Journal of Biomolecular NMR

, Volume 39, Issue 3, pp 197–211 | Cite as

Temperature-dependent sensitivity enhancement of solid-state NMR spectra of α-synuclein fibrils

  • Kathryn D. Kloepper
  • Donghua H. Zhou
  • Ying Li
  • Kem A. Winter
  • Julia M. George
  • Chad M. Rienstra
Article

Abstract

The protein α-synuclein (AS) is the primary fibrillar component of Lewy bodies, the pathological hallmark of Parkinson’s disease. Wild-type human AS and the three mutant forms linked to Parkinson’s disease (A53T, A30P, and E46K) all form fibrils through a nucleation-dependent pathway; however, the biophysical details of these fibrillation events are not yet well understood. Atomic-level structural insight is required in order to elucidate the potential role of AS fibrils in Parkinson’s disease. Here we show that low temperature acquisition of magic-angle spinning NMR spectra of wild type AS fibrils-greatly enhances spectral sensitivity, enabling the detection of a substantially larger number of spin systems. At 0 ± 3°C sample temperature, cross polarization (CP) experiments yield weak signals. Lower temperature spectra (−40 ± 3°C) demonstrated several times greater signal intensity, an effect further amplified in 3D 15N–13C–13C experiments, which are required to perform backbone assignments on this sample. Thus 3D experiments enabled assignments of most amino acids in the rigid part of the fibril (approximately residues 64 to 94), as well as tentative site-specific assignments for T22, V26, A27, Y39, G41, S42, H50, V52, A53, T54, V55, V63, A107, I112, and S129. Most of these signals were not observed in 2D or 3D spectra at 0 ± 3°C. Spectra acquired at low temperatures therefore permitted more complete chemical shift assignments. Observation of the majority of residues in AS fibrils represents an important step towards solving the 3D structure.

Keywords

Magic-angle spinning Amyloid Protein structure Chemical shift assignments Multidimensional Parkinson’s disease 

