluminescence maximum of 615 nm, a narrow emission bandwidth, and favorable kinetic properties. Also, we developed a luciferase, containing the changes Val241Ile, Gly246Ala, and Phe250Ser, whose emission maximum was blue-shifted to 549 nm, providing a set of enzymes whose bioluminescence maxima were separated by 66 nm. Model studies demonstrated that in assays using a set of optical filters, the luciferases could be detected at the attomole level and seven orders of magnitude higher. In addition, in the presence of the Ser284Thr enzyme serving as a control, green light emission could be measured over a 10,000-fold range. The results presented here with the P. pyralis mutants provide evidence that simultaneous multiple analyte assay development is feasible with these novel proteins that require only a single substrate.
- Published
- 2005
- Full Text
- View/download PDF
47. NMR assignment of the backbone resonances of the firefly luciferase C-terminal 14.3 kDa domain.
- Author
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Branchini BR, Gonzalez SA, and Magyar RA
- Subjects
- Magnetic Resonance Spectroscopy, Protein Structure, Secondary, Luciferases, Firefly chemistry
- Published
- 2005
- Full Text
- View/download PDF
48. Synthesis of an N-acyl sulfamate analog of luciferyl-AMP: a stable and potent inhibitor of firefly luciferase.
- Author
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Branchini BR, Murtiashaw MH, Carmody JN, Mygatt EE, and Southworth TL
- Subjects
- Animals, Drug Stability, Kinetics, Molecular Mimicry, Peptide Fragments analysis, Peptide Hydrolases metabolism, Protein Conformation, Structure-Activity Relationship, Sulfonic Acids pharmacology, Adenosine Monophosphate analogs & derivatives, Firefly Luciferin analogs & derivatives, Luciferases, Firefly antagonists & inhibitors, Sulfonic Acids chemical synthesis
- Abstract
In the first of two half-reactions resulting in the emission of visible light, firefly luciferase forms luciferyl-adenylate from its natural substrates beetle luciferin and Mg-ATP. The acyl-adenylate is subsequently oxidized producing the light emitter oxyluciferin in an electronically excited state. In vitro, under mild conditions of temperature and pH, the acyl-adenylate intermediate is readily hydrolyzed and susceptible to oxidation. We report here the multi-step synthesis and physical and enzymatic characterization of an N-acyl sulfamate analog of luciferyl-adenylate, 5'-O-[(N-dehydroluciferyl)-sulfamoyl]-adenosine (compound 5). This represents the first example of a stable and potent (Ki = 340 nM) reversible inhibitor of firefly luciferase activity based on the structure of the natural acyl-adenylate intermediate. Additionally, we present the results of limited proteolysis studies that demonstrate that the binding of the novel acyl-adenylate analog protects luciferase from proteolysis. The findings presented here are interpreted in the context of the hypothesis that luciferase and the other enzymes in a large superfamily of adenylate-forming proteins adopt two conformations to catalyze two different partial reactions. We anticipate that the novel N-acyl sulfamate analog will be a valuable reagent in future studies designed to elucidate the role of conformational changes in firefly luciferase catalyzed bioluminescence.
- Published
- 2005
- Full Text
- View/download PDF
49. Mutagenesis evidence that the partial reactions of firefly bioluminescence are catalyzed by different conformations of the luciferase C-terminal domain.
