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Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment

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Abstract

Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive. One of the viable alternatives, successfully used for producing proteins for solution NMR studies, is yeast expression systems, particularly Pichia pastoris. We report on successful implementation and optimization of isotope labeling protocols, previously used for soluble secreted proteins, to produce homogeneous samples of a eukaryotic seven-transmembrane helical protein, rhodopsin from Leptosphaeria maculans. Even in shake-flask cultures, yields exceeded 5 mg of purified uniformly 13C,15N-labeled protein per liter of culture. The protein was stable (at least several weeks at 5°C) and functionally active upon reconstitution into lipid membranes at high protein-to-lipid ratio required for solid-state NMR. The samples gave high-resolution 13C and 15N solid-state magic angle spinning NMR spectra, amenable to a detailed structural analysis. We believe that similar protocols can be adopted for challenging mammalian targets, which often resist characterization by other structural methods.

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Abbreviations

7TM:

Seven-transmembrane

ASR:

Anabaena sensory rhodopsin

BR:

Bacteriorhodopsin

BMD:

Buffered minimal dextrose

BMM:

Buffered minimal methanol

CHES:

N-Cyclohexyl-2-aminoethanesulfonic acid

DMPC:

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

DMPA:

1,2-dimyristoyl-sn-glycero-3-phosphate

DTT:

Dithiothreitol

E. coli :

Escherichia coli

EDTA:

Ethylenediaminetetraacetic acid

FTIR:

Fourier-transform infrared

GPCRs:

G-protein coupled receptors

LR:

Leptosphaeria rhodopsin

MALDI TOF:

Matrix-assisted laser desorption/ionization time-of-flight

MAS:

Magic angle spinning

NR:

Neurospora rhodopsin

P. pastoris :

Pichia pastoris

PMSF:

Phenylmethylsulfonyl fluoride

PR:

Proteorhodopsin

SDS–PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SR-II:

Sensory rhodopsin II

ssNMR:

Solid-state NMR

YPD:

Yeast peptone dextrose

References

  • Abdulaev NG, Popp MP, Smith WC, Ridge KD (1997) Functional expression of bovine opsin in the methylotrophic yeast Pichia pastoris. Protein Expr Purif 10:61–69

    Article  Google Scholar 

  • Ahuja S, Hornak V, Yan EC, Syrett N, Goncalves JA, Hirshfeld A, Ziliox M, Sakmar TP, Sheves M, Reeves PJ, Smith SO, Eilers M (2009) Helix movement is coupled to displacement of the second extracellular loop in rhodopsin activation. Nat Struct Mol Biol 16:168–175

    Article  Google Scholar 

  • Aller SG, Yu J, Ward A, Weng Y, Chittaboina S, Zhuo R, Harrell PM, Trinh YT, Zhang Q, Urbatsch IL, Chang G (2009) Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding. Science 323:1718–1722

    Article  ADS  Google Scholar 

  • Andre N, Cherouati N, Prual C, Steffan T, Zeder-Lutz G, Magnin T, Pattus F, Michel H, Wagner R, Reinhart C (2006) Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen. Protein Sci 15:1115–1126

    Article  Google Scholar 

  • Berger C, Ho JT, Kimura T, Hess S, Gawrisch K, Yeliseev A (2010) Preparation of stable isotope-labeled peripheral cannabinoid receptor CB2 by bacterial fermentation. Protein Expr Purif 70:236–247

    Article  Google Scholar 

  • Bieszke JA, Spudich EN, Scott KL, Borkovich KA, Spudich JL (1999) A eukaryotic protein, NOP-1, binds retinal to form an archaeal rhodopsin-like photochemically reactive pigment. Biochemistry 38:14138–14145

    Article  Google Scholar 

  • Bokoch MP, Zou Y, Rasmussen SG, Liu CW, Nygaard R, Rosenbaum DM, Fung JJ, Choi HJ, Thian FS, Kobilka TS, Puglisi JD, Weis WI, Pardo L, Prosser RS, Mueller L, Kobilka BK (2010) Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor. Nature 463:108–112

