The Experts below are selected from a list of 705 Experts worldwide ranked by ideXlab platform

Bruce R. Branchini - One of the best experts on this subject based on the ideXlab platform.

  • Mutagenesis and Structural Studies Reveal the Basis for the Activity and Stability Properties That Distinguish the Photinus Luciferases scintillans and pyralis.
    Biochemistry, 2019
    Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Brian P Huta, Allison Racela, Ketan D Patel, Andrew M Gulick
    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...

  • red emitting chimeric firefly luciferase for in vivo imaging in low atp cellular environments
    Analytical Biochemistry, 2017
    Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Dawn Kohrt, Franceine S Welcome, Catherine M Florentine, Emma R Henricks, Demetria B Debartolo, Elisa Michelini, Luca Cevenini
    Abstract:

    Beetle luciferases have been adapted for live cell imaging where bioluminescence is dependent on the cellular availability of ATP, O2, 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.

  • probing bioluminescence resonance energy transfer in quantum rod luciferase nanoconjugates
    ACS Nano, 2016
    Co-Authors: Rabeka Alam, Bruce R. Branchini, Danielle M Fontaine, Liliana M Karam, Tennyson L Doane, Kaitlin Coopersmith, Mathew M Maye
    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 bindin...

  • a Photinus pyralis and luciola italica chimeric firefly luciferase produces enhanced bioluminescence
    Biochemistry, 2014
    Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Audrey L Davis, Curran E Behney, Martha H Murtiashaw
    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.

  • A Photinus pyralis and Luciola italica Chimeric Firefly Luciferase Produces Enhanced Bioluminescence
    2014
    Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Curran E Behney, Audrey L. Davis, Martha H Murtiashaw
    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

Tara L Southworth - One of the best experts on this subject based on the ideXlab platform.

  • Mutagenesis and Structural Studies Reveal the Basis for the Activity and Stability Properties That Distinguish the Photinus Luciferases scintillans and pyralis.
    Biochemistry, 2019
    Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Brian P Huta, Allison Racela, Ketan D Patel, Andrew M Gulick
    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...

  • red emitting chimeric firefly luciferase for in vivo imaging in low atp cellular environments
    Analytical Biochemistry, 2017
    Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Dawn Kohrt, Franceine S Welcome, Catherine M Florentine, Emma R Henricks, Demetria B Debartolo, Elisa Michelini, Luca Cevenini
    Abstract:

    Beetle luciferases have been adapted for live cell imaging where bioluminescence is dependent on the cellular availability of ATP, O2, 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.

  • a Photinus pyralis and luciola italica chimeric firefly luciferase produces enhanced bioluminescence
    Biochemistry, 2014
    Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Audrey L Davis, Curran E Behney, Martha H Murtiashaw
    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.

  • A Photinus pyralis and Luciola italica Chimeric Firefly Luciferase Produces Enhanced Bioluminescence
    2014
    Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Curran E Behney, Audrey L. Davis, Martha H Murtiashaw
    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

  • novel heterocyclic analogues of firefly luciferin
    Biochemistry, 2012
    Co-Authors: Carolyn C Woodroofe, Tara L Southworth, Curran E Behney, Poncho Meisenheimer, Dieter Klaubert, Yumi Kovic, Justin C Rosenberg, Bruce R. Branchini
    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 benzoth...

Sarah E Lower - One of the best experts on this subject based on the ideXlab platform.

  • on the inference of a southern origin of the north american firefly Photinus pyralis
    bioRxiv, 2019
    Co-Authors: Ana Catalan, Sebastian Hoehna, Sarah E Lower, Pablo Duchen
    Abstract:

    The firefly Photinus pyralis inhabits a wide range of latitudinal and ecological niches, with populations living from temperate to tropical habitats. Its ample geographic distribution makes this species an ideal system for the study of local adaptation and demographic inference of wild populations. Therefore, in this study we modelled and inferred different demographic scenarios for North American populations of P. pyralis, collected from Texas to New Jersey. To do this, we used a combination of ABC techniques (for multi-population/colonization analyses), and likelihood inference (dadi) for single-population demographic inference, which proved useful with our RAD data. We uncovered that the most ancestral North American population lays in Texas, which further colonized the Central region of the US and more recently the North Eastern coast. Our study confidently rejects a demographic scenario where the North Eastern populations colonized more southern populations until reaching Texas. Our results suggest that P. pyralis originated in Central- or South America, followed by migration events that populated northern latitudes. Finally, modelling the demographic history of North American P. pyralis serves as a null model of nucleotide diversity patterns, which will inform future studies of adaptation, not only in P. pyralis, but also in other North American taxa.

