Abstract
We describe the expression of the hepatitis C virus nonstructural protein 4B (NS4B), which is an integral membrane protein, in a wheat germ cell-free system, the subsequent purification and characterization of NS4B and its insertion into proteoliposomes in amounts sufficient for multidimensional solid-state NMR spectroscopy. First spectra of the isotopically [2H,13C,15N]-labeled protein are shown to yield narrow 13C resonance lines and a proper, predominantly α-helical fold. Clean residue-selective leucine, isoleucine and threonine-labeling is demonstrated. These results evidence the suitability of the wheat germ-produced integral membrane protein NS4B for solid-state NMR. Still, the proton linewidth under fast magic angle spinning is broader than expected for a perfect sample and possible causes are discussed.
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References
Agarwal V, Penzel S, Székely K et al (2014) De novo 3D structure determination from sub-milligram protein samples by solid-state 100 kHz MAS NMR spectroscopy. Angew Chem Int Ed Engl 53:12253–12256. doi:10.1002/anie.201405730
Andréll J, Tate CG (2013) Overexpression of membrane proteins in mammalian cells for structural studies. Mol Membr Biol. doi:10.3109/09687688.2012.703703
Barbet-Massin E, Pell AJ, Retel JS et al (2014) Rapid proton-detected NMR assignment for proteins with fast magic angle spinning. J Am Chem Soc 136:12489–12497. doi:10.1021/ja507382j
Betton J-M (2004) High throughput cloning and expression strategies for protein production. Biochimie 86:601–605. doi:10.1016/j.biochi.2004.07.004
Böckmann A, Gardiennet C, Verel R et al (2009) Characterization of different water pools in solid-state NMR protein samples. J Biomol NMR 45:319–327. doi:10.1007/s10858-009-9374-3
Böckmann A, Ernst M, Meier BH (2015) Spinning proteins, the faster, the better? J Magn Reson 253:71–79. doi:10.1016/j.jmr.2015.01.012
Briggs ELA, Gomes R, Elhussein M et al (2015) Interaction between the NS4B amphipathic helix, AH2, and charged lipid headgroups alters membrane morphology and AH2 oligomeric state—implications for the Hepatitis C virus life cycle. BBA Biomembranes 1848:1–8. doi:10.1016/j.bbamem.2015.04.015
Brown LS, Ladizhansky V (2015) Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy. Protein Sci 24:1333–1346. doi:10.1002/pro.2700
Egger D, Wölk B, Gosert R et al (2002) Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex. J Virol 76:5974–5984
Einav S, Elazar M, Danieli T, Glenn JS (2004) A nucleotide binding motif in hepatitis C virus (HCV) NS4B mediates HCV RNA replication. J Virol 78:11288–11295. doi:10.1128/JVI.78.20.11288-11295.2004
Emami S, Fan Y, Munro R et al (2013) Yeast-expressed human membrane protein aquaporin-1 yields excellent resolution of solid-state MAS NMR spectra. J Biomol NMR 55:147–155. doi:10.1007/s10858-013-9710-5
Endo Y, Sawasaki T (2006) Cell-free expression systems for eukaryotic protein production. Curr Opin Biotech 17:373–380. doi:10.1016/j.copbio.2006.06.009
Fogeron M-L, Badillo A, Jirasko V et al (2015a) Wheat germ cell-free expression: two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins. Protein Expr Purif 105:39–46. doi:10.1016/j.pep.2014.10.003
Fogeron M-L, Paul D, Jirasko V et al (2015b) Functional expression, purification, characterization, and membrane reconstitution of non-structural protein 2 from hepatitis C virus. Protein Expr Purif. doi:10.1016/j.pep.2015.08.027
Fu R, Gill RL, Gill RL Jr et al (2015) Spherical nanoparticle supported lipid bilayers for the structural study of membrane geometry-sensitive molecules. J Am Chem Soc 137:14031–14034. doi:10.1021/jacs.5b08303
Gosert R, Egger D, Lohmann V et al (2003) Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons. J Virol 77:5487–5492. doi:10.1128/JVI.77.9.5487-5492.2003
