Isolation of Escherichia coli Mannitol Permease,EIImtl, Trapped in Amphipol A8-35 and Fluorescein-Labeled A8-35 |
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Authors: | Milena Opa?i? Fabrice Giusti Jean-Luc Popot Jaap Broos |
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Institution: | 1. 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 2. Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands
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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 EIImtl, 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 EIImtl, 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 EIImtl soluble and functional in the absence of detergent. The specific phosphorylation activity of an APol-trapped Trp-less EIImtl 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 EIImtl 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. |
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