  • molecular variation across populations of a widespread north american firefly Photinus pyralis reveals that coding changes do not underlie flash color variation or associated visual sensitivity
    BMC Evolutionary Biology, 2018
    Co-Authors: Sarah E Lower, Kathrin F Stangerhall, David W Hall
    Abstract:

    BACKGROUND: Genes underlying signal production and reception are expected to evolve to maximize signal detection in specific environments. Fireflies vary in their light signal color both within and between species, and thus provide an excellent system in which to study signal production and reception in the context of signaling environments. Differences in signal color have been hypothesized to be due to variation in the sequence of luciferase, the enzyme that catalyzes the light reaction. Similarly, differences in visual sensitivity, which are expected to match signal color, have been hypothesized to be due to variation in the sequence of opsins, the protein component of visual pigments. Here we investigated (1) whether sequence variation in luciferase correlates with variation in signal color and (2) whether sequence variation in opsins correlates with inferred matching visual sensitivity across populations of a widespread North American firefly species, Photinus pyralis. We further tested (3) whether selection has acted on these loci by examining their population-level differentiation relative to the distribution of differentiation derived from a genome-wide sample of loci generated by double-digest RADseq. RESULTS: We found virtually no coding variation in luciferase or opsins. However, there was extreme divergence in non-coding variation in luciferase across populations relative to a panel of random genomic loci. CONCLUSIONS: The absence of protein variation at both loci challenges the paradigm that variation in signal color and visual sensitivity in fireflies is exclusively due to coding variation in luciferase and opsin genes. Instead, flash color variation within species must involve other mechanisms, such as abdominal pigmentation or regulation of light organ physiology. Evidence for selection at non-coding variation in luciferase suggests that selection is targeting luciferase regulation and may favor differ expression levels across populations.

  • Molecular variation across populations of a widespread North American firefly, Photinus pyralis , reveals that coding changes do not underlie flash color variation or associated visual sensitivity
    BMC evolutionary biology, 2018
    Co-Authors: Sarah E Lower, Kathrin F. Stanger-hall, David W Hall
    Abstract:

    Genes underlying signal production and reception are expected to evolve to maximize signal detection in specific environments. Fireflies vary in their light signal color both within and between species, and thus provide an excellent system in which to study signal production and reception in the context of signaling environments. Differences in signal color have been hypothesized to be due to variation in the sequence of luciferase, the enzyme that catalyzes the light reaction. Similarly, differences in visual sensitivity, which are expected to match signal color, have been hypothesized to be due to variation in the sequence of opsins, the protein component of visual pigments. Here we investigated (1) whether sequence variation in luciferase correlates with variation in signal color and (2) whether sequence variation in opsins correlates with inferred matching visual sensitivity across populations of a widespread North American firefly species, Photinus pyralis. We further tested (3) whether selection has acted on these loci by examining their population-level differentiation relative to the distribution of differentiation derived from a genome-wide sample of loci generated by double-digest RADseq. We found virtually no coding variation in luciferase or opsins. However, there was extreme divergence in non-coding variation in luciferase across populations relative to a panel of random genomic loci. The absence of protein variation at both loci challenges the paradigm that variation in signal color and visual sensitivity in fireflies is exclusively due to coding variation in luciferase and opsin genes. Instead, flash color variation within species must involve other mechanisms, such as abdominal pigmentation or regulation of light organ physiology. Evidence for selection at non-coding variation in luciferase suggests that selection is targeting luciferase regulation and may favor differ expression levels across populations.

  • firefly genomes illuminate parallel origins of bioluminescence in beetles
    bioRxiv, 2018
    Co-Authors: Timothy R Fallon, Sarah E Lower, Chingho Chang, Manabu Besshouehara, Gavin J Martin, Adam J Bewick, Megan G Behringer, Humberto J Debat, Isaac Wong
    Abstract:

    Abstract Fireflies and their fascinating luminous courtships have inspired centuries of scientific study. Today firefly luciferase is widely used in biotechnology, but the evolutionary origin of their bioluminescence remains unclear. To shed light on this long-standing question, we sequenced the genomes of two firefly species that diverged over 100 million-years-ago: the North American Photinus pyralis and Japanese Aquatica lateralis. We also sequenced the genome of a related click-beetle, the Caribbean Ignelater luminosus, with bioluminescent biochemistry near-identical to fireflies, but anatomically unique light organs, suggesting the intriguing but contentious hypothesis of parallel gains of bioluminescence. Our analyses support two independent gains of bioluminescence between fireflies and click-beetles, and provide new insights into the genes, chemical defenses, and symbionts that evolved alongside their luminous lifestyle. One Sentence Summary: Comparative analyses of the first linkage-group-resolution genomes of fireflies and related bioluminescent beetles address long-standing questions of the origin and evolution of bioluminescence and its associated traits.

Danielle M Fontaine - One of the best experts on this subject based on the ideXlab platform.

  • Mutagenesis and Structural Studies Reveal the Basis for the Activity and Stability Properties That Distinguish the Photinus Luciferases scintillans and pyralis.
    Biochemistry, 2019
    Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Brian P Huta, Allison Racela, Ketan D Patel, Andrew M Gulick
    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...

  • red emitting chimeric firefly luciferase for in vivo imaging in low atp cellular environments
    Analytical Biochemistry, 2017
    Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Dawn Kohrt, Franceine S Welcome, Catherine M Florentine, Emma R Henricks, Demetria B Debartolo, Elisa Michelini, Luca Cevenini
    Abstract:

    Beetle luciferases have been adapted for live cell imaging where bioluminescence is dependent on the cellular availability of ATP, O2, 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.

