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Bruce R. Branchini - One of the best experts on this subject based on the ideXlab platform.
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Mutagenesis and Structural Studies Reveal the Basis for the Activity and Stability Properties That Distinguish the Photinus Luciferases scintillans and Pyralis.
Biochemistry, 2019Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Brian P Huta, Allison Racela, Ketan D Patel, Andrew M GulickAbstract: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...
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mutagenesis and structural studies reveal the basis for the activity and stability properties that distinguish the photinus luciferases scintillans and Pyralis
Biochemistry, 2019Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Brian P Huta, Allison Racela, Ketan D Patel, Andrew M GulickAbstract:: 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.
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cloning of the orange light producing luciferase from photinus scintillans a new proposal on how bioluminescence color is determined
Photochemistry and Photobiology, 2017Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Martha H Murtiashaw, Alex Mcgurk, Munya Talukder, Rakhshi Qureshi, Deniz Yetil, Jesse A Sundlov, Andrew M GulickAbstract: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 5 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. This article is protected by copyright. All rights reserved.
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Cloning of the Orange Light-Producing Luciferase from Photinus scintillans-A New Proposal on how Bioluminescence Color is Determined.
Photochemistry and photobiology, 2017Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Martha H Murtiashaw, Alex Mcgurk, Rakhshi Qureshi, Deniz Yetil, Jesse A Sundlov, Munya H. Talukder, Andrew M GulickAbstract: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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an enhanced chimeric firefly luciferase inspired enzyme for atp detection and bioluminescence reporter and imaging applications
Analytical Biochemistry, 2015Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Munya Talukder, Elisa Michelini, Aldo Roda, Dawn Kohrt, Luca Cevenini, Martha J GrosselAbstract: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.
Andrew M Gulick - One of the best experts on this subject based on the ideXlab platform.
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Mutagenesis and Structural Studies Reveal the Basis for the Activity and Stability Properties That Distinguish the Photinus Luciferases scintillans and Pyralis.
Biochemistry, 2019Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Brian P Huta, Allison Racela, Ketan D Patel, Andrew M GulickAbstract: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...
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mutagenesis and structural studies reveal the basis for the activity and stability properties that distinguish the photinus luciferases scintillans and Pyralis
Biochemistry, 2019Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Brian P Huta, Allison Racela, Ketan D Patel, Andrew M GulickAbstract:: 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.
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cloning of the orange light producing luciferase from photinus scintillans a new proposal on how bioluminescence color is determined
Photochemistry and Photobiology, 2017Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Martha H Murtiashaw, Alex Mcgurk, Munya Talukder, Rakhshi Qureshi, Deniz Yetil, Jesse A Sundlov, Andrew M GulickAbstract: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 5 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. This article is protected by copyright. All rights reserved.
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Cloning of the Orange Light-Producing Luciferase from Photinus scintillans-A New Proposal on how Bioluminescence Color is Determined.
Photochemistry and photobiology, 2017Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Martha H Murtiashaw, Alex Mcgurk, Rakhshi Qureshi, Deniz Yetil, Jesse A Sundlov, Munya H. Talukder, Andrew M GulickAbstract: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.
Natalia Nikolaevna Ugarova - One of the best experts on this subject based on the ideXlab platform.
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Fluorescent Properties of Firefly Luciferases and Their Complexes with Luciferin
Bioscience Reports, 2000Co-Authors: Ekaterina I. Dementieva, Elena A. Fedorchuk, Lubov Yu. Brovko, Alexander P. Savitskii, Natalia Nikolaevna UgarovaAbstract:Fluorescence of luciferases from Luciola mingrelica (single tryptophanresidue, Trp-419) and Photinus Pyralis (two tryptophan residues, Trp-417,Trp-426) was studied. Analysis of quenching of tryptophan fluorescenceshowed that the tryptophan residue conserved in all luciferases is notaccessible for charged quenchers, which is explained by the presence ofpositively and negatively charged amino acid residues in the close vicinityto it. An effective energy transfer from tryptophan to luciferin wasobserved during quenching of tryptophan fluorescence of both luciferaseswith luciferin. From the data on the energy transfer, the distance betweenthe luciferin molecule and Trp-417 (419) in the luciferin–luciferasecomplex was calculated: 11–15 Å for P. Pyralis and 12–17Å for L. mingrelica luciferases. The role of the conserved Trp residuein the catalysis is discussed.
