Biomolecular NMR Assignments

, Volume 8, Issue 2, pp 349–356 | Cite as

Solid-state NMR sequential assignments of the amyloid core of full-length Sup35p

  • Anne K. Schütz
  • Birgit Habenstein
  • Nina Luckgei
  • Luc Bousset
  • Yannick Sourigues
  • Anders B. Nielsen
  • Ronald MelkiEmail author
  • Anja BöckmannEmail author
  • Beat H. MeierEmail author


Sup35p is a yeast prion and is responsible for the [PSI +] trait in Saccharomyces cerevisiae. With 685 amino acids, full-length soluble and fibrillar Sup35p are challenging targets for structural biology as they cannot be investigated by X-ray crystallography or NMR in solution. We present solid-state NMR studies of fibrils formed by the full-length Sup35 protein. We detect an ordered and rigid core of the protein that gives rise to narrow and strong peaks, while large parts of the protein show either static disorder or dynamics on time scales which interfere with dipolar polarization transfer or shorten the coherence lifetime. Thus, only a small subset of resonances is observed in 3D spectra. Here we describe in detail the sequential assignments of the 22 residues for which resonances are observed in 3D spectra: their chemical shifts mostly corresponding to β-sheet secondary structure. We suspect that these residues form the amyloid core of the fibril.


Sup35p Fibrils Solid-state NMR Assignments Secondary structure 



We thank Dr. Christian Wasmer for help with recording the NMR spectra. This work was supported by the Agence Nationale de la Recherche (ANR-12-BS08-0013-01), the ETH Zurich, the Swiss National Science Foundation (Grant 200020_124611) and the Centre National de la Recherche Scientifique. We also acknowledge support from the European Commission under the Seventh Framework Programme (FP7), contract Bio-NMR 261863.


