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Saman Hosseinkhani - One of the best experts on this subject based on the ideXlab platform.

  • Bifunctional role of leucine 300 of Firefly Luciferase in structural rigidity
    International journal of biological macromolecules, 2017
    Co-Authors: Farzad Yousefi, Farangis Ataei, Mojtaba Mortazavi, Saman Hosseinkhani
    Abstract:

    Abstract Firefly Luciferase is susceptible to thermal inactivation, thereby its intracellular half-life decreased. Previous reports indicated that L 300 R mutation (LRR mutant) in E 354 R/Arg 356 double mutant (ERR mutant) from Lampyris turkestanicus Luciferase has increased its thermal stability and rigidity through induction of some ionic bonds with Asp 270 and 271. Disruption of the deduced ionic bonds in an ultra-rigid mutant of Firefly Luciferase did not reverse the flexibility of the protein. In this study, we investigated the effects of this residue to find the truth behind an extraordinary increase in thermal stability and rigidity of Luciferase after replacement of leucine 300 by arginine based on previous reports. For this purpose, L 300 R, L 300 K and L 300 E mutations were performed to compare the effects of these mutations on the native Firefly Luciferase. In spite of increase of intrinsic fluorescence of the mutants a slight increase in thermostability and retention of kinetic properties was observed. Based on our results, we can conclude that L 300 R mutation in LRR mutant accompanying with alteration in a flexible loop (352–359) increased thermostability and rigidity of Luciferase.

  • Hydrophobin-1 promotes thermostability of Firefly Luciferase.
    FEBS Journal, 2016
    Co-Authors: Azadeh Lohrasbi-nejad, Masoud Torkzadeh-mahani, Saman Hosseinkhani
    Abstract:

    : The thermal sensitivity of Firefly Luciferase limits its use in certain applications. Firefly Luciferase has hydrophobic sites on its surface, which lead to aggregation and inactivation of the enzyme at temperatures over 30 °C. We have successfully stabilized Firefly Luciferase at high temperatures with the assistance of a unique protein, hydrophobin-1 (HFB1). HFB1 is a small secretory protein belonging to class II of hydrophobins with a low molecular weight (7.5 kDa) and distinct functional hydrophobic patch on its surface. The interaction of HFB1 with hydrophobic sites on the surface of Luciferase was confirmed by extrinsic fluorescence studies using 8-anilino-1-naphthalenesulfonic acid (ANS) as a hydrophobic reporter probe. Calculation of thermodynamic parameters of heat inactivation of Luciferase shows that conformational changes and flexibility of enzyme decreased in the presence of HFB1, and thermostability of the HFB1-treated enzyme increased. Furthermore, the addition of HFB1 into the enzymatic solution leads to an increase in catalytic efficiency of Luciferase and subsequently improves the utility of the enzyme as an ATP detector.

  • The Effect of Surface Charge Saturation on Heat-induced Aggregation of Firefly Luciferase.
    Photochemistry and photobiology, 2015
    Co-Authors: Jamileh Gharanlar, Saman Hosseinkhani, Reza H. Sajedi, Parichehr Yaghmaei
    Abstract:

    We present here the effect of Firefly Luciferase surface charge saturation and the presence of some additives on its thermal-induced aggregation. Three mutants of Firefly Luciferase prepared by introduction of surface Arg residues named as 2R, 3R and 5R have two, three and five additional arginine residues substituted at their surface compared to native Luciferase; respectively. Turbidimetric study of heat-induced aggregation indicates that all three mutants were reproducibly aggregated at higher rates relative to wild type in spite of their higher thermostability. Among them, 2R had most evaluated propensity to heat-induced aggregation. Therefore, the hydrophilization followed by appearing of more substituted arginine residues with positive charge on the Firefly Luciferase surface was not reduced its thermal aggregation. Nevertheless, at the same condition in the presence of charged amino acids, e.g. Arg, Lys and Glu, as well as a hydrophobic amino acid, e.g. Val, the heat-induced aggregation of wild type and mutants of Firefly Luciferases was markedly decelerated than those in the absence of additives. On the basis of obtained results it seems, relinquishment of variety in charge of amino acid side chains, they via local interactions with proteins cause to decrease rate and extent of their thermal aggregation.