- Author
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Branchini BR, Southworth TL, Murtiashaw MH, Wilkinson SR, Khattak NF, Rosenberg JC, and Zimmer M
- Subjects
- Adenosine Monophosphate chemistry, Amino Acid Motifs genetics, Amino Acid Substitution genetics, Animals, Catalysis, Coenzyme A chemistry, Kinetics, Luciferases, Firefly isolation & purification, Models, Molecular, Oxidation-Reduction, Peptide Fragments isolation & purification, Protein Conformation, Protein Structure, Tertiary genetics, Fireflies enzymology, Luciferases, Firefly chemistry, Luciferases, Firefly genetics, Luminescence, Mutagenesis, Site-Directed, Peptide Fragments chemistry, Peptide Fragments genetics
- Abstract
Firefly luciferase catalyzes two sequential partial reactions resulting in the emission of light. The enzyme first catalyzes the adenylation of substrate luciferin with Mg-ATP followed by the multistep oxidation of the adenylate to form the light emitter oxyluciferin in an electronically excited state. The beetle luciferases are members of a large superfamily, mainly comprised of nonbioluminescent enzymes that activate carboxylic acid substrates to form acyl-adenylate intermediates. Recently, the crystal structure of a member of this adenylate-forming family, acetyl-coenzyme A (CoA) synthetase, was determined in complex with an unreactive analogue of its acyl-adenylate and CoA [Gulick, A. M., Starai, V. J., Horswill, A. R., Homick, K. M., and Escalante-Semerena, J. C. (2003) Biochemistry 42, 2866-2873]. This structure presented a new conformation for this enzyme family, in which a significant rotation of the C-terminal domain brings residues of a conserved beta-hairpin motif to interact with the active site. We have undertaken a mutagenesis approach to study the roles of key residues of the equivalent beta-hairpin motif in Photinus pyralis luciferase (442IleLysTyrLysGlyTyrGlnVal449) in the overall production of light and the individual adenylation and oxidation partial reactions. Our results strongly suggest that Lys443 is critical for efficient catalysis of the oxidative half-reaction. Additionally, we provide evidence that Lys443 and Lys529, located on opposite sides of the C-terminal domain and conserved in all firefly luciferases, are each essential for only one of the partial reactions of firefly bioluminescence, supporting the proposal that the superfamily enzymes may adopt two different conformations to catalyze the two half-reactions.
- Published
- 2005
- Full Text
- View/download PDF
50. An alternative mechanism of bioluminescence color determination in firefly luciferase.
- Author
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Branchini BR, Southworth TL, Murtiashaw MH, Magyar RA, Gonzalez SA, Ruggiero MC, and Stroh JG
- Subjects
- Animals, Coleoptera genetics, Color, Firefly Luciferin chemistry, Luciferases genetics, Luciferases isolation & purification, Methylation, Models, Molecular, Molecular Structure, Protein Conformation, Spectrum Analysis, Coleoptera chemistry, Coleoptera metabolism, Luciferases chemistry, Luciferases metabolism, Luminescent Measurements
- Abstract
Beetle luciferases (including those of the firefly) use the same luciferin substrate to naturally display light ranging in color from green (lambda(max) approximately 530 nm) to red (lambda(max) approximately 635 nm). In a recent communication, we reported (Branchini, B. R., Murtiashaw, M. H., Magyar, R. A., Portier, N. C., Ruggiero, M. C., and Stroh, J. G. (2002) J. Am. Chem. Soc. 124, 2112-2113) that the synthetic adenylate of firefly luciferin analogue D-5,5-dimethylluciferin was transformed into the emitter 5,5-dimethyloxyluciferin in bioluminescence reactions catalyzed by luciferases from Photinus pyralis and the click beetle Pyrophorus plagiophthalamus. 5,5-Dimethyloxyluciferin is constrained to exist in the keto form and fluoresces mainly in the red. However, bioluminescence spectra revealed that green light emission was produced by the firefly enzyme, and red light was observed with the click beetle protein. These results, augmented with steady-state kinetic studies, were taken as experimental support for mechanisms of firefly bioluminescence color that require only a single keto form of oxyluciferin. We report here the results of mutagenesis studies designed to determine the basis of the observed differences in bioluminescence color with the analogue adenylate. Mutants of P. pyralis luciferase putative active site residues Gly246 and Phe250, as well as corresponding click beetle residues Ala243 and Ser247 were constructed and characterized using bioluminescence emission spectroscopy and steady state kinetics with adenylate substrates. Based on an analysis of these and recently reported (Branchini, B. R., Southworth, T. L., Murtiashaw, M. H., Boije, H., and Fleet, S. E. (2003) Biochemistry 42, 10429-10436) data, we have developed an alternative mechanism of bioluminescence color. The basis of the mechanism is that luciferase modulates emission color by controlling the resonance-based charge delocalization of the anionic keto form of the oxyluciferin excited state.
- Published
- 2004
- Full Text
- View/download PDF
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