Skip to main content
SpringerLink
Log in

Folding and stability of outer membrane protein A (OmpA) from Escherichia coli in an amphipathic polymer, amphipol A8-35

  • ORIGINAL PAPER
  • Published:
European Biophysics Journal Aims and scope Submit manuscript

Abstract

Amphipols are a class of amphipathic polymers designed to maintain membrane proteins in aqueous solutions in the absence of detergents. Denatured β-barrel membrane proteins, like outer membrane proteins OmpA from Escherichia coli and FomA from Fusobacterium nucleatum, can be folded by dilution of the denaturant urea in the presence of amphipol A8-35. Here, the folding kinetics and stability of OmpA in A8-35 have been investigated. Folding is well described by two parallel first-order processes, whose half-times, ~5 and ~70 min, respectively, are independent of A8-35 concentration. The faster process contributed ~55–64 % to OmpA folding. Folding into A8-35 was faster than into dioleoylphosphatidylcholine bilayers and complete at ratios as low as ~0.17 g/g A8-35/OmpA, corresponding to ~1–2 A8-35 molecules per OmpA. Activation energies were determined from the temperature dependence of folding kinetics, monitored both by electrophoresis, which reports on the formation of stable OmpA tertiary structure, and by fluorescence spectroscopy, which reflects changes in the environment of tryptophan side chains. The two methods yielded consistent estimates, namely ~5–9 kJ/mol for the fast process and ~29–37 kJ/mol for the slow one, which is lower than is observed for OmpA folding into dioleoylphosphatidylcholine bilayers. Folding and unfolding titrations with urea demonstrated that OmpA folding into A8-35 is reversible and that amphipol-refolded OmpA is thermodynamically stable at room temperature. Comparison of activation energies for folding and unfolding in A8-35 versus detergent indicates that stabilization of A8-35-trapped OmpA against denaturation by urea is a kinetic, not a thermodynamic phenomenon.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Notes

  1. The molecular mass of A8-35 was previously reported to be 8–10 kDa, depending on the source of polyacrylic acid. This estimate was based on size exclusion chromatography analyses in aqueous solvent using polyethyleneglycol polymers as standards of molecular mass (Gohon et al. 2006; Tribet et al. 1996). A recent reexamination of this issue, using absolute calibration, has yielded the revised number-averaged mass 〈M n〉 ≈ 4.3 kDa (Giusti and Rieger, personal communication). This value will be used in the present article. This revision does not affect any of the conclusions that have been published thus far regarding the mass, composition and properties of A8-35 particles and TMP/A8-35 complexes.

Abbreviations

A8-35:

A specific type of amphipol

APol:

Amphipol

Borate:

Sodium tetraborate-10-hydrate

diC10PC:

1,2-Dicapryl-sn-glycero-3-phosphocholine

diC12PC:

1,2-Dilauroyl-sn-glycero-3-phosphocholine

diC12PG:

1,2-Dilauroyl-sn-glycero-3-phosphoglycerol

DOPC:

1,2-Dioleoyl-sn-glycero-3-phosphocholine

EDTA:

Ethylenediaminetetraacetic acid

FomA:

Major outer membrane protein from Fusobacterium nucleatum

KTSE:

Kinetics of tertiary structure formation determined by electrophoresis

LDAO:

N-Lauryl-N,N-dimethylammonium-N-oxide

MOMP:

Major outer membrane protein from Chlamydia trachomatis

OMP:

Outer membrane protein

OmpA:

Outer membrane protein A from Escherichia coli

PAGE:

Polyacrylamide gel electrophoresis

SDS:

Sodium dodecyl sulfate

TMP:

Transmembrane protein

Tris:

Tris-(hydroxymethyl)-aminomethane

References

  • Althoff T, Mills DJ, Popot JL, Kühlbrandt W (2011) Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1. EMBO J 30:4652–4664

    Article  CAS  PubMed  Google Scholar 

  • Andersen KK, Wang H, Otzen DE (2012) A kinetic analysis of the folding and unfolding of OmpA in urea and guanidinium chloride: single and parallel pathways. Biochemistry 51:8371–8383

    Article  CAS  PubMed  Google Scholar 

  • Arora A, Rinehart D, Szabo G, Tamm LK (2000) Refolded outer membrane protein A of Escherichia coli forms ion channels with two conductance states in planar lipid bilayers. J Biol Chem 275:1594–1600

