title: Structural and functional effects of circular permutation on firefly luciferase: in vitro assay of caspase 3/7.
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.
authors.affiliation: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 14115-175, Iran.
authors.forename: Roya
authors.initials: R
authors.lastname: Cheraghi
authors.forename: Saman
authors.initials: S
authors.lastname: Hosseinkhani
authors.forename: Jamshid
authors.initials: J
authors.lastname: Davoodi
authors.forename: Mahboobeh
authors.initials: M
authors.lastname: Nazari
authors.forename: Zahra
authors.initials: Z
authors.lastname: Amini-Bayat
authors.forename: Hossein
authors.initials: H
authors.lastname: Karimi
authors.forename: Marie
authors.initials: M
authors.lastname: Shamseddin
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Gheidari
mesh_terms: D020816:Amino Acid Motifs; D053148:Caspase 3 / Q000737:chemistry; D053179:Caspase 7 / Q000737:chemistry; D002942:Circular Dichroism; D057075:Enzyme Assays; D007700:Kinetics; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D059748:Proteolysis; D013050:Spectrometry, Fluorescence
chemical_list: D049409:Luciferases, Firefly; D053148:Caspase 3; D053179:Caspase 7