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Immobilization of recombinant firefly luciferase. Physicochemical properties and application.
Biochemistry, 1998Co-Authors: Irina Lundovskikh, Ekaterina I. Dementieva, Natalia Nikolaevna UgarovaAbstract:: Immobilization of the recombinant Luciola mingrelica and Photinus Pyralis firefly luciferases on BrCN-activated Sepharose was investigated. The catalytic properties and analytical characteristics of the immobilized recombinant and native luciferases were comparatively studied. The catalytic properties of the immobilized recombinant L. mingrelica luciferase are close to those of the native luciferase, but the former enzyme appeared to be significantly more stable. The immobilized recombinant luciferases can be used for ATP assay within the 0.01-10000 nM range.
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kinetics of luciferase gene expression from fireflies photinus Pyralis and luciola mingrelica in escherichia coli cells ii the role of ph in the biosynthesis of proteins and their transformation into active conformers
Biochemistry, 1994Co-Authors: O V Leonteva, G D Kutuzova, V B Turovetskiĭ, Natalia Nikolaevna UgarovaAbstract:: The Photinus Pyralis and Luciola mingrelica luciferases genes expression kinetics has been studied, repectively, on the pME61 and pJG lambda plasmids with a thermoinducible lambda PR promoter in the E. coli strain CA under conditions of a pH shift. An increase in the enzyme activity after a pH shift has been revealed. The maximal amount of the firefly Ph. Pyralis luciferase reaches 5.3%, while the maximal amount of the L. mingrelica enzyme reaches 4.9% of the total amount of cellular proteins in case of pHout shift to 9.0 according to the SDS gel electrophoresis data; that in case of a pHout shift to 8.5 is 4.9% and 4.5%, respectively. The enzyme activation correlates well with the dynamics of pHout and pHin.
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kinetics of luciferase gene expression from fireflies photinus Pyralis and luciola mingrelica in escherichia coli cells i kinetics of the transformation of recombinant luciferase into enzymatically active conformers
Biochemistry, 1994Co-Authors: G D Kutuzova, O V Leonteva, E A Skripkin, Natalia Nikolaevna UgarovaAbstract:: The Photinus Pyralis and Luciola mingrelica luciferases genes expression has been studied on the pME61 or pJG lambda plasmids with a thermoinducible lambda Pr promoter in the E. coli strain CA using three independent methods: SDS gel electrophoresis to quantify the synthesized luciferase protein, EIA to quantify the enzyme native conformer and activity measurements. The cultures were incubated by the temperature schemes 28 degrees-42 degrees-21 degrees C and 28 degrees-21 degrees C. The maximal amount of the Ph. Pyralis protein reached 4.5%, while that of L. mingrelica luciferase was 4.1% of the total amount of the cellular proteins after 10-hr incubation. The amount of the native conformer and the luciferase activity began to grow after a long induction period, reaching the maximal level by hour 50-60 after the thermoinduction. The increase in the enzyme activity correlated well with the increase in the ATP content in the cell. The observed "low-temperature induction" of the enzyme activity is interpreted as a protein transition to an active conformation. This transformation is considerably behind the protein biosynthesis process. Intracellular metabolic reactions have been shown to play an active part in these conformational changes.
Tara L Southworth - One of the best experts on this subject based on the ideXlab platform.
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Mutagenesis and Structural Studies Reveal the Basis for the Activity and Stability Properties That Distinguish the Photinus Luciferases scintillans and Pyralis.
Biochemistry, 2019Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Brian P Huta, Allison Racela, Ketan D Patel, Andrew M GulickAbstract: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...
