Cellular and Molecular Life Sciences

, Volume 71, Issue 24, pp 4895–4910 | Cite as

Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination

  • Coilín Boland
  • Dianfan Li
  • Syed Tasadaque Ali Shah
  • Stefan Haberstock
  • Volker Dötsch
  • Frank Bernhard
  • Martin Caffrey
Research Article

Abstract

Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a three-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high-quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipid mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28-Å resolution. The quality of cellular and cell-free-expressed kinase samples has been evaluated systematically by comparing (1) spectroscopic properties, (2) purity and oligomer formation, (3) lipid content and (4) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free-expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved.

Keywords

Cell-free Crystallisation In meso Lipid cubic phase Macromolecular crystallography Membrane protein structure 

Abbreviations

ADP

Adenosine diphosphate

ARII

Acetabularia acetabulum rhodopsin II

ATP

Adenosine triphosphate

C12E8

n-octaethylene glycol monododecyl ether

C8E4

n-octyl tetraethylene glycol monoether

CL

Cardiolipin

CTP

Cytidine triphosphate

CV

Column volume

DgkA

Diacylglycerol kinase

DDM

n-dodecyl-β-d-maltopyranoside

DM

n-decyl-β-d-maltopyranoside

DNA

Deoxyribonucleic acid

DTT

DL-dithiothreitol

EDTA

Ethylenediaminetetraacetic acid

EGTA

Ethyleneglycoltetraacetic acid

GFP

Green fluorescent protein

GTP

Guanosine triphosphate

HCl

Hydrochloric acid

HEPES

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HRP

Horseradish peroxidase

hVDAC1

Human voltage-dependent anion channel 1

IPTG

Isopropyl β-D-1-thiogalactopyranoside

kDa

Kilodalton

LCP

Lipid cubic phase

LDAO

Lauryldimethylamine N-oxide

MAG

Monoacylglycerol

MPD

2-methyl-2,4-pentanediol

MWCO

Molecular weight cut off

NADH

Nicotinamide adenine dinucleotide

OD

Optical density

PCR

Polymerase chain reaction

PE

Phosphatidylethanolamine

PEG

Polyethyleneglycol

PEP

Phosphenolpyruvic acid

PK

Pyruvate kinase

PG

Phosphatidylglycerol

PIPES

Piperazine-1,4-bis(2-ethanesulfonic acid

PMSF

Phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluoride

RMSD

Root-mean-square deviation

RNA

Ribonucleic acid

SEC

Size-exclusion chromatography

TCEP

tris (2-carboxyethyl) phosphine hydrochloride

TLC

Thin-layer chromatography

tRNA

Transfer ribonucleic acid

UTP

Uridine triphosphate

YPTG

Yeast phosphate tryptone glucose

References

  1. 1.
    Schwarz D, Junge F, Durst F, Frolich N, Schneider B, Reckel S, Sobhanifar S, Dötsch V, Bernhard F (2007) Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems. Nat Protoc 2:2945–2957PubMedCrossRefGoogle Scholar
  2. 2.
    Chen YJ, Pornillos O, Lieu S, Ma C, Chen AP, Chang G (2007) X-ray structure of EmrE supports dual topology model. Proc Natl Acad Sci USA 104:18999–19004PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Wada T, Shimono K, Kikukawa T, Hato M, Shinya N, Kim SY, Kimura-Someya T, Shirouzu M, Tamogami J, Miyauchi S, Jung KH, Kamo N, Yokoyama S (2011) Crystal structure of the eukaryotic light-driven proton-pumping rhodopsin, Acetabularia rhodopsin II, from marine alga. J Mol Biol 411:986–998PubMedCrossRefGoogle Scholar
  4. 4.
    Walsh JP, Bell RM (1986) sn-1,2-Diacylglycerol kinase of Escherichia coli. Structural and kinetic analysis of the lipid cofactor dependence. J Biol Chem 261:5062–5069Google Scholar
  5. 5.
    Raetz CRH, Newman KF (1979) Diglyceride kinase mutants of Escherichia coli: inner membrane association of 1,2-diglyceride and its relation to synthesis of membrane-derived oligosaccharides. J Bacteriol 137:860–868PubMedCentralPubMedGoogle Scholar
  6. 6.
    Badola P, Sanders CR (1997) Escherichia coli diacylglycerol kinase is an evolutionarily optimized membrane enzyme and catalyzes direct phosphoryl transfer. J Biol Chem 272:24176–24182PubMedCrossRefGoogle Scholar
  7. 7.
    Savage DF, Anderson CL, Robles-Colmenares Y, Newby ZE, Stroud RM (2007) Cell-free complements in vivo expression of the E. coli membrane proteome. Protein Sci 16:966–976PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Li D, Lyons J, Pye VE, Vogeley L, Aragão D, Kenyon CP, Shah ST, Doherty C, Aherne M, Caffrey M (2013) Crystal structure of the integral membrane diacylglycerol kinase. Nature 497:521–524PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Caffrey M, Lyons J, Smyth T, Hart DJ (2009) Current topics in membranes, DeLucas L (ed), Academic Press, Burlington, pp 83–108Google Scholar
