Advertisement

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
Article

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.

Keywords

Membrane protein Fluorescent amphipol Fluorescence quenching Förster resonance energy transfer 

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

Notes

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).

References

  1. Althoff T, Mills DJ, Popot J-L, Kühlbrandt W (2011) Assembly of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1. EMBO J 30:4652–4664CrossRefGoogle Scholar
  2. Banères J-L, Popot J-L, Mouillac B (2011) New advances in production and functional folding of G protein-coupled receptors. Trends Biotechnol 29:314–322CrossRefGoogle Scholar
  3. Bowie JU (2001) Stabilizing membrane proteins. Curr Opin Struct Biol 11:397–402CrossRefGoogle Scholar
  4. Broos J, ter Veld F, Robillard GT (1999) Membrane protein–ligand interactions in Escherichia coli vesicles and living cells monitored via a biosynthetically incorporated tryptophan analogue. Biochemistry 38:9798–9803CrossRefGoogle Scholar
  5. Broos J, Strambini GB, Gonnelli M, Vos EPP, Koolhof M, Robillard GT (2000) Sensitive monitoring of the dynamics of a membrane-bound transport protein by tryptophan phosphorescence spectroscopy. Biochemistry 39:10877–10883CrossRefGoogle Scholar
  6. Broos J, Gabellieri E, van Boxel GI, Jackson JB, Strambini GB (2003) 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 278:47578–47584CrossRefGoogle Scholar
  7. Broos J, Maddalena F, Hesp BH (2004) In vivo synthesized proteins with monoexponential fluorescence decay kinetics. J Am Chem Soc 126:22–23CrossRefGoogle Scholar
  8. Catoire LJ, Zoonens M, van Heijenoort C, Giusti F, Popot J-L, Guittet E (2009) Inter- and intramolecular contacts in a membrane protein/surfactant complex observed by hetero-nuclear dipole-to-dipole cross-relaxation. J Magn Res 197:91–95CrossRefGoogle Scholar
  9. Champeil P, Menguy T, Tribet C, Popot J-L, le Maire M (2000) Interaction of amphipols with the sarcoplasmic reticulum Ca2+-ATPase. J Biol Chem 275:18623–18637CrossRefGoogle Scholar
  10. Charvolin D, Picard M, Huang L-S, Berry EA, Popot J-L (2014) Solution behavior and crystallization of cytochrome bc 1 in the presence of amphipols. J Membr Biol. doi: 10.1007/s00232-014-9694-4 CrossRefGoogle Scholar
  11. Dahmane T, Rappaport F, Popot J-L (2013) Amphipol-assisted folding of bacteriorhodopsin in the presence and absence of lipids. Functional consequences. Eur Biophys J 42:85–101CrossRefGoogle Scholar
  12. Dijkstra DS, Broos J, Robillard GT (1996) Membrane proteins and impure detergents: procedures to purify membrane proteins to a degree suitable for tryptophan fluorescence spectroscopy. Anal Biochem 240:142–147CrossRefGoogle Scholar
  13. Etzkorn M, Raschle T, Hagn F, Gelev V, Rice AJ, Walz T, Wagner G (2013) Cell-free expressed bacteriorhodopsin in different soluble membrane mimetics: biophysical properties and NMR accessibility. Structure 21:394–401CrossRefGoogle Scholar
  14. Etzkorn M, Zoonens M, Catoire LJ, Popot J-L, Hiller S (2014) How amphipols embed membrane proteins: global solvent accessibility and interaction with a flexible protein terminus. J Membr Biol. doi: 10.1007/s00232-014-9657-9 CrossRefGoogle Scholar
  15. Garavito RM, Ferguson-Miller S (2001) Detergents as tools in membrane biochemistry. J Biol Chem 276:32403–32406CrossRefGoogle Scholar