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mutagenesis and structural studies reveal the basis for the activity and stability properties that distinguish the photinus luciferases scintillans and Pyralis
Biochemistry, 2019Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Brian P Huta, Allison Racela, Ketan D Patel, Andrew M GulickAbstract:: 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.
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cloning of the orange light producing luciferase from photinus scintillans a new proposal on how bioluminescence color is determined
Photochemistry and Photobiology, 2017Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Martha H Murtiashaw, Alex Mcgurk, Munya Talukder, Rakhshi Qureshi, Deniz Yetil, Jesse A Sundlov, Andrew M GulickAbstract: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 5 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. This article is protected by copyright. All rights reserved.
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Cloning of the Orange Light-Producing Luciferase from Photinus scintillans-A New Proposal on how Bioluminescence Color is Determined.
Photochemistry and photobiology, 2017Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Martha H Murtiashaw, Alex Mcgurk, Rakhshi Qureshi, Deniz Yetil, Jesse A Sundlov, Munya H. Talukder, Andrew M GulickAbstract: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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an enhanced chimeric firefly luciferase inspired enzyme for atp detection and bioluminescence reporter and imaging applications
Analytical Biochemistry, 2015Co-Authors: Bruce R. Branchini, Tara L Southworth, Danielle M Fontaine, Munya Talukder, Elisa Michelini, Aldo Roda, Dawn Kohrt, Luca Cevenini, Martha J GrosselAbstract: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.
Saman Hosseinkhani - One of the best experts on this subject based on the ideXlab platform.
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Increase of segmental mobility through insertion of a flexible linker in split point of firefly luciferase.
International journal of biological macromolecules, 2016Co-Authors: Parisa Bahmani, Saman HosseinkhaniAbstract:The crystal structure of Photinus Pyralis luciferase shows a unique molecular architecture consisting of a large N-terminal domain and a small C-terminal domain which is separated by a wide cleft. Protein engineering methods attempts to design the peptide linkers that make a connection between different protein domains or subunits to allow for separating domains and improve kinetics and structural features of proteins. In regard to this; introduction of a flexible linker at split point of luciferase which has a strong self-association activity, may leads to conformational change and improve general flexibility of protein. In this study, two flexible linkers in the split point of luciferase are introduced in order to test the effect of linker on flexibility of luciferase activity. Glycine-rich linkers are introduced into P. Pyralis firefly luciferase to make two separate mutant enzymes. Enzymatic properties of mutant and native forms were measured using luminescence assay. Results show that lengthening of luciferase domains through insertion of a flexible linker did not affect bioluminescence emission spectra. Also adding linkers do not have remarkable effect on thermostability. The Km values of mutants were increased compared to native form, indicating lower affinity of mutants toward substrates.
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Simple and Rapid Immobilization of Firefly Luciferase on Functionalized Magnetic Nanoparticles; a Try to Improve Kinetic Properties and Stability
2015Co-Authors: Mehdi Ebrahimi, Saman Hosseinkhani, Akbar Heydari, Jafar AkbariAbstract:We expressed and purified a recombinant P. Pyralis luciferase with N-terminal His-tags. The silanized Ni or Cu-loaded magnetic particles were prepared and used to assemble the His-tagged P. Pyralis luciferase. This enzyme immobilized on functionalized magnetic nanoparticles (MNPs) via electrostatic interactions of His-tag with Ni2+/Cu2+ ions on the surface of MNPs using simple one step method. These particles were also used for purification of recombinant luciferase from crude extract of cell lysate. Effect of incubation time and amount of MNPs in bioluminescent activity were investigated to determine optimum condition for immobilization. Several properties of immobilized luciferase were studied and compared with free enzyme. Immobilization has shown different effects on Km for ATP and luciferin. In both immobilized form, Km(ATP) was increased while Km(luciferin) was shown decreases. Optimal temperature of both immobilized luciferase increased to 30 oC while thermal stabilities have not shown significant differences compared to free enzyme. Both immobilized form inactivated after five consecutive reaction cycles.
