title: Hydrophobin-1 promotes thermostability of firefly luciferase.
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.
authors.affiliation: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Azadeh
authors.initials: A
authors.lastname: Lohrasbi-Nejad
authors.affiliation: Department of Biotechnology, Research institute for Science and High Technology and Environmental Sciences, Graduated University of Advanced Technology, Kerman, Iran.
authors.forename: Masoud
authors.initials: M
authors.lastname: Torkzadeh-Mahani
authors.affiliation: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Saman
authors.initials: S
authors.lastname: Hosseinkhani
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D002417:Cattle; D004795:Enzyme Stability; D006358:Hot Temperature; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000378:metabolism*; D008163:Luminescent Measurements; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D013696:Temperature
chemical_list: C515594:8-anilino-1-naphthalenesulfonic acid; D000817:Anilino Naphthalenesulfonates; D012710:Serum Albumin, Bovine; D049409:Luciferases, Firefly
keywords: firefly luciferase; hydrophobic sites; hydrophobin-1; interaction; stabilization