  10. 10.
    Coleman BE, Cwynar V, Hart DJ, Havas F, Mohan JM, Patterson S, Ridenour S, Schmidt M, Smith E, Wells AJ (2004) Modular approach to the synthesis of unsaturated 1-monoacyl glycerols. Synlett 8:1339–1342Google Scholar
  11. 11.
    Li D, Caffrey M (2011) Lipid cubic phase as a membrane mimetic for integral membrane protein enzymes. Proc Natl Acad Sci USA 108:8639–8644PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Sanders CR, Czerski L, Vinogradova O, Badola P, Song D, Smith SO (1996) Escherichia coli diacylglycerol kinase is an alpha-helical polytopic membrane protein and can spontaneously insert into preformed lipid vesicles. Biochemistry 35:8610–8618PubMedCrossRefGoogle Scholar
  13. 13.
    Tanzer ML, Gilvarg C (1959) Creatine and creatine kinase measurement. J Biol Chem 234:3201–3204PubMedGoogle Scholar
  14. 14.
    Chen AH, Hummel B, Qiu H, Caffrey M (1998) A simple mechanical mixer for small viscous lipid-containing samples. Chem Phys Lipids 95:11–21CrossRefGoogle Scholar
  15. 15.
    Rouser G, Fleische S, Yamamoto A (1970) Two dimensional then layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 5:494–496PubMedCrossRefGoogle Scholar
  16. 16.
    Folch J, Lees M, Stanley GHS (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226:497–509PubMedGoogle Scholar
  17. 17.
    Louis-Jeune C, Andrade-Navarro MA, Perez-Iratxeta C (2012) Prediction of protein secondary structure from circular dichroism using theoretically derived spectra. Proteins 80:374–381PubMedCrossRefGoogle Scholar
  18. 18.
    Caffrey M, Porter C (2010) Crystallizing membrane proteins for structure determination using lipidic mesophases. J Vis Exp 45:e1712Google Scholar
  19. 19.
    Li D, Boland C, Walsh K, Caffrey M (2012) Use of a robot for high-throughput crystallization of membrane proteins in lipidic mesophases. J Vis Exp 67:e4000PubMedGoogle Scholar
  20. 20.
    Li D, Boland C, Aragao D, Walsh K, Caffrey M (2012) Harvesting and cryo-cooling crystals of membrane proteins grown in lipidic mesophases for structure determination by macromolecular crystallography. J Vis Exp 67:e4001PubMedGoogle Scholar
  21. 21.
    Winter G (2010) xia2: an expert system for macromolecular crystallography data reduction. J Appl Cryst 43:186–190CrossRefGoogle Scholar
  22. 22.
    Kabsch W (2010) XDS. Acta Crystallogr D66:125–132Google Scholar
  23. 23.
    Evans P (2006) Scaling and assessment of data quality. Acta Crystallogr D62:72–82Google Scholar
  24. 24.
    McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr 40:658–674PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D66:486–501Google Scholar
  26. 26.
    Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D66:213–221Google Scholar
  27. 27.
    Van Horn WD, Kim HJ, Ellis CD, Hadziselimovic A, Sulistijo ES, Karra MD, Tian CL, Sonnichsen FD, Sanders CR (2009) Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase. Science 324:1726–1729PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Bogdanov M, Dowham W (2002) Biochemistry of lipids, lipoproteins and membranes. Vance DE, Vance JE (eds) Elsevier, Amsterdam, pp 1–35Google Scholar
  29. 29.
    Berrier C, Guilvout I, Bayan N, Park KH, Mesneau A, Chami M, Pugsley AP, Ghazi A (2011) Coupled cell-free synthesis and lipid vesicle insertion of a functional oligomeric channel MscL MscL does not need the insertase YidC for insertion in vitro. Biochim Biophys Acta 1808:41–46PubMedCrossRefGoogle Scholar
  30. 30.
    Bratbak G, Dundas I (1984) Bacterial dry matter content and biomass estimations. Appl Environ Microbiol 48:755–757PubMedCentralPubMedGoogle Scholar
  31. 31.
    Neidhardt FC (1996) Escherichia coli and Salmonella: cellular and molecular biology. Neidhardt FC (ed) ASM Press, Washington, pp 1–62Google Scholar
  32. 32.
    Pornillos O, Chen YJ, Chen AP, Chang G (2005) X-ray structure of the EmrE multidrug transporter in complex with a substrate. Science 310:1950–1953PubMedCrossRefGoogle Scholar
  33. 33.
    Li D, Shah ST, Caffrey M (2013) Host lipid and temperature as important screening variables for crystallizing integral membrane proteins in lipidic mesophases. Trials with diacylglycerol kinase. Cryst Growth Des 13:2846–2857Google Scholar