    Article  ADS  Google Scholar 

  • Buensanteai N, Mukherjee PK, Horwitz BA, Cheng C, Dangott LJ, Kenerley CM (2010) Expression and purification of biologically active Trichoderma virens proteinaceous elicitor Sm1 in Pichia pastoris. Protein Expr Purif 72:131–138

    Article  Google Scholar 

  • Choi BK, Bobrowicz P, Davidson RC, Hamilton SR, Kung DH, Li H, Miele RG, Nett JH, Wildt S, Gerngross TU (2003) Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc Natl Acad Sci USA 100:5022–5027

    Article  ADS  Google Scholar 

  • Chow BY, Han X, Dobry AS, Qian X, Chuong AS, Li M, Henninger MA, Belfort GM, Lin Y, Monahan PE, Boyden ES (2010) High-performance genetically targetable optical neural silencing by light-driven proton pumps. Nature 463:98–102

    Article  ADS  Google Scholar 

  • de Jong LA, Grunewald S, Franke JP, Uges DR, Bischoff R (2004) Purification and characterization of the recombinant human dopamine D2S receptor from Pichia pastoris. Protein Expr Purif 33:176–184

    Article  Google Scholar 

  • Egorova-Zachernyuk TA, Bosman GJ, Pistorius AM, DeGrip WJ (2009) Production of yeastolates for uniform stable isotope labelling in eukaryotic cell culture. Appl Microbiol Biotechnol 84:575–581

    Article  Google Scholar 

  • Egorova-Zachernyuk TA, Bosman GJ, Degrip WJ (2011) Uniform stable-isotope labeling in mammalian cells: formulation of a cost-effective culture medium. Appl Microbiol Biotechnol 85:397–406

    Google Scholar 

  • Etzkorn M, Martell S, Andronesi OC, Seidel K, Engelhard M, Baldus M (2007) Secondary structure, dynamics, and topology of a seven-helix receptor in native membranes, studied by solid-state NMR spectroscopy. Angew Chem Int Ed Engl 46:459–462

    Article  Google Scholar 

  • Fan Y, Shi L, Brown LS (2007) Structural basis of diversification of fungal retinal proteins probed by site-directed mutagenesis of Leptosphaeria rhodopsin. FEBS Lett 581:2557–2561

    Article  Google Scholar 

  • Furutani Y, Bezerra AG, Waschuk SA, Sumii M, Brown LS, Kandori H (2004) FTIR spectroscopy of the K photointermediate of Neurospora rhodopsin: structural changes of the retinal, protein, and water molecules after photoisomerization. Biochemistry 43:9636–9646

    Article  Google Scholar 

  • Furutani Y, Sumii M, Fan Y, Shi LC, Waschuk SA, Brown LS, Kandori H (2006) Conformational coupling between the cytoplasmic carboxylic acid and the retinal in a fungal light-driven proton pump. Biochemistry 45:15349–15358

    Article  Google Scholar 

  • Gautier A, Mott HR, Bostock MJ, Kirkpatrick JP, Nietlispach D (2010) Structure determination of the seven-helix transmembrane receptor sensory rhodopsin II by solution NMR spectroscopy. Nat Struct Mol Biol 17:768–774

    Article  Google Scholar 

  • Goncalves JA, Ahuja S, Erfani S, Eilers M, Smith SO (2010) Structure and function of G protein-coupled receptors using NMR spectroscopy. Prog Nucl Magn Reson Spectrosc 57:159–180

    Article  Google Scholar 

  • Gourdon P, Alfredsson A, Pedersen A, Malmerberg E, Nyblom M, Widell M, Berntsson R, Pinhassi J, Braiman M, Hansson O, Bonander N, Karlsson G, Neutze R (2008) Optimized in vitro and in vivo expression of proteorhodopsin: a seven-transmembrane proton pump. Protein Expr Purif 58:103–113

    Article  Google Scholar 

  • Grisshammer R (2009) Purification of recombinant G-protein-coupled receptors. Methods Enzymol 463:631–645

