title: Contribution of dynamic and static quenchers for the study of protein conformation in ionic liquids by steady-state fluorescence spectroscopy.
abstract: The study of protein conformation in ionic liquids (ILs) is crucial to understand enzymatic activity. Steady-state fluorescence is a proven, rapid and easy method to evaluate the protein structure in aqueous solutions, but it is discussed when used in ILs. In this work, the structure of the formate dehydrogenase from Candida boidinii (FDH, EC: 1.2.1.2) in three imidazolium-based ILs (dimethylimidazolium dimethylphosphate [MMIm][Me(2)PO(4)], 1-butyl-3-methylimidazolium acetate [BMIm][CH(3)COO], and dimethylimidazolium methylphosphonate [MMIm][CH(3)HPO(2)(OCH(3))]) is studied by fluorescence spectroscopy. The UV-vis spectroscopic analysis shows that the decrease of the FDH fluorescence is not only due to the high light absorption of these ILs. The Stern-Volmer analysis clearly shows that these ILs are quenchers of the indole fluorescence, while this quenching property is not found when imidazole is used. Fluorescence spectra of the FDH in the presence of the ILs show that a maximal ionic liquid concentration (MILc), which could be used for steady-state fluorescence study, should be defined. Therefore, FDH conformation could not be directly related to the decrease of its fluorescence in ILs. Nevertheless, the structure of the FDH could be evaluated with dynamic and static quenchers like iodide or acrylamide, used below the MILc, demonstrating the relevance of this parameter. The Stern-Volmer constants (K(SV)(Q)), calculated in the presence of the different ILs, demonstrate that these ILs are strong denaturing agents, each one acting with a different mechanism. This report provides a suitable and easy-to-apply method to study any enzyme structures in ILs by steady-state fluorescence.
authors: ICBMS, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, CNRS UMR 5246, Université Lyon 1, Villeurbanne, France.
authors: Mourad
authors: M
authors: Bekhouche
authors: Loïc J
authors: LJ
authors: Blum
authors: Bastien
authors: B
authors: Doumèche
mesh_terms: D000465:Algorithms; D002175:Candida / Q000201:enzymology; D005560:Formate Dehydrogenases / Q000737:chemistry* / Q000378:metabolism; D007093:Imidazoles / Q000737:chemistry; D052578:Ionic Liquids / Q000737:chemistry*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
chemical_list: C525963:1-butyl-3-methylimidazolium; D007093:Imidazoles; D052578:Ionic Liquids; D005560:Formate Dehydrogenases; C020644:Formate dehydrogenase (cytochrome)