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

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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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Abbreviations

2D:

Two dimensional

A8-35:

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

A8-75:

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

APol:

Amphipol

Btot :

Total amount of binding sites

C10E5 :

Decylpentaethylene glycol ether

C10-PEG:

Decylpoly(ethyleneglycol) 300

CBB:

Coomassie brillant blue

DABCO:

1,4-Diazabicyclo[2.2.2]octane

DCI:

N,N-Dicyclohexylcarbodiimide

DMF:

Dimethylformamide

DOC:

Deoxycholate

DTT:

Dithiothreitol

EIImtl :

Dimeric mannitol permease from the inner membrane of Escherichia coli

FAPol:

Fluorescently-labeled A8-35

FAPolfluo :

Fluorescein-labeled A8-35

FITC:

Fluorescein isothiocyanate

FRET:

Förster resonance energy transfer

IIAmtl, IIBmtl :

Cytoplasmic A and B domains of EIImtl, respectively

IICmtl :

Transmembrane C domain of EIImtl

ISO:

Inside-out

KD :

Dissociation constant

MP:

Membrane protein

NBD:

7-Nitrobenz-2-oxa-1,3-diazol-4-yl

NTA:

Nitrilotriacetic acid

PAA:

Poly(acrylic acid)

SDS-PAGE:

Sodium dodecylsulfate-polyacrylamide gel electrophoresis

TL:

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

TMHI:

The first putative transmembrane helix of IICmtl

tOmpA:

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

Trp:

Tryptophan

UAPol:

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

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Acknowledgments

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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Authors and Affiliations

  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

    Milena Opačić, Fabrice Giusti & Jean-Luc Popot

  2. Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands

    Milena Opačić & Jaap Broos

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  1. Milena Opačić
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  2. Fabrice Giusti
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  3. Jean-Luc Popot
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  4. Jaap Broos
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Correspondence to Jean-Luc Popot or Jaap Broos.

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Opačić, M., Giusti, F., Popot, JL. et al. Isolation of Escherichia coli Mannitol Permease, EIImtl, Trapped in Amphipol A8-35 and Fluorescein-Labeled A8-35. J Membrane Biol 247, 1019–1030 (2014). https://doi.org/10.1007/s00232-014-9691-7

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