    Article  Google Scholar 

  • Hanson MA, Stevens RC (2009) Discovery of new GPCR biology: one receptor structure at a time. Structure 17:8–14

    Article  Google Scholar 

  • Ho JD, Yeh R, Sandstrom A, Chorny I, Harries WE, Robbins RA, Miercke LJ, Stroud RM (2009) Crystal structure of human aquaporin 4 at 1.8 A and its mechanism of conductance. Proc Natl Acad Sci USA 106:7437–7442

    Article  ADS  Google Scholar 

  • Horsefield R, Norden K, Fellert M, Backmark A, Tornroth-Horsefield S, Terwisscha van Scheltinga AC, Kvassman J, Kjellbom P, Johanson U, Neutze R (2008) High-resolution X-ray structure of human aquaporin 5. Proc Natl Acad Sci USA 105:13327–13332

    Article  ADS  Google Scholar 

  • Hu JGG, Griffin RG, Herzfeld J (1997) Interactions between the protonated Schiff base and its counterion in the photointermediates of bacteriorhodopsin. J Am Chem Soc 119:9495–9498

    Article  Google Scholar 

  • Idnurm A, Howlett BJ (2001) Characterization of an opsin gene from the ascomycete Leptosphaeria maculans. Genome 44:167–171

    Article  Google Scholar 

  • Ikeda D, Furutani Y, Kandori H (2007) FTIR study of the retinal Schiff base and internal water molecules of proteorhodopsin. Biochemistry 46:5365–5373

    Article  Google Scholar 

  • Jaroniec CP, Lansing JC, Tounge BA, Belenky M, Herzfeld J, Griffin RG (2001) Measurement of dipolar couplings in a uniformly C-13, N-15-labeled membrane protein: distances between the Schiff base and aspartic acids in the active site of bacteriorhodopsin. J Am Chem Soc 123:12929–12930

    Article  Google Scholar 

  • Kim TK, Zhang R, Feng W, Cai J, Pierce W, Song ZH (2005) Expression and characterization of human CB1 cannabinoid receptor in methylotrophic yeast Pichia pastoris. Protein Expr Purif 40:60–70

    Article  Google Scholar 

  • Kim HJ, Howell SC, Van Horn WD, Jeon YH, Sanders CR (2009) Recent advances in the application of solution NMR spectroscopy to multi-span integral membrane proteins. Prog Nucl Magn Reson Spectrosc 55:335–360

    Article  Google Scholar 

  • Klein-Seetharaman J, Yanamala NVK, Javeed F, Reeves PJ, Getmanova EV, Loewen MC, Schwalbe H, Khorana HG (2004) Differential dynamics in the G protein-coupled receptor rhodopsin revealed by solution NMR. Proc Natl Acad Sci USA 101:3409–3413

    Article  ADS  Google Scholar 

  • Kobilka B, Schertler GF (2008) New G-protein-coupled receptor crystal structures: insights and limitations. Trends Pharmacol Sci 29:79–83

    Article  Google Scholar 

  • Kofuku Y, Yoshiura C, Ueda T, Terasawa H, Hirai T, Tominaga S, Hirose M, Maeda Y, Takahashi H, Terashima Y, Matsushima K, Shimada I (2009) Structural basis of the interaction between chemokine stromal cell-derived factor-1/CXCL12 and its G-protein-coupled receptor CXCR4. J Biol Chem 284:35240–35250

    Article  Google Scholar 

  • Lansing JC, Hu JGG, Belenky M, Griffin RG, Herzfeld J (2003) Solid-state NMR investigation of the buried X-proline peptide bonds of bacteriorhodopsin. Biochemistry 42:3586–3593

    Article  Google Scholar 

  • Laroche Y, Storme V, De Meutter J, Messens J, Lauwereys M (1994) High-level secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast, Pichia pastoris. Biotechnology (NY) 12:1119–1124