  • Structural and functional effects of circular permutation on Firefly Luciferase: in vitro assay of caspase 3/7.
    International journal of biological macromolecules, 2013
    Co-Authors: Roya Cheraghi, Saman Hosseinkhani, Jamshid Davoodi, Mahboobeh Nazari, Zahra Amini-bayat, Hossein Karimi, Marie Shamseddin, Fatemeh Gheidari
    Abstract:

    Abstract Bioluminescence reaction, which uses luciferin, Mg 2+ -ATP and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by the Luciferase and emit visible light. One of the most promising applications of Firefly Luciferase is biosensors. In order to develop an apoptosis biosensor based on caspase 3/7, we have generated 3 forms of circularly permuted variants of Photinus pyralis Firefly Luciferase and a relatively good tolerance toward disruption of the polypeptide chain by introduction of new termini were found. Two forms of circular permuted Luciferases showed significant activity enhancement in comparison with control after exposure to caspase 3. Moreover, the effect of circular permutation and also the length of inserted peptide (caspase 3/7 recognition sites) in structure of Firefly Luciferase were analyzed using circular dichroism and fluorescence spectroscopy.

  • Water-miscible ionic liquids as novel effectors for the Firefly Luciferase reaction
    Engineering in Life Sciences, 2012
    Co-Authors: Ali Reza Noori, Saman Hosseinkhani, Akbar Heydari, Parisa Ghiasi, Jafar Akbari
    Abstract:

    Ionic liquids (IL) are used as a new class of solvents for various reactions. Especially using IL in biocatalysis in an aqueous milieu has attracted considerable attention because enzymes show remarkable differences in their catalytic features in IL-containing reaction media. Firefly Luciferase is widely used in many analytical techniques, because light production of Firefly Luciferase is one of the most sensitive analytical measures in the ultrasensitive detection of adenosine-5′-triphosphate, e.g. for measuring microbial contamination and monitoring gene expression, as well as for monitoring tumor growth and metastasis in whole animals. Firefly Luciferase is an unstable enzyme and its inactivation can lead to low sensitivity in the above-mentioned assays. The present study addresses the comparative influence of six different water-immiscible IL, the 3-methylimidazolium derivatives [BMIM]Cl, [HMIM]Cl, [BMIM]Br, [EMIM]Br, [HMIM]Br, and [BMIM]BF4, on the kinetic properties, structural stability, and function of Firefly Luciferase from Photinus pyralis using circular dichroism, fluorescence spectroscopy, and a bioluminescence assay. The incubation of Luciferase with various IL showed that, with the exception of [BMIM]BF4, the activity and stability of Luciferase was considerably increased in the presence of IL, compared to Luciferase in aqueous medium. Moreover, Km for the substrate adenosine-5′-triphosphate in the presence of IL (except for [BMIM]BF4) decreased while Km for luciferin remained constant.

Sanjiv Sam Gambhir - One of the best experts on this subject based on the ideXlab platform.

  • Firefly Luciferase enzyme fragment complementation for imaging in cells and living animals
    Analytical Chemistry, 2005
    Co-Authors: Ramasamy Paulmurugan, Sanjiv Sam Gambhir
    Abstract:

    We identified different fragments of the Firefly Luciferase gene based on the crystal structure of Firefly Luciferase. These split reporter genes which encode for protein fragments, unlike the fragments currently used for studying protein−protein interactions, can self-complement and provide Luciferase enzyme activity in different cell lines in culture and in living mice. The comparison of the fragment complementation associated recovery of Firefly Luciferase enzyme activity with intact Firefly Luciferase was estimated for different fragment combinations and ranged from 0.01 to 4% of the full Firefly Luciferase activity. Using a cooled optical charge-coupled device camera, the analysis of Firefly Luciferase fragment complementation in transiently transfected subcutaneous 293T cell implants in living mice showed significant detectable enzyme activity upon injecting d-luciferin, especially from the combinations of fragments identified (Nfluc and Cfluc are the N and C fragments of the Firefly Luciferase gene...