  34. 34.
    Blesneac I, Ravaud S, Juillan-Binard C, Barret LA, Zoonens M, Polidori A, Miroux B, Pucci B, Pebay-Peyroula E (2012) Production of UCP1 a membrane protein from the inner mitochondrial membrane using the cell free expression system in the presence of a fluorinated surfactant. Biochim Biophys Acta 1818:798–805PubMedCrossRefGoogle Scholar
  35. 35.
    Roos C, Kai L, Proverbio D, Ghoshdastider U, Filipek S, Dötsch V, Bernhard F (2013) Co-translational association of cell-free expressed membrane proteins with supplied lipid bilayers. Mol Membr Biol 30:75–89PubMedCrossRefGoogle Scholar
  36. 36.
    Roos C, Zocher M, Muller D, Munch D, Schneider T, Sahl HG, Scholz F, Wachtveitl J, Ma Y, Proverbio D, Henrich E, Dötsch V, Bernhard F (2012) Characterization of co-translationally formed nanodisc complexes with small multidrug transporters, proteorhodopsin and with the E. coli MraY translocase. Biochim Biophys Acta 1818:3098–3106PubMedCrossRefGoogle Scholar
  37. 37.
    Walden H (2010) Selenium incorporation using recombinant techniques. Acta Crystallogr D66:352–357Google Scholar
  38. 38.
    Ogara M, Adams GM, Gong WM, Kobayashi R, Blumenthal RM, Cheng XD (1997) Expression, purification, mass spectrometry, crystallization and multiwavelength anomalous diffraction of selenomethionyl PvuII DNA methyltransferase (cytosine-N4-specific). Eur J Biochem 247:1009–1018CrossRefGoogle Scholar
  39. 39.
    Suchanek M, Radzikowska A, Thiele C (2005) Photo-leucine and photo-methionine allow identification of protein–protein interactions in living cells. Nat Methods 2:261–267PubMedCrossRefGoogle Scholar
  40. 40.
    Gubbens J, Kim SJ, Yang ZY, Johnson AE, Skach WR (2010) In vitro incorporation of nonnatural amino acids into protein using tRNA(Cys)-derived opal, ochre, and amber suppressor tRNAs. RNA 16:1660–1672PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Goto Y, Katoh T, Suga H (2011) Flexizymes for genetic code reprogramming. Nature Prot 6:779–790CrossRefGoogle Scholar
  42. 42.
    Verardi R, Traaseth NJ, Masterson LR, Vostrikov VV, Veglia G (2012) Isotope labeling for solution and solid-state NMR spectroscopy of membrane proteins. Adv Exp Med Biol 992:35–62Google Scholar
  43. 43.
    Bayrhuber M, Meins T, Habeck M, Becker S, Giller K, Villinger S, Vonrhein C, Griesinger C, Zweckstetter M, Zeth K (2008) Structure of the human voltage-dependent anion channel. Proc Natl Acad Sci USA 105:15370–15375PubMedCentralPubMedCrossRefGoogle Scholar
  44. 44.
    Hiller S, Garces RG, Malia TJ, Orekhov VY, Colombini M, Wagner G (2008) Solution structure of the integral human membrane protein VDAC-1 in detergent micelles. Science 321:1206–1210PubMedCentralPubMedCrossRefGoogle Scholar
  45. 45.
    Deniaud A, Liguori L, Blesneac I, Lenormand JL, Pebay-Peyroula E (2010) Crystallization of the membrane protein hVDAC1 produced in cell-free system. Biochim Biophys Acta 1798:1540–1546PubMedCrossRefGoogle Scholar
  46. 46.
    Rajesh S, Knowles T, Overduin M (2011) Production of membrane proteins without cells or detergents. New Biotechnol 28:250–254CrossRefGoogle Scholar
  47. 47.
    Uhlemann EME, Pierson HE, Fillingame RH, Dmitriev OY (2012) Cell-free synthesis of membrane subunits of ATP synthase in phospholipid bicelles: NMR shows subunit a fold similar to the protein in the cell membrane. Prot Sci 21:279–288CrossRefGoogle Scholar
  48. 48.
    Botting CH, Randall RE (1995) Reporter enzyme-nitrilotriacetic acid-nickel conjugates: reagents for detecting histidine-tagged proteins. Biotechniques 19:362–363PubMedGoogle Scholar
  49. 49.
    Lomize MA, Pogozheva ID, Joo H, Mosberg HI, Lomize AL (2012) OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic Acids Res 40:D370–D376PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Basel 2014

Authors and Affiliations

  • Coilín Boland
    • 1
  • Dianfan Li
    • 1
  • Syed Tasadaque Ali Shah
    • 1
  • Stefan Haberstock
    • 2
  • Volker Dötsch
    • 2
  • Frank Bernhard
    • 2
  • Martin Caffrey
    • 1
  1. 1.Membrane Structural and Functional Group, School of Medicine and School of Biochemistry and ImmunologyTrinity College DublinDublinIreland
  2. 2.Centre of Biomolecular Magnetic Resonance, Institute of Biophysical ChemistryJohann Wolfgang Goethe-University of FrankfurtFrankfurtGermany

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