Isolation of Escherichia Coli Mannitol Permease, EIImtl, Trapped in Amphipol A8-35 and Fluorescein-labeled A8-35

Overview

Journal J Membr Biol

Date 2014 Jun 23

PMID 24952466

Citations 3

Authors

Milena Opacic

Fabrice Giusti

Jean-Luc Popot

Jaap Broos

Affiliations

Soon will be listed here.

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.

Citing Articles

Amphipols for each season.

Zoonens M, Popot J J Membr Biol. 2014; 247(9-10):759-96.

PMID: 24969706 PMC: 4282167. DOI: 10.1007/s00232-014-9666-8.

Synthesis of an oligonucleotide-derivatized amphipol and its use to trap and immobilize membrane proteins.

Le Bon C, Della Pia E, Giusti F, Lloret N, Zoonens M, Martinez K Nucleic Acids Res. 2014; 42(10):e83.

PMID: 24744236 PMC: 4041424. DOI: 10.1093/nar/gku250.

Synthesis, characterization and applications of a perdeuterated amphipol.

Giusti F, Rieger J, Catoire L, Qian S, Calabrese A, Watkinson T J Membr Biol. 2014; 247(9-10):909-24.

PMID: 24652511 DOI: 10.1007/s00232-014-9656-x.

References

Giusti F, Rieger J, Catoire L, Qian S, Calabrese A, Watkinson T . Synthesis, characterization and applications of a perdeuterated amphipol. J Membr Biol. 2014; 247(9-10):909-24. DOI: 10.1007/s00232-014-9656-x. View

Opacic M, Hesp B, Fusetti F, Dijkstra B, Broos J . Structural investigation of the transmembrane C domain of the mannitol permease from Escherichia coli using 5-FTrp fluorescence spectroscopy. Biochim Biophys Acta. 2011; 1818(3):861-8. DOI: 10.1016/j.bbamem.2011.11.001. View

Broos J, Strambini G, Gonnelli M, Vos E, Koolhof M, Robillard G . Sensitive monitoring of the dynamics of a membrane-bound transport protein by tryptophan phosphorescence spectroscopy. Biochemistry. 2000; 39(35):10877-83. DOI: 10.1021/bi000803z. View

Robillard G, Broos J . Structure/function studies on the bacterial carbohydrate transporters, enzymes II, of the phosphoenolpyruvate-dependent phosphotransferase system. Biochim Biophys Acta. 1999; 1422(2):73-104. DOI: 10.1016/s0304-4157(99)00002-7. View

Seybold P, Gouterman M, Callis J . Calorimetric, photometric and lifetime determinations of fluorescence yields of fluorescein dyes. Photochem Photobiol. 1969; 9(3):229-42. DOI: 10.1111/j.1751-1097.1969.tb07287.x. View

Champeil P, Menguy T, Tribet C, Popot J, LE MAIRE M . Interaction of amphipols with sarcoplasmic reticulum Ca2+-ATPase. J Biol Chem. 2000; 275(25):18623-37. DOI: 10.1074/jbc.M000470200. View

Lolkema J, Kuiper H, Robillard G . Mannitol-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli: physical size of enzyme IImtl and its domains IIBA and IIC in the active state. Biochemistry. 1993; 32(6):1396-400. DOI: 10.1021/bi00057a002. View

Baneres J, Popot J, Mouillac B . New advances in production and functional folding of G-protein-coupled receptors. Trends Biotechnol. 2011; 29(7):314-22. DOI: 10.1016/j.tibtech.2011.03.002. View

Robillard G, Boer H, van Weeghel R, Wolters G, Dijkstra A . Expression and characterization of a structural and functional domain of the mannitol-specific transport protein involved in the coupling of mannitol transport and phosphorylation in the phosphoenolpyruvate-dependent phosphotransferase system of.... Biochemistry. 1993; 32(37):9553-62. DOI: 10.1021/bi00088a006. View

10.

Legler P, Cai M, Peterkofsky A, Clore G . Three-dimensional solution structure of the cytoplasmic B domain of the mannitol transporter IImannitol of the Escherichia coli phosphotransferase system. J Biol Chem. 2004; 279(37):39115-21. DOI: 10.1074/jbc.M406764200. View

11.

Popot J, Althoff T, Bagnard D, Baneres J, Bazzacco P, Billon-Denis E . Amphipols from A to Z. Annu Rev Biophys. 2011; 40:379-408. DOI: 10.1146/annurev-biophys-042910-155219. View

12.

Garavito R, Ferguson-Miller S . Detergents as tools in membrane biochemistry. J Biol Chem. 2001; 276(35):32403-6. DOI: 10.1074/jbc.R100031200. View

13.

Zoonens M, Giusti F, Zito F, Popot J . Dynamics of membrane protein/amphipol association studied by Förster resonance energy transfer: implications for in vitro studies of amphipol-stabilized membrane proteins. Biochemistry. 2007; 46(36):10392-404. DOI: 10.1021/bi7007596. View

14.

Koning R, Keegstra W, Oostergetel G, Robillard G, Brisson A . The 5 A projection structure of the transmembrane domain of the mannitol transporter enzyme II. J Mol Biol. 1999; 287(5):845-51. DOI: 10.1006/jmbi.1999.2650. View

15.

LE MAIRE M, Champeil P, MOLLER J . Interaction of membrane proteins and lipids with solubilizing detergents. Biochim Biophys Acta. 2000; 1508(1-2):86-111. DOI: 10.1016/s0304-4157(00)00010-1. View

16.

Gohon Y, Giusti F, Prata C, Charvolin D, Timmins P, Ebel C . Well-defined nanoparticles formed by hydrophobic assembly of a short and polydisperse random terpolymer, amphipol A8-35. Langmuir. 2006; 22(3):1281-90. DOI: 10.1021/la052243g. View

17.

Robillard G, Blaauw M . Enzyme II of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: protein-protein and protein-phospholipid interactions. Biochemistry. 1987; 26(18):5796-803. DOI: 10.1021/bi00392a032. View

18.

Etzkorn M, Raschle T, Hagn F, Gelev V, Rice A, Walz T . Cell-free expressed bacteriorhodopsin in different soluble membrane mimetics: biophysical properties and NMR accessibility. Structure. 2013; 21(3):394-401. PMC: 3595385. DOI: 10.1016/j.str.2013.01.005. View

19.

Broos J, Gabellieri E, van Boxel G, Jackson J, Strambini G . Tryptophan phosphorescence spectroscopy reveals that a domain in the NAD(H)-binding component (dI) of transhydrogenase from Rhodospirillum rubrum has an extremely rigid and conformationally homogeneous protein core. J Biol Chem. 2003; 278(48):47578-84. DOI: 10.1074/jbc.M309287200. View

20.

Perlmutter J, Popot J, Sachs J . Molecular dynamics simulations of a membrane protein/amphipol complex. J Membr Biol. 2014; 247(9-10):883-95. DOI: 10.1007/s00232-014-9690-8. View