John S. O’neill - One of the best experts on this subject based on the ideXlab platform.

Naohiro Kato - One of the best experts on this subject based on the ideXlab platform.

  • mathematical model of the Firefly Luciferase complementation assay reveals a non linear relationship between the detected luminescence and the affinity of the protein pair being analyzed
    PLOS ONE, 2016
    Co-Authors: Renee Dale, Hiroshi Ueda, Yuki Ohmuromatsuyama, Naohiro Kato
    Abstract:

    The Firefly Luciferase complementation assay is widely used as a bioluminescent reporter technology to detect protein-protein interactions in vitro, in cellulo, and in vivo. Upon the interaction of a protein pair, complemented Firefly Luciferase emits light through the adenylation and oxidation of its substrate, luciferin. Although it has been suggested that kinetics of light production in the Firefly Luciferase complementation assay is different from that in full length Luciferase, the mechanism behind this is still not understood. To quantitatively understand the different kinetics and how changes in affinity of a protein pair affect the light emission in the assay, a mathematical model of the in vitro Firefly Luciferase complementation assay was constructed. Analysis of the model finds that the change in kinetics is caused by rapid dissociation of the protein pair, low adenylation rate of luciferin, and increased affinity of adenylated luciferin to the enzyme. The model suggests that the affinity of the protein pair has an exponential relationship with the light detected in the assay. This relationship causes the change of affinity in a protein pair to be underestimated. This study underlines the importance of understanding the molecular mechanism of the Firefly Luciferase complementation assay in order to analyze protein pair affinities quantitatively.

  • Truly quantitative analysis of the Firefly Luciferase complementation assay
    Current Plant Biology, 2016
    Co-Authors: Renee Dale, Naohiro Kato
    Abstract:

    Abstract Luciferase complementation assays detect protein-protein interactions within living cells using bioluminescence. Since the first report using plant cells was published in 2007, over 100 peer-reviewed articles have been published describing the detection of protein-protein interactions within plant cells by the assays. The assays have also been used to analyze networks of protein-protein interactions in plants. Although the assays have a high dynamic range, they remain qualitative with respect to determining the affinities of interactions. In this article, we first summarize the Luciferase complementation assays developed in the past years. We then describe the mechanism of the Firefly Luciferase complementation that is most widely used in plants, and the reason it is qualitative rather than quantitative using a mathematical model. Finally, we discuss possible procedures to quantitatively determine the affinity of a protein pair using the Firefly Luciferase complementation assay.

Ramasamy Paulmurugan - One of the best experts on this subject based on the ideXlab platform.

  • Firefly Luciferase enzyme fragment complementation for imaging in cells and living animals
    Analytical Chemistry, 2005
    Co-Authors: Ramasamy Paulmurugan, Sanjiv Sam Gambhir
    Abstract:

    We identified different fragments of the Firefly Luciferase gene based on the crystal structure of Firefly Luciferase. These split reporter genes which encode for protein fragments, unlike the fragments currently used for studying protein−protein interactions, can self-complement and provide Luciferase enzyme activity in different cell lines in culture and in living mice. The comparison of the fragment complementation associated recovery of Firefly Luciferase enzyme activity with intact Firefly Luciferase was estimated for different fragment combinations and ranged from 0.01 to 4% of the full Firefly Luciferase activity. Using a cooled optical charge-coupled device camera, the analysis of Firefly Luciferase fragment complementation in transiently transfected subcutaneous 293T cell implants in living mice showed significant detectable enzyme activity upon injecting d-luciferin, especially from the combinations of fragments identified (Nfluc and Cfluc are the N and C fragments of the Firefly Luciferase gene...