    Article  Google Scholar 

  • Li Y, Berthold DA, Gennis RB, Rienstra CM (2008) Chemical shift assignment of the transmembrane helices of DsbB, a 20-kDa integral membrane enzyme, by 3D magic-angle spinning NMR spectroscopy. Protein Sci 17:199–204

    Article  Google Scholar 

  • Long SB, Campbell EB, Mackinnon R (2005) Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 309:897–903

    Article  ADS  Google Scholar 

  • Lopez JJ, Shukla AK, Reinhart C, Schwalbe H, Michel H, Glaubitz C (2008) The structure of the neuropeptide bradykinin bound to the human G-protein coupled receptor bradykinin B2 as determined by solid-state NMR spectroscopy. Angew Chem Int Ed Engl 47:1668–1671

    Article  Google Scholar 

  • Luca S, White JF, Sohal AK, Filippov DV, van Boom JH, Grisshammer R, Baldus M (2003) The conformation of neurotensin bound to its G protein-coupled receptor. Proc Natl Acad Sci USA 100:10706–10711

    Article  ADS  Google Scholar 

  • Lundstrom K, Wagner R, Reinhart C, Desmyter A, Cherouati N, Magnin T, Zeder-Lutz G, Courtot M, Prual C, Andre N, Hassaine G, Michel H, Cambillau C, Pattus F (2006) Structural genomics on membrane proteins: comparison of more than 100 GPCRs in 3 expression systems. J Struct Func Genomics 7:77–91

    Article  Google Scholar 

  • Massou S, Puech V, Talmont F, Demange P, Lindley ND, Tropis M, Milon A (1999) Heterologous expression of a deuterated membrane-integrated receptor and partial deuteration in methylotrophic yeasts. J Biomol NMR 14:231–239

    Article  Google Scholar 

  • McCusker EC, Bane SE, O’Malley MA, Robinson AS (2007) Heterologous GPCR expression: a bottleneck to obtaining crystal structures. Biotechnol Prog 23:540–547

    Article  Google Scholar 

  • Metz G, Siebert F, Engelhard M (1992) High-resolution solid state 13C NMR of bacteriorhodopsin: characterization of [4–13C]Asp resonances. Biochemistry 31:455–462

    Article  Google Scholar 

  • Molina DM, Wetterholm A, Kohl A, McCarthy AA, Niegowski D, Ohlson E, Hammarberg T, Eshaghi S, Haeggstrom J, Nordlund PR (2007) Structural basis for synthesis of inflammatory mediators by human leukotriene C-4 synthase. Nature 448:U613–U616

    Article  ADS  Google Scholar 

  • Morgan WD, Kragt A, Feeney J (2000) Expression of deuterium-isotope-labelled protein in the yeast Pichia pastoris for NMR studies. J Biomol NMR 17:337–347

    Article  Google Scholar 

  • Mustafi D, Palczewski K (2009) Topology of class A G protein-coupled receptors: insights gained from crystal structures of rhodopsins, adrenergic and adenosine receptors. Mol Pharmacol 75:1–12

    Article  Google Scholar 

  • O’Leary JM, Radcliffe CM, Willis AC, Dwek RA, Rudd PM, Downing AK (2004) Identification and removal of O-linked and non-covalently linked sugars from recombinant protein produced using Pichia pastoris. Protein Expr Purif 38:217–227

    Article  Google Scholar 

  • Oberg F, Ekvall M, Nyblom M, Backmark A, Neutze R, Hedfalk K (2009) Insight into factors directing high production of eukaryotic membrane proteins; production of 13 human AQPs in Pichia pastoris. Mol Membr Biol 26:215–227

    Article  Google Scholar 

  • Park SH, Prytulla S, De Angelis AA, Brown JM, Kiefer H, Opella SJ (2006) High-resolution NMR spectroscopy of a GPCR in aligned bicelles. J Am Chem Soc 128:7402–7403

    Article  Google Scholar 

  • Pfleger N, Lorch M, Woerner AC, Shastri S, Glaubitz C (2008) Characterisation of Schiff base and chromophore in green proteorhodopsin by solid-state NMR. J Biomol NMR 40:15–21