    Article  CAS  PubMed  Google Scholar 

  • Banères JL, Popot JL, Mouillac B (2011) New advances in production and functional folding of G-protein-coupled receptors. Trends Biotechnol 29:314–322

    Article  PubMed  Google Scholar 

  • Bazzacco P, Billon-Denis E, Sharma KS, Catoire LJ, Mary S, Le Bon C, Point E, Banères JL, Durand G, Zito F, Pucci B, Popot JL (2012) Nonionic homopolymeric amphipols: application to membrane protein folding, cell-free synthesis, and solution nuclear magnetic resonance. Biochemistry 51:1416–1430

    Article  CAS  PubMed  Google Scholar 

  • Buchanan SK (1999) β-barrel proteins from bacterial outer membranes: structure, function and refolding. Curr Opin Struct Biol 9:455–461

    Article  CAS  PubMed  Google Scholar 

  • Bulieris PV, Behrens S, Holst O, Kleinschmidt JH (2003) Folding and insertion of the outer membrane protein OmpA is assisted by the chaperone Skp and by lipopolysaccharide. J Biol Chem 278:9092–9099

    Article  CAS  PubMed  Google Scholar 

  • Catoire LJ, Zoonens M, van Heijenoort C, Giusti F, Popot JL, Guittet E (2009) Inter- and intramolecular contacts in a membrane protein/surfactant complex observed by heteronuclear dipole-to-dipole cross-relaxation. J Magn Reson 197:91–95

    Article  CAS  PubMed  Google Scholar 

  • Catoire LJ, Zoonens M, van Heijenoort C, Giusti F, Guittet E, Popot JL (2010) Solution NMR mapping of water-accessible residues in the transmembrane β-barrel of OmpX. Eur Biophys J 39:623–630

    Article  CAS  PubMed  Google Scholar 

  • Champeil P, Menguy T, Tribet C, Popot JL, le Maire M (2000) Interaction of amphipols with sarcoplasmic reticulum Ca2+-ATPase. J Biol Chem 275:18623–18637

    Article  CAS  PubMed  Google Scholar 

  • Dahmane T, Damian M, Mary S, Popot JL, Banères JL (2009) Amphipol-assisted in vitro folding of G protein-coupled receptors. Biochemistry 48:6516–6521

    Article  CAS  PubMed  Google Scholar 

  • Dahmane T, Giusti F, Catoire LJ, Popot JL (2011) Sulfonated amphipols: synthesis, properties, and applications. Biopolymers 95:811–823

    Article  CAS  PubMed  Google Scholar 

  • Dahmane T, Rappaport F, Popot JL (2013) Amphipol-assisted folding of bacteriorhodopsin in the presence or absence of lipids: functional consequences. Eur Biophys J. doi:10.1007/s00249-012-0839-z

  • Dornmair K, Kiefer H, Jähnig F (1990) Refolding of an integral membrane protein. OmpA of Escherichia coli. J Biol Chem 265:18907–18911

    CAS  PubMed  Google Scholar 

  • Gianni S, Travaglini-Allocatelli C, Cutruzzola F, Brunori M, Shastry MC, Roder H (2003) Parallel pathways in cytochrome c 551 folding. J Mol Biol 330:1145–1152

    Article  CAS  PubMed  Google Scholar 

  • Giusti F, Popot JL, Tribet C (2012) Well-defined critical association concentration and rapid adsorption at the air/water interface of a short amphiphilic polymer, amphipol A8-35: a study by Förster resonance energy transfer and dynamic surface tension measurements. Langmuir 28:10372–10380

    Article  CAS  PubMed  Google Scholar 

  • 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 334:318–334

    Article  CAS  PubMed  Google Scholar 

  • Gohon Y, Giusti F, Prata C, Charvolin D, Timmins P, Ebel C, Tribet C, Popot JL (2006) Well-defined nanoparticles formed by hydrophobic assembly of a short and polydisperse random terpolymer, amphipol A8-35. Langmuir 22:1281–1290

    Article  CAS  PubMed  Google Scholar 

  • He S, Wang B, Chen H, Tang C, Feng Y (2012) Preparation and antimicrobial properties of gemini surfactant-supported triiodide complex system. ACS Appl Mater Interfaces 4:2116–2123

    Article  CAS  PubMed  Google Scholar 

  • Hong H, Tamm LK (2004) Elastic coupling of integral membrane protein stability to lipid bilayer forces. Proc Natl Acad Sci USA 101:4065–4070