  1. Böckmann A, Meier BH (2010) Prions: en route from structural models to structures. Prion 4(2):72–79CrossRefGoogle Scholar
  2. Böckmann A, Gardiennet C, Verel R, Hunkeler A, Loquet A, Pintacuda G, Emsley L, Meier BH, Lesage A (2009) Characterization of different water pools in solid-state NMR protein samples. J Biomol NMR 45(3):319–327CrossRefGoogle Scholar
  3. Comellas G, Lemkau LR, Nieuwkoop AJ, Kloepper KD, Ladror DT, Ebisu R, Woods WS, Lipton AS, George JM, Rienstra CM (2011) Structured regions of α-synuclein fibrils include the early-onset parkinsons disease mutation sites. J Mol Biol 411:881–895CrossRefGoogle Scholar
  4. Cox BS (1965) PSI, a cytoplasmic suppressor of super-suppressor in yeast. Heredity 20(4):505–521CrossRefGoogle Scholar
  5. Debelouchina GT, Platt GW, Bayro MJ, Radford SE, Griffin RG (2010) Magic angle spinning NMR analysis of beta(2)-microglobulin amyloid fibrils in two distinct morphologies. J Am Chem Soc 132(30):10414–10423CrossRefGoogle Scholar
  6. DePace AH, Santoso A, Hillner P, Weissman JS (1998) A critical role for amino-terminal glutamine/asparagine repeats in the formation and propagation of a yeast prion. Cell 93(7):1241–1252CrossRefGoogle Scholar
  7. Fitzpatrick AWP, Debelouchina GT, Bayro MJ, Clare DK, Caporini MA, Bajaj VS, Jaroniec CP, Wang L, Ladizhansky V, Müller SA, MacPhee CE, Waudby CA, Mott HR, De Simone A, Knowles TPJ, Saibil HR, Vendruscolo M, Orlova EV, Griffin RG, Dobson CM (2013) Atomic structure and hierarchical assembly of a cross-β amyloid fibril. Proc Natl Acad Sci USA 110:5468–5473 ADSCrossRefGoogle Scholar
  8. Fogh R, Ionides J, Ulrich E, Boucher W, Vranken W, Linge JP, Habeck M, Rieping W, Bhat TN, Westbrook J, Henrick K, Gilliland G, Berman H, Thornton J, Nilges M, Markley J, Laue E (2002) The CCPN project: an interim report on a data model for the NMR community. Nat Struct Biol 9(6):416–418CrossRefGoogle Scholar
  9. Gath J, Habenstein B, Bousset L, Melki R, Meier BH, Böckmann A (2012) Solid-state NMR sequential assignments of α-synuclein. Biomol NMR Assign 6(1):51–55CrossRefGoogle Scholar
  10. Habenstein B, Wasmer C, Bousset L, Sourigues Y, Schütz A, Loquet A, Meier BH, Melki R, Böckmann A (2011) Extensive de novo solid-state NMR assignments of the 33 kDa C-terminal domain of the Ure2 prion. J Biomol NMR 51(3):235–243CrossRefGoogle Scholar
  11. Heise H, Celej MS, Becker S, Riede D, Pelah A, Kumar A, Jovin TM, Baldus M (2008) Solid-state NMR reveals structural differences between fibrils of wild-type and disease-related A53T mutant alpha-synuclein. J Mol Biol 380(3):444–450CrossRefGoogle Scholar
  12. Krishnan R, Lindquist S (2005) Structural insights into a yeast prion illuminate nucleation and strain diversity. Nature 435(7043):765–772ADSCrossRefGoogle Scholar
  13. Krzewska J, Melki R (2006) Molecular chaperones and the assembly of the prion Sup35p, an in vitro study. EMBO J 25(4):822–833CrossRefGoogle Scholar
  14. Krzewska J, Tanaka M, Burston SG, Melki R (2007) Biochemical and functional analysis of the assembly of full-length Sup35p and its prion-forming domain. J Biol Chem 282(3):1679–1686CrossRefGoogle Scholar
  15. Lewandowski JR, van der Wel PCA, Rigney M, Grigorieff N, Griffin RG (2011) Structural complexity of a composite amyloid fibril. J Am Chem Soc 133(37):14686–14698CrossRefGoogle Scholar
  16. Loquet A, Bousset L, Gardiennet C, Sourigues Y, Wasmer C, Habenstein B, Schütz A, Meier BH, Melki R, Böckmann A (2009) Prion fibrils of Ure2p assembled under physiological conditions contain highly ordered, natively folded modules. J Mol Biol 394(1):108–118CrossRefGoogle Scholar
  17. Loquet A, Giller K, Becker S, Lange A (2010) Supramolecular interactions probed by 13C–13C solid-state NMR spectroscopy. J Am Chem Soc 132(43):15164–15166CrossRefGoogle Scholar
  18. Luckgei N, Schütz AK, Bousset L, Habenstein B, Sourigues Y, Gardiennet C, Meier BH, Melki R, Böckmann A (2013a) The conformation of the prion domain of Sup35p in isolation and in the full-length protein is different (submitted)Google Scholar
  19. Luckgei N, Schütz AK, Habenstein B, Bousset L, Sourigues Y, Melki R, Meier BH, Böckmann A (2013b) Solid-state NMR sequential assignments of the amyloid core of Sup35pN. doi: 10.1007/s12104-013-9518-y