    Article  Google Scholar 

  • Pickford AR, O’Leary JM (2004) Isotopic labeling of recombinant proteins from the methylotrophic yeast Pichia pastoris. Methods Mol Biol 278:17–33

    Google Scholar 

  • Ratnala VR, Kiihne SR, Buda F, Leurs R, de Groot HJ, DeGrip WJ (2007) Solid-state NMR evidence for a protonation switch in the binding pocket of the H1 receptor upon binding of the agonist histamine. J Am Chem Soc 129:867–872

    Article  Google Scholar 

  • Renault M, Cukkemane A, Baldus M (2010) Solid-state NMR spectroscopy on complex biomolecules. Angew Chem Int Ed Engl 49:8346–8357

    Article  Google Scholar 

  • Rodriguez E, Krishna NR (2001) An economical method for (15)N/(13)C isotopic labeling of proteins expressed in Pichia pastoris. J Biochem 130:19–22

    Article  Google Scholar 

  • Sarramegna V, Demange P, Milon A, Talmont F (2002) Optimizing functional versus total expression of the human mu-opioid receptor in Pichia pastoris. Protein Expr Purif 24:212–220

    Article  Google Scholar 

  • Sarramegna V, Muller I, Milon A, Talmont F (2006) Recombinant G protein-coupled receptors from expression to renaturation: a challenge towards structure. Cell Mol Life Sci 63:1149–1164

    Article  Google Scholar 

  • Schmidt P, Berger C, Scheidt HA, Berndt S, Bunge A, Beck-Sickinger AG, Huster D (2010) A reconstitution protocol for the in vitro folded human G protein-coupled Y2 receptor into lipid environment. Biophys Chem 150:29–36

    Article  Google Scholar 

  • Shi L, Ahmed MA, Zhang W, Whited G, Brown LS, Ladizhansky V (2009a) Three-dimensional solid-state NMR study of a seven-helical integral membrane proton pump—structural insights. J Mol Biol 386:1078–1093

    Article  Google Scholar 

  • Shi L, Lake EM, Ahmed MA, Brown LS, Ladizhansky V (2009b) Solid-state NMR study of proteorhodopsin in the lipid environment: secondary structure and dynamics. Biochim Biophys Acta 1788:2563–2574

    Article  Google Scholar 

  • Shi L, Kawamura I, Jung KH, Brown LS, Ladizhansky V (2010) Conformation of a seven-helical transmembrane photosensor in the lipid environment. Angew Chem Int Ed Engl (in press). doi: 10.1002/anie.201004422

  • Shukla AK, Haase W, Reinhart C, Michel H (2007) Heterologous expression and characterization of the recombinant bradykinin B2 receptor using the methylotrophic yeast Pichia pastoris. Protein Expr Purif 55:1–8

    Article  Google Scholar 

  • Singh S, Hedley D, Kara E, Gras A, Iwata S, Ruprecht J, Strange PG, Byrne B (2010) A purified C-terminally truncated human adenosine A(2A) receptor construct is functionally stable and degradation resistant. Protein Expr Purif 74:80–87

    Article  Google Scholar 

  • Sumii M, Furutani Y, Waschuk SA, Brown LS, Kandori H (2005) Strongly hydrogen-bonded water molecule present near the retinal chromophore of Leptosphaeria rhodopsin, the bacteriorhodopsin-like proton pump from a eukaryote. Biochemistry 44:15159–15166

    Article  Google Scholar 

  • Takahashi H, Shimada I (2010) Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells. J Biomol NMR 46:3–10

    Article  Google Scholar 

  • Talmont F (2009) Monitoring the human beta1, beta2, beta3 adrenergic receptors expression and purification in Pichia pastoris using the fluorescence properties of the enhanced green fluorescent protein. Biotechnol Lett 31:49–55

    Article  Google Scholar 

  • Tao X, Avalos JL, Chen J, MacKinnon R (2009) Crystal structure of the eukaryotic strong inward-rectifier K+ channel Kir2.2 at 3.1 A resolution. Science 326:1668–1674