    Article  CAS  PubMed  Google Scholar 

  • Kiefer H (2003) In vitro folding of α-helical membrane proteins. Biochim Biophys Acta 1610:57–62

    Article  CAS  PubMed  Google Scholar 

  • Kleinschmidt JH (2003) Membrane protein folding on the example of outer membrane protein A of Escherichia coli. Cell Mol Life Sci 60:1547–1558

    Article  CAS  PubMed  Google Scholar 

  • Kleinschmidt JH (2006) Folding kinetics of the outer membrane proteins OmpA and FomA into phospholipid bilayers. Chem Phys Lipids 141:30–47

    Article  CAS  PubMed  Google Scholar 

  • Kleinschmidt JH, Tamm LK (1996) Folding intermediates of a β-barrel membrane protein. Kinetic evidence for a multi-step membrane insertion mechanism. Biochemistry 35:12993–13000

    Article  CAS  PubMed  Google Scholar 

  • Kleinschmidt JH, Tamm LK (1999) Time-resolved distance determination by tryptophan fluorescence quenching: probing intermediates in membrane protein folding. Biochemistry 38:4996–5005

    Article  CAS  PubMed  Google Scholar 

  • Kleinschmidt JH, Tamm LK (2002) Secondary and tertiary structure formation of the β-barrel membrane protein OmpA is synchronized and depends on membrane thickness. J Mol Biol 324:319–330

    Article  CAS  PubMed  Google Scholar 

  • Kleinschmidt JH, den Blaauwen T, Driessen A, Tamm LK (1999a) Outer membrane protein A of E. coli inserts and folds into lipid bilayers by a concerted mechanism. Biochemistry 38:5006–5016

    Article  CAS  PubMed  Google Scholar 

  • Kleinschmidt JH, Wiener MC, Tamm LK (1999b) Outer membrane protein A of E. coli folds into detergent micelles, but not in the presence of monomeric detergent. Protein Sci 8:2065–2071

    Article  CAS  PubMed  Google Scholar 

  • Kleinschmidt JH, Bulieris PV, Qu J, Dogterom M, den Blaauwen T (2011) Association of neighboring β–strands of outer membrane protein A in lipid bilayers revealed by site directed fluorescence quenching. J Mol Biol 407:316–332

    Article  CAS  PubMed  Google Scholar 

  • Knowles TJ, Scott-Tucker A, Overduin M, Henderson IR (2009) Membrane protein architects: the role of the BAM complex in outer membrane protein assembly. Nat Rev Microbiol 7:206–214

    Article  CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Leney AC, McMorran LM, Radford SE, Ashcroft AE (2012) Amphipathic polymers enable the study of functional membrane proteins in the gas phase. Anal Chem 84:9841–9847

    Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    CAS  PubMed  Google Scholar 

  • Marsh D, Shanmugavadivu B, Kleinschmidt JH (2006) Membrane elastic fluctuations and the insertion and tilt of β-barrel proteins. Biophys J 91:227–232

    Article  CAS  PubMed  Google Scholar 

  • Pace CN (1990) Measuring and increasing protein stability. Trends Biotechnol 8:93–98

    Article  CAS  PubMed  Google Scholar 

  • Pace CN, Shaw KL (2000) Linear extrapolation method of analyzing solvent denaturation curves. Proteins Suppl 4:1–7

    Google Scholar 

  • Pace CN, Hebert EJ, Shaw KL, Schell D, Both V, Krajcikova D, Sevcik J, Wilson KS, Dauter Z, Hartley RW, Grimsley GR (1998) Conformational stability and thermodynamics of folding of ribonucleases Sa, Sa2 and Sa3. J Mol Biol 279:271–286

    Article  CAS  PubMed  Google Scholar 

  • Patel GJ, Behrens-Kneip S, Holst O, Kleinschmidt JH (2009) The periplasmic chaperone Skp facilitates targeting, insertion and folding of OmpA into lipid membranes with a negative membrane surface potential. Biochemistry 48:10235–10245

    Article  CAS  PubMed  Google Scholar 

  • Pautsch A, Schulz GE (1998) Structure of the outer membrane protein A transmembrane domain. Nat Struct Biol 5:1013–1017