  16. Giusti F, Rieger J, Catoire L, Qian S, Calabrese AN, Watkinson TG, Pembouong G, Casiraghi M, Radford SE, Ashcroft AE, Popot J-L (2014) Synthesis, characterization and applications of a perdeuterated amphipol. J Membr Biol. doi: 10.1007/s00232-014-9656-x CrossRefGoogle Scholar
  17. Gohon Y, Popot J-L (2003) Membrane protein–surfactant complexes. Curr Opin Colloid Interface Sci 8:15–22CrossRefGoogle Scholar
  18. Gohon Y, Pavlov G, Timmins P, Tribet C, Popot J-L, Ebel C (2004) Partial specific volume and solvent interactions of amphipol A8-35. Anal Biochem 334:318–334CrossRefGoogle Scholar
  19. Gohon Y, Giusti F, Prata C, Charvolin D, Timmins P, Ebel C, Tribet C, Popot J-L (2006) Well-defined nanoparticles formed by hydrophobic assembly of a short and polydisperse random terpolymer, amphipol A8-35. Langmuir 22:1281–1290CrossRefGoogle Scholar
  20. Gohon Y, Dahmane T, Ruigrok R, Schuck P, Charvolin D, Rappaport F, Timmins P, Engelman DM, Tribet C, Popot J-L, Ebel C (2008) Bacteriorhodopsin/amphipol complexes: structural and functional properties. Biophys J 94:3523–3537CrossRefGoogle Scholar
  21. Huynh KW, Cohen MR, Moiseenkova-Bell VY (2014) Application of amphipols for structure-functional analysis of TRP channels. J Membr Biol. doi: 10.1007/s00232-014-9684-6 CrossRefGoogle Scholar
  22. Koning RI, Keegstra W, Oostergetel GT, Schuurman-Wolters G, Robillard GT, Brisson A (1999) The 5 Å projection structure of the transmembrane domain of the mannitol transporter enzyme II. J Mol Biol 287:845–851CrossRefGoogle Scholar
  23. Le Bon C, Popot J-L, Giusti F (2014) Labeling and functionalizing amphipols for biological applications. J Membr Biol. doi: 10.1007/s00232-014-9655-y CrossRefGoogle Scholar
  24. le Maire M, Champeil P, Møller JV (2000) Interaction of membrane proteins and lipids with solubilizing detergents. Biochim Biophys Acta 1508:86–111CrossRefGoogle Scholar
  25. Legler PM, Cai ML, Peterkofsky A, Clore GM (2004) Three-dimensional solution structure of the cytoplasmic B domain of the mannitol transporter II(mtl) of the Escherichia coli phosphotransferase system. J Biol Chem 279:39115–39121CrossRefGoogle Scholar
  26. Liao M, Cao E, Julius D, Cheng Y (2013) Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 504:107–112CrossRefGoogle Scholar
  27. Liao M, Cao E, Julius D, Cheng Y (2014) Single particle electron cryo-microscopy of a mammalian ion channel. Curr Opin Struct Biol 27:1–7CrossRefGoogle Scholar
  28. Liu TQ, Callis PR, Hesp BH, de Groot M, Buma WJ, Broos J (2005) Ionization potentials of fluoroindoles and the origin of nonexponential tryptophan fluorescence decay in proteins. J Am Chem Soc 127:4104–4113CrossRefGoogle Scholar
  29. Lolkema JS, Kuiper H, ten Hoeve Duurkens RH, Robillard GT (1993) 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 32:1396–1400CrossRefGoogle Scholar
  30. Martinez KL, Gohon Y, Corringer P-J, Tribet C, Mérola F, Changeux J-P, Popot J-L (2002) Allosteric transitions of Torpedo acetylcholine receptor in lipids, detergent and amphipols: molecular interactions vs. physical constraints. FEBS Lett 528:251–256CrossRefGoogle Scholar
  31. Mary S, Damian M, Rahmeh R, Mouillac B, Marie J, Granier S, Banères J-L (2014) Amphipols in G protein-coupled receptor pharmacology: what are they good for? J Membr Biol. doi: 10.1007/s00232-014-9665-9 CrossRefGoogle Scholar
  32. Opačić M, Vos EPP, Hesp BH, Broos J (2010) Localization of the substrate binding site in the homodimeric mannitol transporter, EIImtl, of Escherichia coli. J Biol Chem 285:25324–25331CrossRefGoogle Scholar