Gouttenoire J, Castet V, Montserret R et al (2009a) Identification of a novel determinant for membrane association in hepatitis C virus nonstructural protein 4B. J Virol 83:6257–6268. doi:10.1128/JVI.02663-08
Gouttenoire J, Montserret R, Kennel A et al (2009b) An amphipathic-helix at the C terminus of hepatitis C virus nonstructural protein 4B mediates membrane association. J Virol 83:11378–11384. doi:10.1128/JVI.01122-09
Gouttenoire J, Penin F, Moradpour D (2010) Hepatitis C virus nonstructural protein 4B: a journey into unexplored territory. Rev Med Virol 20:117–129. doi:10.1002/rmv.640
Gouttenoire J, Montserret R, Paul D et al (2014) Aminoterminal amphipathic α-helix AH1 of hepatitis C virus nonstructural protein 4B possesses a dual role in RNA replication and virus production. PLoS Pathog 10:e1004501–e1004517. doi:10.1371/journal.ppat.1004501
Hagn F, Etzkorn M, Raschle T, Gerhard Wagner (2013) Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins. J Am Chem Soc 135:1919–1925. doi:10.1021/ja310901f
Hansen SK, Bertelsen K, Paaske B et al (2015) Solid-state NMR methods for oriented membrane proteins. Prog Nucl Magn Reson Spectrosc 88–89:48–85. doi:10.1016/j.pnmrs.2015.05.001
Huber M, Hiller S, Schanda P et al (2011) A proton-detected 4D solid-state NMR experiment for protein structure determination. ChemPhysChem 12:915–918. doi:10.1002/cphc.201100062
Kainosho M, Torizawa T, Iwashita Y et al (2006) Optimal isotope labelling for NMR protein structure determinations. Nature 440:52–57. doi:10.1038/nature04525
Kaur H, Lakatos A, Spadaccini R et al (2015) The ABC exporter MsbA probed by solid state NMR—challenges and opportunities. Biol Chem. doi:10.1515/hsz-2015-0119
Klammt C, Maslennikov I, Bayrhuber M et al (2012) Facile backbone structure determination of human membrane proteins by NMR spectroscopy. Nat Methods 9:834–839. doi:10.1038/nmeth.2033
Kunert B, Gardiennet C, Lacabanne D et al (2014) Efficient and stable reconstitution of the ABC transporter BmrA for solid-state NMR studies. Front Mol Biosci 1:1–11. doi:10.3389/fmolb.2014.00005
le Maire M, Viel A, Moller JV (1989) Size exclusion chromatography and universal calibration of gel columns. Anal Biochem 177:50–56. doi:10.1016/0003-2697(89)90012-2
Lundin M, Lindström H, Grönwall C, Persson MAA (2006) Dual topology of the processed hepatitis C virus protein NS4B is influenced by the NS5A protein. J Gen Virol 87:3263–3272. doi:10.1099/vir.0.82211-0
Mandal A, Hoop CL, DeLucia M et al (2015) Structural changes and proapoptotic peroxidase activity of cardiolipin-bound mitochondrial cytochrome c. Biophys J 109:1873–1884. doi:10.1016/j.bpj.2015.09.016
McDermott A (2009) Structure and dynamics of membrane proteins by magic angle spinning solid-state NMR. Annu Rev Biophys 38:385–403. doi:10.1146/annurev.biophys.050708.133719
Mehler M, Eckert CE, Busche A et al (2015) Assembling a correctly folded and functional heptahelical membrane protein by protein trans-splicing. J Biol Chem. doi:10.1074/jbc.M115.681205
Moradpour D, Penin F (2013) Hepatitis C virus proteins: from structure to function. Curr Top Microbiol Immunol 369:113–142. doi:10.1007/978-3-642-27340-7_5
Morita EH, Shimizu M, Ogasawara T et al (2004) A novel way of amino acid-specific assignment in (1)H-(15)N HSQC spectra with a wheat germ cell-free protein synthesis system. J Biomol NMR 30:37–45. doi:10.1023/B:JNMR.0000042956.65678.b8
Noirot C, Habenstein B, Bousset L et al (2011) Wheat-germ cell-free production of prion proteins for solid-state NMR structural studies. New Biotechnol 28:232–238. doi:10.1016/j.nbt.2010.06.016
Opitz C, Isogai S, Grzesiek S (2015) An economic approach to efficient isotope labeling in insect cells using homemade N-15-, C-13- and H-2-labeled yeast extracts. J Biomol NMR 62:373–385. doi:10.1007/s10858-015-9954-3