    Article  CAS  PubMed  Google Scholar 

  • Perlmutter JD, Drasler WJ 2nd, Xie W, Gao J, Popot JL, Sachs JN (2011) All-atom and coarse-grained molecular dynamics simulations of a membrane protein stabilizing polymer. Langmuir 27:10523–10537

    Article  CAS  PubMed  Google Scholar 

  • Picard M, Dahmane T, Garrigos M, Gauron C, Giusti F, le Maire M, Popot JL, Champeil P (2006) Protective and inhibitory effects of various types of amphipols on the Ca2+-ATPase from sarcoplasmic reticulum: a comparative study. Biochemistry 45:1861–1869

    Article  CAS  PubMed  Google Scholar 

  • Pocanschi CL, Apell H-J, Puntervoll P, Høgh BT, Jensen HB, Welte W, Kleinschmidt JH (2006a) The major outer membrane protein of Fusobacterium nucleatum (FomA) folds and inserts into lipid bilayers via parallel folding pathways. J Mol Biol 355:548–561

    Article  CAS  PubMed  Google Scholar 

  • Pocanschi CL, Dahmane T, Gohon Y, Rappaport F, Apell H-J, Kleinschmidt JH, Popot J-L (2006b) Amphipathic polymers: tools to fold integral membrane proteins to their active form. Biochemistry 45:13954–13961

    Article  CAS  PubMed  Google Scholar 

  • Pocanschi CL, Patel GJ, Marsh D, Kleinschmidt JH (2006c) Curvature elasticity and refolding of OmpA in large unilamellar vesicles. Biophys J 91:L75–L78

    Article  CAS  PubMed  Google Scholar 

  • Popot JL (2010) Amphipols, nanodiscs, and fluorinated surfactants: three nonconventional approaches to studying membrane proteins in aqueous solutions. Annu Rev Biochem 79:737–775

    Article  CAS  PubMed  Google Scholar 

  • Popot JL, Engelman DM (2000) Helical membrane protein folding, stability, and evolution. Annu Rev Biochem 69:881–922

    Article  CAS  PubMed  Google Scholar 

  • Popot JL, Berry EA, Charvolin D, Creuzenet C, Ebel C, Engelman DM, Flötenmeyer M, Giusti F, Gohon Y, 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 60:1559–1574

    Article  CAS  PubMed  Google Scholar 

  • Popot JL, Althoff T, Bagnard D, Banerès JL, Bazzacco P, Billon-Denis E, Catoire LJ, Champeil P, Charvolin D, Cocco MJ, Cremel 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, Sachs JN, Tribet C, van Heijenoort C, Wien F, Zito F, Zoonens M (2011) Amphipols from A to Z. Annu Rev Biophys 40:379–408

    Article  CAS  PubMed  Google Scholar 

  • Qu J, Mayer C, Behrens S, Holst O, Kleinschmidt JH (2007) The trimeric periplasmic chaperone Skp of Escherichia coli forms 1:1 complexes with outer membrane proteins via hydrophobic and electrostatic interactions. J Mol Biol 374:91–105

    Article  CAS  PubMed  Google Scholar 

  • Rodionova NA, Tatulian SA, Surrey T, Jähnig F, Tamm LK (1995) Characterization of two membrane-bound forms of OmpA. Biochemistry 34:1921–1929

    Article  CAS  PubMed  Google Scholar 

  • Roumestand C, Boyer M, Guignard L, Barthe P, Royer CA (2001) Characterization of the folding and unfolding reactions of a small β-barrel protein of novel topology, the MTCP1 oncogene product P13. J Mol Biol 312:247–259

    Article  CAS  PubMed  Google Scholar 

  • Royer CA, Mann CJ, Matthews CR (1993) Resolution of the fluorescence equilibrium unfolding profile of trp aporepressor using single tryptophan mutants. Protein Sci 2:1844–1852

    Article  CAS  PubMed  Google Scholar 

  • Sanders CR, Kuhn Hoffmann A, Gray DN, Keyes MH, Ellis CD (2004) French swimwear for membrane proteins. Chembiochem 5:423–426

    Article  CAS  PubMed  Google Scholar 

  • Schweizer M, Hindennach I, Garten W, Henning U (1978) Major proteins of the Escherichia coli outer cell envelope membrane. Interaction of protein II with lipopolysaccharide. Eur J Biochem 82:211–217