  33. Opačić M, Hesp BH, Fusetti F, Dijkstra BW, Broos J (2012) Structural investigation of the transmembrane C domain of the mannitol permease from Escherichia coli using 5-FTrp fluorescence spectroscopy. Biochim Biophys Acta 1818:861–868CrossRefGoogle Scholar
  34. Perlmutter JD, Drasler WJ, Xie W, Gao J, Popot J-L, Sachs JN (2011) All-atom and coarse-grained molecular dynamics simulations of a membrane protein stabilizing poly-mer. Langmuir 27:10523–10537CrossRefGoogle Scholar
  35. Perlmutter JD, Popot J-L, Sachs JN (2014) Molecular dynamics simulations of a membrane protein/amphipol complex. J Membr Biol. doi: 10.1007/s00232-014-9690-8 CrossRefGoogle Scholar
  36. Picard M, Dahmane T, Garrigos M, Gauron C, Giusti F, le Maire M, Popot J-L, Champeil P (2006) Protective and inhibitory effects of various types of amphipols on the Ca2+-ATPase from sarcoplasmic reticulum: a comparative study. Biochemistry 45:1861–1869CrossRefGoogle Scholar
  37. Pocanschi C, Popot J-L, Kleinschmidt JH (2013) Folding and stability of outer membrane protein A (OmpA) from Escherichia coli in an amphipathic polymer, amphipol A8-35. Eur Biophys J 42:103–118CrossRefGoogle Scholar
  38. Popot J-L (2010) Amphipols, nanodiscs, and fluorinated surfactants: three non-conventional approaches to studying membrane proteins in aqueous solutions. Annu Rev Biochem 79:737–775CrossRefGoogle Scholar
  39. Popot J-L, Berry EA, Charvolin D, Creuzenet C, Ebel C, Engelman DM, Flötenmeyer M, Giusti F, Gohon Y, Hervé P, Hong Q, Lakey JH, Leonard K, Shuman HA, Timmins P, Warschawski DE, Zito F, Zoonens M, Pucci B, Tribet C (2003) Amphipols: polymeric surfactants for membrane biology research. Cell Mol Life Sci 60:1559–1574CrossRefGoogle Scholar
  40. Popot J-L, Althoff T, Bagnard D, Banères J-L, Bazzacco P, Billon-Denis E, Catoire LJ, Champeil P, Charvolin D, Cocco MJ, Crémel G, Dahmane T, de la Maza LM, Ebel C, Gabel F, Giusti F, Gohon Y, Goormaghtigh E, Guittet E, Kleinschmidt JH, Kühlbrandt W, Le Bon C, Martinez KL, Picard M, Pucci B, Rappaport F, Sachs JN, Tribet C, van Heijenoort C, Wien F, Zito F, Zoonens M (2011) Amphipols from A to Z. Annu Rev Biophys 40:379–408CrossRefGoogle Scholar
  41. Robillard GT, Blaauw M (1987) Enzyme II of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: protein-protein and protein-phospholipid interactions. Biochemistry 26:5796–5803CrossRefGoogle Scholar
  42. Robillard GT, Broos J (1999) Structure/function studies on the bacterial carbohydrate transporters, enzymes II, of the phosphoenolpyruvate-dependent phosphotransferase system. Biochim Biophys Acta 1422:73–104CrossRefGoogle Scholar
  43. Robillard GT, Boer H, van Weeghel RP, Wolters G, Dijkstra A (1993) 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 Escherichia coli. Biochemistry 32:9553–9562CrossRefGoogle Scholar
  44. Rosenbusch JP (2001) Stability of membrane proteins: relevance for the selection of appropriate methods for high-resolution structure determinations. J Struct Biol 136:144–157CrossRefGoogle Scholar
  45. Seybold PG, Gouterman M, Callis J (1969) Calorimetric, photometric and lifetime determinations of fluorescence yields of fluorescein dyes. Photochem Photobiol 9:229–242CrossRefGoogle Scholar
  46. Sugiyama JE, Mahmoodian S, Jacobson GR (1991) Membrane topology analysis of Escherichia coli mannitol permease by using a nested-deletion method to create mtlA-phoA fusions. Proc Natl Acad Sci USA 88:9603–9607CrossRefGoogle Scholar
