Synergistic Mutations Produce Blue-Shifted Bioluminescence in Firefly Luciferase

Branchini, Bruce R.; Ablamsky, Danielle M.; Rosenman, Julie M.; Uzasci, Lerna; Southworth, Tara L.; Zimmer, Marc

doi:10.1021/bi7015052

Cited by 55 publications

(62 citation statements)

References 57 publications

(96 reference statements)

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“…Small changes in emission color can no doubt be regulated by changes in polarity at the active site as we previously documented . Possibly, the click beetle and railroad worm Lucs have more highly stabilized H‐bond networks or operate by an entirely different mechanism.…”

Section: Resultsmentioning

confidence: 61%

Cloning of the Orange Light‐Producing Luciferase from Photinus scintillans—A New Proposal on how Bioluminescence Color is Determined

Branchini

Southworth

Fontaine

et al. 2017

Photochem & Photobiology

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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.

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Section: Resultsmentioning

confidence: 61%

Cloning of the Orange Light‐Producing Luciferase from Photinus scintillans—A New Proposal on how Bioluminescence Color is Determined

Branchini

Southworth

Fontaine

et al. 2017

Photochem & Photobiology

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“…Mutations at all of these target sites have been shown to perturb D-luciferin binding (and thus light emission), while preserving the overall structural integrity of the enzyme. 9,30,62 …”

Section: Resultsmentioning

confidence: 99%

Orthogonal Luciferase–Luciferin Pairs for Bioluminescence Imaging

et al. 2017

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Bioluminescence imaging with luciferase-luciferin pairs is widely used in biomedical research. Several luciferases have been identified in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for imaging in vivo utilize the same substrate, and therefore cannot easily differentiate multiple cell types in a single subject. To develop a broader set of distinguishable probes, we crafted custom luciferins that can be selectively processed by engineered luciferases. Libraries of mutant enzymes were iteratively screened with sterically modified luciferins, and orthogonal enzyme-substrate “hits” were identified. These tools produced light when complementary enzyme-substrate partners interacted both in vitro and in cultured cell models. Based on their selectivity, these designer pairs will bolster multi-component imaging and enable the direct interrogation of cell networks not currently possible with existing tools. Our screening platform is also general and will expedite the identification of more unique luciferases and luciferins, further expanding the bioluminescence toolkit.

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“…Imaging with Fluc has also been historically limited to visualizing one feature at a time, owing to a lack of distinguishable probes . To craft improved bioluminescent tools, many groups have relied on Fluc mutagenesis to isolate brighter and spectrally resolved luciferases . A complementary and potentially more general approach involves modulating the luciferin itself.…”

Section: Introductionmentioning

confidence: 99%

Pyridone Luciferins and Mutant Luciferases for Bioluminescence Imaging

2018

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New applications for bioluminescence imaging require an expanded set of luciferase enzymes and luciferin substrates. Here, we report two novel luciferins for use in vitro and in cells. These molecules comprise regioisomeric pyridone cores that can be accessed from a common synthetic route. The analogues exhibited unique emission spectra with firefly luciferase, although photon intensities remained weak. Enhanced light outputs were achieved by using mutant luciferase enzymes. One of the luciferin-luciferase pairs produced light on par with native probes in live cells. The pyridone analogues and complementary luciferases add to a growing set of designer probes for bioluminescence imaging.

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Synergistic Mutations Produce Blue-Shifted Bioluminescence in Firefly Luciferase

Cited by 55 publications

References 57 publications

Cloning of the Orange Light‐Producing Luciferase from Photinus scintillans—A New Proposal on how Bioluminescence Color is Determined

Cloning of the Orange Light‐Producing Luciferase from Photinus scintillans—A New Proposal on how Bioluminescence Color is Determined

Orthogonal Luciferase–Luciferin Pairs for Bioluminescence Imaging

Pyridone Luciferins and Mutant Luciferases for Bioluminescence Imaging

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