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Step-wise addition of disulfide bridge in firefly luciferase controls color shift through a flexible loop: a thermodynamic perspective
Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology, 2012Co-Authors: Mahboobeh Nazari, Saman Hosseinkhani, Leila HassaniAbstract:Multi-color bioluminescence is developed using the introduction of single/double disulfide bridges in firefly luciferase. The bioluminescence reaction, which uses luciferin, Mg2+-ATP and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by the luciferase and emits visible light. The bioluminescence color of firefly luciferases is determined by the luciferase sequence and assay conditions. It has been proposed that the stability of a protein may increase through the introduction of a disulfide bridge that decreases the configurational entropy of unfolding. Single and double disulfide bridges are introduced into Photinus Pyralis firefly luciferase to make separate mutant enzymes with a single/double bridge (C81–A105C, L306C–L309C, P451C–V469C; C81–A105C/P451C–V469C, and A296C–A326C/P451C–V469C). By introduction of disulfide bridges using site-directed mutagenesis in Photinus Pyralis luciferase the color of emitted light was changed to red or kept in different extents. The bioluminescence color shift occurred with displacement of a critical loop in the luciferase structure without any change in green emitter mutants. Thermodynamic analysis revealed that among mutants, L306C–L309C shows a remarkable stability against urea denaturation and also a considerable increase in kinetic stability and a clear shift in bioluminescence spectra towards red.
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Delicate balance of electrostatic interactions and disulfide bridges in thermostability of firefly luciferase.
International journal of biological macromolecules, 2012Co-Authors: Somayeh Karimzadeh, Maryam Moradi, Saman HosseinkhaniAbstract:The wild type Photinus Pyralis luciferase does not have any disulfide bridge. Disulfide bridges are determinant in inherent stability of protein at moderate temperatures. Meanwhile, arginin is responsible for thermostability at higher temperatures. In this study, by concomitant introduction of disulfide bridge and a surface arginin in a mutant (A296C–A326C/I232R), the contribution of disulfide bridge introduction and surface hydrophilic residue on activity and global stability of P. Pyralis luciferase is investigated. In addition to the mentioned mutant; I232R, A296C–A326C and wild type luciferases are characterized. Though addition of Arg caused stability against proteolysis but in combination with disulfide bridge resulted in decreased thermal stability compared to A296C–A326C mutant. In spite of long distance of two different mutations (A296C–A326C and I232R) from each other in the three-dimensional structure, combination of their effects on the stability of luciferase was not cumulative.
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Relationship between stability and bioluminescence color of firefly luciferase
Photochemical and Photobiological Sciences, 2010Co-Authors: Parvaneh Maghami, Saman Hosseinkhani, Bijan Ranjbar, Atiyeh Ghasemi, Ali Moradi, Pooria GillAbstract:Firefly luciferase catalyzes the oxidation of luciferin in the presence of ATP, Mg2+ and molecular oxygen. The bioluminescence color of firefly luciferases is identified by the luciferase structure and assay conditions. Amongst different types of beetles, luciferase from Phrixotrix railroad worm (PhRE) with a unique additional residue (Arg353) naturally emits red bioluminescence color. By insertion of Arg356 in luciferase of Lampyris turkestanicus, corresponding to Arg353 in Phrixotrix hirtus, the color of the emitted light was changed to red. To understand the effect of this position on the bioluminescence color shift, four residues with similar sizes but different charges (Arg, Lys, Glu, and Gln) were inserted into Photinus Pyralis luciferase. Comparison of mutants with native luciferase shows that mutation brought an increase in the content of secondary structure and globular compactness of (P. pylalis) luciferase. Comparative study of chemical denaturation of native and mutant luciferases by activity measurement, intrinsic and extrinsic fluorescence, circular dichroism, and DSC techniques revealed that insertion of positively charged residues (Arg, Lys) in the flexible loop (352–358) plays a significant role on the stability of (P. Pyralis) luciferase and changes the light color to red.