  47. Tehei M, Perlmutter J, Giusti F, Sachs J, Zaccai G, Popot J-L (2014) Thermal fluctuations in amphipol A8-35 measured by neutron scattering. J Membr Biol (submitted)Google Scholar
  48. Tribet C, Audebert R, Popot J-L (1996) Amphipols: polymers that keep membrane proteins soluble in aqueous solutions. Proc Natl Acad Sci USA 93:15047–15050CrossRefGoogle Scholar
  49. Tribet C, Diab C, Dahmane T, Zoonens M, Popot J-L, Winnik FM (2009) Thermodynamic characterization of the exchange of detergents and amphipols at the surfaces of integral membrane proteins. Langmuir 25:12623–12634CrossRefGoogle Scholar
  50. Tsybovsky Y, Orban T, Molday RS, Taylor D, Palczewski K (2013) Molecular organization and ATP-induced conformational changes of ABCA4, the photoreceptor-specific ABC transporter. Structure 21:854–860CrossRefGoogle Scholar
  51. van Montfort RLM, Pijning T, Kalk KH, Hangyi I, Kouwijzer MLCE, Robillard GT, Dijkstra BW (1998) The structure of the Escherichia coli phosphotransferase IIA(mannitol) reveals a novel fold with two conformations of the active site. Structure 6:377–388CrossRefGoogle Scholar
  52. Veldhuis G (2006) Mechanism of the mannitol transporter from Escherichia coli—substrate probing and oligomeric structure. Ph.D. Thesis, University of Groningen, pp 77–89Google Scholar
  53. Veldhuis G, Broos J, Poolman B, Scheek RM (2005a) Stoichiometry and substrate affinity of the mannitol transporter, Enzymell(mtl), from Escherichia coli. Biophys J 89:201–210CrossRefGoogle Scholar
  54. Veldhuis G, Gabellieri E, Vos EPP, Poolman B, Strambini GB, Broos J (2005b) Substrate-induced conformational changes in the membrane-embedded IICmtl-domain of the mannitol permease from Escherichia coli, EnzymeII(mtl), probed by tryptophan phosphorescence spectroscopy. J Biol Chem 280:35148–35156CrossRefGoogle Scholar
  55. Vervoort EB, Bultema JB, Schuurman-Wolters GK, Geertsma ER, Broos J, Poolman B (2005) The first cytoplasmic loop of the mannitol permease from Escherichia coli is accessible for sulfhydryl reagents from the periplasmic side of the membrane. J Mol Biol 346:733–743CrossRefGoogle Scholar
  56. Vos EPP, Bokhove M, Hesp BH, Broos J (2009a) Structure of the cytoplasmic loop between putative helices II and III of the mannitol permease of Escherichia coli: a tryptophan and 5-fluorotryptophan spectroscopy study. Biochemistry 48:5284–5290CrossRefGoogle Scholar
  57. Vos EPP, ter Horst R, Poolman B, Broos J (2009b) Domain complementation studies reveal residues critical for the activity of the mannitol permease from Escherichia coli. Biochim Biophys Acta 1788:581–586CrossRefGoogle Scholar
  58. Zoonens M, Popot J-L (2014) Amphipols for each season. J Membr Biol. doi: 10.1007/s00232-014-9666-8 CrossRefGoogle Scholar
  59. Zoonens M, Catoire LJ, Giusti F, Popot J-L (2005) NMR study of a membrane protein in detergent-free aqueous solution. Proc Natl Acad Sci USA 102:8893–8898CrossRefGoogle Scholar
  60. Zoonens M, Giusti F, Zito F, Popot J-L (2007) Dynamics of membrane protein/amphipol association studied by Förster resonance energy transfer. Implications for in vitro studies of amphipol-stabilized membrane proteins. Biochemistry 46:10392–10404CrossRefGoogle Scholar
  61. Zoonens M, Zito F, Martinez KL, Popot J-L (2014) Amphipols: a general introduction and some protocols. In: Mus-Veteau I (ed) Membrane protein production for structural analysis. SpringerGoogle Scholar

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

Personalised recommendations