  • probing bioluminescence resonance energy transfer in quantum rod luciferase nanoconjugates
    ACS Nano, 2016
    Co-Authors: Rabeka Alam, Bruce R. Branchini, Danielle M Fontaine, Liliana M Karam, Tennyson L Doane, Kaitlin Coopersmith, Mathew M Maye
    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 bindin...

  • a Photinus pyralis and luciola italica chimeric firefly luciferase produces enhanced bioluminescence
    Biochemistry, 2014
    Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Audrey L Davis, Curran E Behney, Martha H Murtiashaw
    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.

  • A Photinus pyralis and Luciola italica Chimeric Firefly Luciferase Produces Enhanced Bioluminescence
    2014
    Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Curran E Behney, Audrey L. Davis, Martha H Murtiashaw
    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

James A H Murray - One of the best experts on this subject based on the ideXlab platform.

  • δflucs brighter Photinus pyralis firefly luciferases identified by surveying consecutive single amino acid deletion mutations in a thermostable variant
    Biotechnology and Bioengineering, 2018
    Co-Authors: Lisa Marie Halliwell, Darran Dafydd Jones, Amit P Jathoul, Jack P Bate, Harley L Worthy, James C Anderson, James A H Murray
    Abstract:

    The bright bioluminescence catalyzed by Photinus pyralis firefly luciferase (Fluc) enables a vast array of life science research such as bio imaging in live animals and sensitive in vitro diagnostics. The effectiveness of such applications is improved using engineered enzymes that to date have been constructed using amino acid substitutions. We describe ΔFlucs: consecutive single amino acid deletion mutants within six loop structures of the bright and thermostable ×11 Fluc. Deletion mutations are a promising avenue to explore new sequence and functional space and isolate novel mutant phenotypes. However, this method is often overlooked and to date there have been no surveys of the effects of consecutive single amino acid deletions in Fluc. We constructed a large semi-rational ΔFluc library and isolated significantly brighter enzymes after finding ×11 Fluc activity was largely tolerant to deletions. Targeting an “omega-loop” motif (T352-G360) significantly enhanced activity, altered kinetics, reduced Km for D-luciferin, altered emission colors, and altered substrate specificity for redshifted analog DL-infraluciferin. Experimental and in silico analyses suggested remodeling of the Ω-loop impacts on active site hydrophobicity to increase light yields. This work demonstrates the further potential of deletion mutations, which can generate useful Fluc mutants and broaden the palette of the biomedical and biotechnological bioluminescence enzyme toolbox.

  • mutagenesis of solvent exposed amino acids in Photinus pyralis luciferase improves thermostability and ph tolerance
    Biochemical Journal, 2006
    Co-Authors: Olga Gandelman, Laurence Carlo Tisi, Christopher R Lowe, James A H Murray
    Abstract:

    2+ , firefly luciferin and molecular oxygen as substrates, leading to the efficient emission of yellow‐green light. We report the identification of novel luciferase mutants which combine improved pH-tolerance and thermostability and that retain the specificactivityofthewild-typeenzyme.Thesewereidentifiedby the mutagenesis of solvent-exposed non-conserved hydrophobic amino acids to hydrophilic residues in Photinus pyralis firefly luciferase followed by in vivo activity screening. Mutants F14R, L35Q, V182K, I232K and F465R were found to be the preferred substitutionsattherespectivepositions.Theeffectsoftheseamino acid replacements are additive, since combination of the five substitutions produced an enzyme with greatly improved pHtolerance and stability up to 45 ◦ C. All mutants, including the mutant with all five substitutions, showed neither a decrease in specificactivityrelativetotherecombinantwild-typeenzyme,nor anysubstantialdifferencesinkineticconstants.Itisenvisagedthat the combined mutant will be superior to wild-type luciferase for many in vitro and in vivo applications.

  • mutagenesis of solvent exposed amino acids in Photinus pyralis luciferase improves thermostability and ph tolerance
    Biochemical Journal, 2006
    Co-Authors: G Erica H Law, Laurence Carlo Tisi, Olga Gandelman, Christopher R Lowe, James A H Murray
    Abstract:

    Firefly luciferase catalyses a two-step reaction, using ATP-Mg2+, firefly luciferin and molecular oxygen as substrates, leading to the efficient emission of yellow-green light. We report the identification of novel luciferase mutants which combine improved pH-tolerance and thermostability and that retain the specific activity of the wild-type enzyme. These were identified by the mutagenesis of solvent-exposed non-conserved hydrophobic amino acids to hydrophilic residues in Photinus pyralis firefly luciferase followed by in vivo activity screening. Mutants F14R, L35Q, V182K, I232K and F465R were found to be the preferred substitutions at the respective positions. The effects of these amino acid replacements are additive, since combination of the five substitutions produced an enzyme with greatly improved pH-tolerance and stability up to 45 degrees C. All mutants, including the mutant with all five substitutions, showed neither a decrease in specific activity relative to the recombinant wild-type enzyme, nor any substantial differences in kinetic constants. It is envisaged that the combined mutant will be superior to wild-type luciferase for many in vitro and in vivo applications.