  20. Nelson R, Sawaya M, Balbirnie M, Madsen A, Riekel C, Grothe R, Eisenberg D (2005) Structure of the cross-beta spine of amyloid-like fibrils. Nature 435(7043):773–778ADSCrossRefGoogle Scholar
  21. Paravastu AK, Leapman RD, Yau W-M, Tycko R (2008) Molecular structural basis for polymorphism in Alzheimer’s beta-amyloid fibrils. Proc Natl Acad Sci USA 105(47):18349–18354ADSCrossRefGoogle Scholar
  22. Sangill R, Rastrupandersen N, Bildsoe H, Jakobsen HJ, Nielsen NC (1994) Optimized spectral editing of 13C MAS NMR spectra of rigid solids using cross-polarization methods. J Magn Reson A 107(1):67–78ADSCrossRefGoogle Scholar
  23. Schuetz A, Wasmer C, Habenstein B, Verel R, Greenwald J, Riek R, Böckmann A, Meier BH (2010) Protocols for the sequential solid-state NMR spectroscopic assignment of a uniformly labeled 25 kDa protein: HET-s(1-227). Chembiochem 11(11):1543–1551CrossRefGoogle Scholar
  24. Shewmaker F, Wickner RB, Tycko R (2006) Amyloid of the prion domain of Sup35p has an in-register parallel beta-sheet structure. Proc Natl Acad Sci USA 103(52):19754–19759ADSCrossRefGoogle Scholar
  25. Stansfield I, Jones K, Kushnirov V, Dagkesamanskaya A, Poznyakovski A, Paushkin S, Nierras C, Cox B, Teravanesyan M, Tuite M (1995) The products of the Sup45 (eRF1) and Sup35 genes interact to mediate translation termination in saccharomyces-cerevisiae. EMBO J 14(17):4365–4373Google Scholar
  26. Stevens TJ, Fogh RH, Boucher W, Higman VA, Eisenmenger F, Bardiaux B, van Rossum B-J, Oschkinat H, Laue ED (2011) A software framework for analysing solid-state MAS NMR data. J Biomol NMR 51(4):437–447CrossRefGoogle Scholar
  27. Toyama B, Kelly M, Gross J, Weissman J (2007) The structural basis of yeast prion strain variants. Nature 449(7159):233–237 ADSCrossRefGoogle Scholar
  28. Tycko R (2006) Solid-state NMR as a probe of amyloid structure. Protein Pept Lett 13:229–234CrossRefGoogle Scholar
  29. van der Wel PC, Hu K, Lewandowski JR, Griffin RG (2006) Dynamic nuclear polarization of amyloidogenic peptide nanocrystals: GNNQQNY, a core segment of the yeast prion protein Sup35p. J Am Chem Soc 128(33):10840–10846CrossRefGoogle Scholar
  30. van der Wel PCA, Lewandowski JR, Griffin RG (2007) Solid-state NMR study of amyloid nanocrystals and fibrils formed by the peptide GNNQQNY from yeast prion protein Sup35p. J Am Chem Soc 129(16):5117–5130CrossRefGoogle Scholar
  31. van der Wel PCA, Lewandowski JR, Griffin RG (2010) Structural characterization of GNNQQNY amyloid fibrils by magic angle spinning NMR. Biochemistry 49(44):9457–9469CrossRefGoogle Scholar
  32. van Melckebeke H, Wasmer C, Lange A, Ab E, Loquet A, Böckmann A, Meier BH (2010) Atomic-resolution three-dimensional structure of HET-s(218–289) amyloid fibrils by solid-state NMR spectroscopy. J Am Chem Soc 132(39):13765–13775CrossRefGoogle Scholar
  33. Vranken W, Boucher W, Stevens T, Fogh R, Pajon A, Llinas P, Ulrich E, Markley J, Ionides J, Laue E (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 59(4):687–696CrossRefGoogle Scholar
  34. Wang Y, Jardetzky O (2002) Probability-based protein secondary structure identification using combined NMR chemical-shift data. Protein Sci 11(4):852–861CrossRefGoogle Scholar
  35. Wasmer C, Lange A, van Melckebeke H, Siemer AB, Riek R, Meier BH (2008) Amyloid fibrils of the HET-s(218–289) prion form a beta solenoid with a triangular hydrophobic core. Science 319(5869):1523–1526ADSCrossRefGoogle Scholar
  36. Wickner RB, Masison DC, Edskes HK (1995) [PSI] and [URE3] as yeast prions. Yeast 11(16):1671–1685CrossRefGoogle Scholar
  37. Wishart DS, Sykes BD (1994) The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J Biomol NMR 4(2):171–180Google Scholar
  38. Wu X, Zilm K (1993) Complete spectral editing in CPMAS NMR. J Magn Reson A 102(2):205–213ADSCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Anne K. Schütz
    • 1
  • Birgit Habenstein
    • 2
  • Nina Luckgei
    • 2
  • Luc Bousset
    • 3
  • Yannick Sourigues
    • 3
  • Anders B. Nielsen
    • 1
  • Ronald Melki
    • 3
    Email author
  • Anja Böckmann
    • 2
    Email author
  • Beat H. Meier
    • 1
    Email author
  1. 1.Physical ChemistryETH ZürichZurichSwitzerland
  2. 2.Institut de Biologie et Chimie des ProtéinesUMR 5086 CNRS/Université de Lyon 1LyonFrance
  3. 3.Laboratoire d’Enzymologie et Biochimie StructuralesUPR 3082 CNRSGif-sur-YvetteFrance

Personalised recommendations