The Journal of Membrane Biology

, Volume 247, Issue 9–10, pp 1019–1030 | Cite as

Isolation of Escherichia coli Mannitol Permease, EIImtl, Trapped in Amphipol A8-35 and Fluorescein-Labeled A8-35

  • Milena Opačić
  • Fabrice Giusti
  • Jean-Luc PopotEmail author
  • Jaap BroosEmail author


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.


Membrane protein Fluorescent amphipol Fluorescence quenching Förster resonance energy transfer 



Two dimensional


Poly(sodium acrylate) based amphipol comprising 35 % of free carboxylate, 25 % of octyl chains, 40 % of isopropyl groups, and whose weight average molar mass is ~4.3 kDa


Poly(sodium acrylate) based amphipol comprising 75 % of free carboxylate, 25 % of octylchains, whose weight average molar mass is ~4 kDa




Total amount of binding sites


Decylpentaethylene glycol ether


Decylpoly(ethyleneglycol) 300


Coomassie brillant blue












Dimeric mannitol permease from the inner membrane of Escherichia coli


Fluorescently-labeled A8-35


Fluorescein-labeled A8-35


Fluorescein isothiocyanate


Förster resonance energy transfer

IIAmtl, IIBmtl

Cytoplasmic A and B domains of EIImtl, respectively


Transmembrane C domain of EIImtl




Dissociation constant


Membrane protein




Nitrilotriacetic acid


Poly(acrylic acid)


Sodium dodecylsulfate-polyacrylamide gel electrophoresis


Trp-less EIImtl, in which the four native Trp residues are replaced by Phe


The first putative transmembrane helix of IICmtl


The transmembrane domain of outer membrane protein A from E. coli




A8-35 grafted with an amino arm

W36, W37, W38, W167, and W188

Single-Trp-containing EIImtl mutants based on Trp-less EIImtl

wt EIImtl

Wild-type EIImtl, with Trp residues at positions 30, 42, 109, and 117



This project was supported by the French Centre National de la Recherche Scientifique, by University Paris-7, and by the “Initiative d’Excellence” program from the French State (Grant “DYNAMO”, ANR-11-LABX-0011-01). M.O. was the recipient of a fellowship from the European International Training Network SBMPs (Structural Biology of Membrane Proteins).


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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Unité Mixte de Recherche 7099, Centre National de la Recherche Scientifique and Université Paris 7, Institut de Biologie Physico-Chimique, CNRS FRC 550ParisFrance
  2. 2.Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology InstituteUniversity of GroningenGroningenThe Netherlands

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