Palomares-Jerez F, Nemesio H, Villalaín J (2012) The membrane spanning domains of protein NS4B from hepatitis C virus. Biochim Biophys Acta 1818:2958–2966. doi:10.1016/j.bbamem.2012.07.022
Paul D, Romero-Brey I, Gouttenoire J et al (2011) NS4B self-interaction through conserved C-terminal elements is required for the establishment of functional hepatitis C virus replication complexes. J Virol 85:6963–6976. doi:10.1128/JVI.00502-11
Penzel S, Smith AA, Agarwal V et al (2015) Protein resonance assignment at MAS frequencies approaching 100 kHz: a quantitative comparison of J-coupling and dipolar- coupling-based transfer methods. J Biomol NMR 63:1–22. doi:10.1007/s10858-015-9975-y
Rai R, Deval J (2011) New opportunities in anti-hepatitis C virus drug discovery: targeting NS4B. Antiviral Res 90:93–101. doi:10.1016/j.antiviral.2011.01.009
Renault M, Cukkemane A, Baldus M (2010) Solid-State NMR spectroscopy on complex biomolecules. Angew Chem Int Ed Engl 49:8346–8357. doi:10.1002/anie.201002823
Rigaud J-L, Pitard B, Lévy D (1995) Reconstitution of membrane proteins into liposomes: application to energy-transducing membrane proteins. BBA Bioenergetics 1231:223–246
Sawasaki T, Ogasawara T, Morishita R, Endo Y (2002) A cell-free protein synthesis system for high-throughput proteomics. Proc Natl Acad Sci USA 99:14652–14657. doi:10.1073/pnas.232580399
Shahid SA, Nagaraj M, Chauhan N et al (2015) Solid-state NMR study of the YadA membrane-anchor domain in the bacterial outer membrane. Angew Chem Int Ed Engl 54:12602–12606. doi:10.1002/anie.201505506
Takai K, Sawasaki T, Endo Y (2010) Practical cell-free protein synthesis system using purified wheat embryos. Nat Protoc 5:227–238. doi:10.1038/nprot.2009.207
Tonelli M, Singarapu KK, Makino S-I et al (2011) Hydrogen exchange during cell-free incorporation of deuterated amino acids and an approach to its inhibition. J Biomol NMR 51:467–476. doi:10.1007/s10858-011-9575-4
Underwood KA, Swartz JR, Puglisi JD (2005) Quantitative polysome analysis identifies limitations in bacterial cell-free protein synthesis. Biotechnol Bioeng 91:425–435. doi:10.1002/bit.20529
van Oers MM, Pijlman GP, Vlak JM (2015) Thirty years of baculovirus–insect cell protein expression: from dark horse to mainstream technology. J Gen Virol 96:6–23. doi:10.1099/vir.0.067108-0
Verel R, Ernst M, Meier BH (2001) Adiabatic dipolar recoupling in solid-state NMR: the DREAM scheme. J Magn Reson 150:81–99
Vinarov DA, Lytle BL, Peterson FC et al (2004) Cell-free protein production and labeling protocol for NMR-based structural proteomics. Nat Methods 1:149–153. doi:10.1038/nmeth716
Vranken WF, Boucher W, Stevens TJ et al (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 59:687–696. doi:10.1002/prot.20449
Ward ME, Wang S, Munro R et al (2015) In situ structural studies of Anabaena Sensory Rhodopsin in the E. coli membrane. Biophys J 108:1683–1696. doi:10.1016/j.bpj.2015.02.018
Acknowledgments
This work was supported by the CNRS and by grants from the French ANRS (France Recherche, Nord & Sud, Sida-HIV et Hépatites), an autonomous agency at INSERM, France, and the ANR (ANR-14-CE09-0024B), the Swiss National Science Foundation (200020_146757, 200020_159707 and 31003A-156030), as well as the DFG TRR83-TP13.
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Marie-Laure Fogeron, Vlastimil Jirasko and Susanne Penzel have contributed equally to this work.
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Fogeron, ML., Jirasko, V., Penzel, S. et al. Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus. J Biomol NMR 65, 87–98 (2016). https://doi.org/10.1007/s10858-016-0040-2
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DOI: https://doi.org/10.1007/s10858-016-0040-2