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.

Activation/Inactivation Role of Ionic Liquids on Formate Dehydrogenase from Pseudomonas sp. 101 and Its Mutated Thermostable Form

“…The fluorescence increase at 1 %, which is shown by the spectra, may be attributed to this bond that involves greater exposure to the solvent of the hydrophobic cavities in which fluorescent residues are present. In contrast, the intensity decrease observed at higher ILs concentrations may be attributed to a damping of the fluorescence signal caused by the IL, as also observed for the standard N‐Ac‐Trp‐O‐Et.…”

Protein stabilization and enzyme activation in ionic liquids: specific ion effects

“…The Yang group obtained up to 86% conversion of corn oil to biodiesel in [BMIM][PF 6 ] catalyzed by Penicillium expansum lipase; however, they obtained no enzymatic activity in other ILs containing anions of MeSO 4 − , OAc − , NO 3 − , and H 2 PO 4 − . Bekhouche et al examined the activity and stability of formate dehydrogenase from Candida boidinii (FDH, EC: 1.2.1.2) in three ILs (i.e. [MMIM][Me 2 PO 4 ], [BMIM][OAc], [MMIM][CH 3 HPO 2 (OCH 3 )]) by activity assays and steady‐state fluorescence spectroscopy (using iodide as the dynamic quencher or acrylamide as the static quencher).…”

Trace Water as Prominent Factor to Induce Peptide Self‐Assembly: Dynamic Evolution and Governing Interactions in Ionic Liquids

“…Third, the hygroscopicity of ILs in air enables water molecules to slowly and controllably permeate into the solvent system, allowing for exploring the interactions of trace water with peptide building blocks, particularly at the early stages of self‐assembly. Indeed, ILs can influence the protein and enzymatic activity, and have been worldwide applied as protein and enzyme stabilizers, biocatalytic reaction media, etc . On the other hand, although the assembly of proteins closely relies on the biological environment such as the cytoplasm, the biological environment is too complex to be completely reconstructed.…”