    Article  CAS  PubMed  Google Scholar 

  • Sharma KS, Durand G, Gabel F, Bazzacco P, Le Bon C, Billon-Denis E, Catoire LJ, Popot JL, Ebel C, Pucci B (2012) Non-ionic amphiphilic homopolymers: synthesis, solution properties, and biochemical validation. Langmuir 28:4625–4639

    Article  CAS  PubMed  Google Scholar 

  • Sugawara E, Steiert M, Rouhani S, Nikaido H (1996) Secondary structure of the outer membrane proteins OmpA of Escherichia coli and OprF of Pseudomonas aeruginosa. J Bacteriol 178:6067–6069

    CAS  PubMed  Google Scholar 

  • Surrey T, Jähnig F (1992) Refolding and oriented insertion of a membrane protein into a lipid bilayer. Proc Natl Acad Sci USA 89:7457–7461

    Article  CAS  PubMed  Google Scholar 

  • Surrey T, Jähnig F (1995) Kinetics of folding and membrane insertion of a β-barrel membrane protein. J Biol Chem 270:28199–28203

    Article  CAS  PubMed  Google Scholar 

  • Tifrea DF, Sun G, Pal S, Zardeneta G, Cocco MJ, Popot JL, de la Maza LM (2011) Amphipols stabilize the Chlamydia major outer membrane protein and enhance its protective ability as a vaccine. Vaccine 29:4623–4631

    Article  CAS  PubMed  Google Scholar 

  • Tribet C, Audebert R, Popot JL (1996) Amphipols: polymers that keep membrane proteins soluble in aqueous solutions. Proc Natl Acad Sci USA 93:15047–15050

    Article  CAS  PubMed  Google Scholar 

  • van Holde KE, Curtis Johnson W, Shing Ho P (2006) Principles of physical biochemistry, 2nd edn. Pearson Prentice Hall, London

    Google Scholar 

  • Vogel H, Jähnig F (1986) Models for the structure of outer-membrane proteins of Escherichia coli derived from Raman spectroscopy and prediction methods. J Mol Biol 190:191–199

    Article  CAS  PubMed  Google Scholar 

  • Walser R, Kleinschmidt JH, Zerbe O (2011) A chimeric GPCR model mimicking the ligand binding site of the human Y1 receptor studied by NMR spectroscopy. Chembiochem 12:1690–1693

    Article  CAS  PubMed  Google Scholar 

  • Walser R, Kleinschmidt JH, Skerra A, Zerbe O (2012) β-Barrel scaffolds for the grafting of extracellular loops from G protein-coupled receptors. Biol Chem 393:1341–1355

    Article  CAS  PubMed  Google Scholar 

  • Weber K, Osborne M (1964) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412

    Google Scholar 

  • Zoonens M, Giusti F, Zito F, Popot JL (2007) Dynamics of membrane protein/amphipol association studied by Förster resonance energy transfer: implications for in vitro studies of amphipol-stabilized membrane proteins. Biochemistry 46:10392–10404

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Particular thanks are due to Fabrice Giusti (CNRS UMR 7099, Paris) for synthesizing the batches of A8-35 used in the present work, as well as for information about the revised estimate of its average molecular mass. This work was supported by grants KL 1024/2-5 and 2-6 from the Deutsche Forschungsgemeinschaft to J.H.K.

Author information

Authors and Affiliations

  1. Fachbereich Biologie, Universität Konstanz, 78457, Konstanz, Germany

    Cosmin L. Pocanschi & Jörg H. Kleinschmidt

  2. Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON, M5S 3H2, Canada

    Cosmin L. Pocanschi

  3. UMR 7099, Centre National de la Recherche Scientifique and Université Paris-7, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005, Paris, France

    Jean-Luc Popot

  4. Abteilung Biophysik, Institut für Biologie, Universität Kassel, Heinrich-Plett-Straße 40, 34132, Kassel, Germany

    Jörg H. Kleinschmidt

Authors
  1. Cosmin L. Pocanschi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jean-Luc Popot
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jörg H. Kleinschmidt
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jörg H. Kleinschmidt.

Additional information

Special issue: Structure, function, folding and assembly of membrane proteins—insight from Biophysics.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pocanschi, C.L., Popot, JL. & Kleinschmidt, J.H. Folding and stability of outer membrane protein A (OmpA) from Escherichia coli in an amphipathic polymer, amphipol A8-35. Eur Biophys J 42, 103–118 (2013). https://doi.org/10.1007/s00249-013-0887-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00249-013-0887-z

Keywords

Search

Navigation