    Article  ADS  Google Scholar 

  • Tapaneeyakorn S, Goddard AD, Oates J, Willis CL, Watts A (2010) Solution- and solid-state NMR studies of GPCRs and their ligands. Biochim Biophys Acta (in press)

  • Tian C, Breyer RM, Kim HJ, Karra MD, Friedman DB, Karpay A, Sanders CR (2005) Solution NMR spectroscopy of the human vasopressin V2 receptor, a G protein-coupled receptor. J Am Chem Soc 127:8010–8011

    Article  Google Scholar 

  • Tikhonova IG, Costanzi S (2009) Unraveling the structure and function of G protein-coupled receptors through NMR spectroscopy. Curr Pharm Des 15:4003–4016

    Article  Google Scholar 

  • Varga K, Aslimovska L, Watts A (2008) Advances towards resonance assignments for uniformly-13C, 15 N enriched bacteriorhodopsin at 18.8 T in purple membranes. J Biomol NMR 41:1–4

    Article  Google Scholar 

  • Vogel R, Martell S, Mahalingam M, Engelhard M, Siebert F (2007) Interaction of a G protein-coupled receptor with a G protein-derived peptide induces structural changes in both peptide and receptor: a Fourier-transform infrared study using isotopically labeled peptides. J Mol Biol 366:1580–1588

    Article  Google Scholar 

  • Wang Y, Jardetzky O (2002) Probability-based protein secondary structure identification using combined NMR chemical-shift data. Protein Sci 11:852–861

    Article  Google Scholar 

  • Waschuk SA, Bezerra AG, Shi L, Brown LS (2005) Leptosphaeria rhodopsin: Bacteriorhodopsin-like proton pump from a eukaryote. Proc Natl Acad Sci USA 102:6879–6883

    Article  ADS  Google Scholar 

  • Werner K, Lehner I, Dhiman HK, Richter C, Glaubitz C, Schwalbe H, Klein-Seetharaman J, Khorana HG (2007) Combined solid state and solution NMR studies of alpha, epsilon-15 N labeled bovine rhodopsin. J Biomol NMR 37:303–312

    Article  Google Scholar 

  • Werner K, Richter C, Klein-Seetharaman J, Schwalbe H (2008) Isotope labeling of mammalian GPCRs in HEK293 cells and characterization of the C-terminus of bovine rhodopsin by high resolution liquid NMR spectroscopy. J Biomol NMR 40:49–53

    Article  MATH  Google Scholar 

  • Wood MJ, Komives EA (1999) Production of large quantities of isotopically labeled protein in Pichia pastoris by fermentation. J Biomol NMR 13:149–159

    Article  Google Scholar 

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Acknowledgments

This research was supported by the University of Guelph (start-up funds to V.L. and L.S.B.), the Natural Sciences and Engineering Research Council of Canada (discovery grants to L.S.B. and to V.L., and doctoral scholarship to Y.F.), the Canada Foundation for Innovation, and the Ontario Innovation Trust. V.L. holds Canada Research Chair in Biophysics, and is a recipient of an Early Researcher Award from the Ontario Ministry of Research and Innovation. L.S. is a recipient of the MITACS Accelerate scholarship, co-funded by Bruker Ltd. We thank Cambridge Isotope Laboratories for the generous gift of isotopically labeled methanol.

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Authors and Affiliations

  1. Department of Physics, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada

    Ying Fan, Lichi Shi, Vladimir Ladizhansky & Leonid S. Brown

  2. Department of Physics and Biophysics Interdepartmental Group, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada

    Ying Fan, Lichi Shi, Vladimir Ladizhansky & Leonid S. Brown

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  1. Ying Fan
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Correspondence to Leonid S. Brown.

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Fan, Y., Shi, L., Ladizhansky, V. et al. Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment. J Biomol NMR 49, 151–161 (2011). https://doi.org/10.1007/s10858-011-9473-9

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