title: Isolation of Escherichia coli mannitol permease, EIImtl, trapped in amphipol A8-35 and fluorescein-labeled A8-35.
abstract: Amphipols (APols) are short amphipathic polymers that keep integral membrane proteins water-soluble while stabilizing them as compared to detergent solutions. In the present work, we have carried out functional and structural studies of a membrane transporter that had not been characterized in APol-trapped form yet, namely EII(mtl), a dimeric mannitol permease from the inner membrane of Escherichia coli. A tryptophan-less and dozens of single-tryptophan (Trp) mutants of this transporter are available, making it possible to study the environment of specific locations in the protein. With few exceptions, the single-Trp mutants show a high mannitol-phosphorylation activity when in membranes, but, as variance with wild-type EII(mtl), some of them lose most of their activity upon solubilization by neutral (PEG- or maltoside-based) detergents. Here, we present a protocol to isolate these detergent-sensitive mutants in active form using APol A8-35. Trapping with A8-35 keeps EII(mtl) soluble and functional in the absence of detergent. The specific phosphorylation activity of an APol-trapped Trp-less EII(mtl) mutant was found to be ~3× higher than the activity of the same protein in dodecylmaltoside. The preparations are suitable both for functional and for fluorescence spectroscopy studies. A fluorescein-labeled version of A8-35 has been synthesized and characterized. Exploratory studies were conducted to examine the environment of specific Trp locations in the transmembrane domain of EII(mtl) using Trp fluorescence quenching by water-soluble quenchers and by the fluorescein-labeled APol. This approach has the potential to provide information on the transmembrane topology of MPs.
authors: Unité Mixte de Recherche 7099, Centre National de la Recherche Scientifique and Université Paris 7, Institut de Biologie Physico-Chimique, CNRS FRC 550, 13 rue Pierre-et-Marie Curie, 75005, Paris, France.
authors: Milena
authors: M
authors: Opačić
authors: Fabrice
authors: F
authors: Giusti
authors: Jean-Luc
authors: JL
authors: Popot
authors: Jaap
authors: J
authors: Broos
mesh_terms: D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000648:ultrastructure; D019793:Fluorescein / Q000032:analysis / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D005590:Fractional Precipitation / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000648:ultrastructure; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000302:isolation & purification* / Q000648:ultrastructure; D011108:Polymers / Q000737:chemistry*; D011437:Propylamines / Q000737:chemistry*; D012995:Solubility; D012996:Solutions; D013048:Specimen Handling / Q000379:methods; D013194:Staining and Labeling; D013501:Surface-Active Agents / Q000737:chemistry*
chemical_list: D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D009004:Monosaccharide Transport Proteins; D011108:Polymers; D011437:Propylamines; D012996:Solutions; D013501:Surface-Active Agents; C499017:amphipol A8-35; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli; D019793:Fluorescein