title: Increase of segmental mobility through insertion of a flexible linker in split point of firefly luciferase.
abstract: 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.
authors.affiliation: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Parisa
authors.initials: P
authors.lastname: Bahmani
authors.affiliation: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran. Electronic address: saman_h@modares.ac.ir.
authors.forename: Saman
authors.initials: S
authors.lastname: Hosseinkhani
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D048888:Fireflies / Q000737:chemistry* / Q000201:enzymology; D015870:Gene Expression; D007700:Kinetics; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D000072417:Protein Domains; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D049409:Luciferases, Firefly
keywords: Firefly luciferase; Flexible liker; Photinus pyralis