References

  1. Apetri MM, Maiti NC, Zagorski MG, Carey PR, Anderson VE (2006) Secondary structure of alpha-synuclein oligomers: Characterization by Raman and atomic force microscopy. J Mol Biol 355:63–71CrossRefGoogle Scholar
  2. Balbach JJ, Ishii Y, Antzutkin ON, Leapman RD, Rizzo NW, Dyda F, Reed J, Tycko R (2000) Amyloid fibril formation by A beta(16–22), a seven-residue fragment of the Alzheimer’s beta-amyloid peptide, and structural characterization by solid state NMR. Biochemistry 39:13748–13759CrossRefGoogle Scholar
  3. Balbach JJ, Petkova AT, Oyler NA, Antzutkin ON, Gordon DJ, Meredith SC, Tycko R (2002) Supramolecular structure in full-length Alzheimer’s beta-amyloid fibrils: Evidence for a parallel beta-sheet organization from solid-state nuclear magnetic resonance. Biophys J 83:1205–1216Google Scholar
  4. Baldus M, Petkova AT, Herzfeld JH, Griffin RG (1998) Cross polarization in the tilted frame: assignment and spectral simplification in heteronuclear spin systems. Mol Phys 95:1197–1207CrossRefADSGoogle Scholar
  5. Bennett AE, Rienstra CM, Auger M, Lakshmi KV, Griffin RG (1995) Heteronuclear decoupling in rotating solids. J Chem Phys 103:6951–6958CrossRefADSGoogle Scholar
  6. Benzinger TLS, Gregory DM, Burkoth TS, Miller-Auer H, Lynn DG, Botto RE, Meredith SC (2000) Two-dimensional structure of beta-amyloid(10–35) fibrils. Biochemistry 39:3491–3499CrossRefGoogle Scholar
  7. Benzinger TLS, Gregory DM, Burkoth TS, Miller-Auer H, Lynn DG, Botto RE, Meredith SC (1998) Propagating structure of Alzheimer’s beta-amyloid ((10–35)) is parallel beta-sheet with residues in exact register. Proc Natl Acad Sci USA 95:13407–13412CrossRefADSGoogle Scholar
  8. Castellani F, van Rossum B, Diehl A, Schubert M, Rehbein K, Oschkinat H (2002) Structure of a protein determined by solid-state magic-angle-spinning NMR spectroscopy. Nature 420:98–102CrossRefADSGoogle Scholar
  9. Conway KA, Harper JD, Lansbury PT (2000) Fibrils formed in vitro from alpha-synuclein and two mutant forms linked to Parkinson’s disease are typical amyloid. Biochemistry 39:2552–2563CrossRefGoogle Scholar
  10. Cornilescu G, Delaglio F, Bax A (1999) Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J Biomol NMR 13:289–302CrossRefGoogle Scholar
  11. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) Nmrpipe – a multidimensional spectral processing system based on Unix pipes. J Biomol NMR 6:277–293CrossRefGoogle Scholar
  12. Del Mar C, Greenbaum EA, Mayne L, Englander SW, Woods VL (2005) Structure and properties of alpha-synuclein and other amyloids determined at the amino acid level. Proc Natl Acad Sci USA 102:15477–15482CrossRefADSGoogle Scholar
  13. Der-Sarkissian A, Jao CC, Chen J, Langen R (2003) Structural organization of alpha-synuclein fibrils studied by site-directed spin labeling. J Biol Chem 278:37530–37535CrossRefGoogle Scholar
  14. Franks WT, Zhou DH, Wylie BJ, Money BG, Graesser DT, Frericks HL, Sahota G, Rienstra CM (2005) Magic-angle spinning solid-state NMR spectroscopy of the beta 1 immunoglobulin binding domain of protein G (GB1): 15N and 13C chemical shift assignments and conformational analysis. J Am Chem Soc 127:12291–12305CrossRefGoogle Scholar
  15. Frericks HL, Zhou DH, Yap LL, Gennis RB, Rienstra CM (2006) Magic-angle spinning solid-state NMR of a 144 kDa membrane protein complex: E-coli cytochrome bo(3) oxidase. J Biomol NMR 36:55–71CrossRefGoogle Scholar
  16. Goddard TD, Kneller DG (2006), Sparky 3.112 University of California, San FranciscoGoogle Scholar
  17. Hartmann SR, Hahn EL (1962) Nuclear double resonance in the rotating frame. Phys Rev 128:2042MATHCrossRefADSGoogle Scholar
  18. Hediger S, Meier BH, Ernst RR (1995) Adiabatic passage Hartmann-Hahn cross-polarization in NMR under magic-angle sample-spinning. Chem Phys Lett 240:449–456CrossRefADSGoogle Scholar
  19. Heise H, Hoyer W, Becker S, Andronesi OC, Riedel D, Baldus M (2005) Molecular-level secondary structure, polymorphism, and dynamics of full-length alpha-synuclein fibrils studied by solid-state NMR. Proc Natl Acad Sci USA 102:15871–15876CrossRefADSGoogle Scholar
  20. Igumenova TI, Wand AJ, McDermott AE (2004) Assignment of the backbone resonances for microcrystalline ubiquitin. J Am Chem Soc 126:5323–5331CrossRefGoogle Scholar
  21. Iwai A, Yoshimoto M, Masliah E, Saitoh T (1995) Non-a-beta component of Alzheimers-disease amyloid (Nac) is amyloidogenic. Biochemistry 34:10139–10145CrossRefGoogle Scholar
  22. Kloepper KD, Woods WS, Winter KA, George JM, Rienstra CM (2006) Preparation of alpha-synuclein fibrils for solid-state NMR: Expression, purification, and incubation of wild-type and mutant forms. Protein Expr Purif 48:112–117CrossRefGoogle Scholar
  23. Kruger R, Kuhn W, Muller T, Woitalla D, Graeber M, Kosel S, Przuntek H, Epplen JT, Schols L, Riess O (1998) Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson’s disease. Nat Genet 18:106–108CrossRefGoogle Scholar
  24. Lange A, Becker S, Seidel K, Giller K, Pongs O, Baldus M (2005) A concept for rapid protein-structure determination by solid-state NMR spectroscopy. Angew Chem Int Ed 44:2089–2092CrossRefGoogle Scholar
  25. Lansbury PT, Costa PR, Griffiths JM, Simon EJ, Auger M, Halverson KJ, Kocisko DA, Hendsch ZS, Ashburn TT, Spencer RGS, Tidor B, Griffin RG (1995) Structural model for the beta-amyloid fibril based on interstrand alignment of an antiparallel-sheet comprising a c-terminal peptide. Nat Struct Biol 2:990–998CrossRefGoogle Scholar
  26. Lewis BA, Rice DM, Olejniczak ET, Dasgupta SK, Herzfeld J, Griffin RG (1984) Deuterium NMR studies of molecular dynamics in bacteriorhodopsin - analysis of lineshapes and intensities for phenylalanine, tyrosine, and leucine sidechains. Biophys J 45:A213–A213CrossRefGoogle Scholar
  27. Li Y, Wylie BJ, Rienstra CM (2006) Selective refocusing pulses in magic-angle spinning NMR: Characterization and applications to multidimensional protein spectroscopy. J Magn Reson 179:206–216CrossRefADSGoogle Scholar
  28. Li Y, Berthold DA, Frericks HL, Gennis RB, Rienstra CM (2007) Partial 13C and 15N chemical shift assignments of the disulfide bond forming enzyme DsbB by 3D magic-angle spinning NMR spectroscopy. Chembiochem 5:434–42CrossRefGoogle Scholar
  29. Marion D, Wuthrich K (1983) Application of phase sensitive two-dimensional correlated spectroscopy (Cosy) for measurements of 1H-1H spin-spin coupling-constants in proteins. Biochem Biophys Res Commun 113:967–974CrossRefGoogle Scholar
  30. Martin RW, Zilm KW (2003) Preparation of protein nanocrystals and their characterization by solid state NMR. J Magn Reson 165:162–174CrossRefADSGoogle Scholar
  31. McDermott A, Polenova T, Bockmann A, Zilm KW, Paulsen EK, Martin RW, Montelione GT (2000) Partial NMR assignments for uniformly (13C, 15N)-enriched BPTI in the solid state. J Biomol NMR 16:209–219CrossRefGoogle Scholar
  32. Miake H, Mizusawa H, Iwatsubo T, Hasegawa M (2002) Biochemical characterization of the core structure of alpha-synuclein filaments. J Biol Chem 277:19213–19219CrossRefGoogle Scholar
  33. Morcombe CR, Gaponenko V, Byrd RA, Zilm KW (2004) Diluting abundant spins by isotope edited radio frequency field assisted diffusion. J Am Chem Soc 126:7196–7197CrossRefGoogle Scholar
  34. Morcombe CR, Zilm KW (2003) Chemical shift referencing in MAS solid state NMR. J Magn Reson 162:479–486CrossRefADSGoogle Scholar
  35. Petkova AT, Ishii Y, Balbach JJ, Antzutkin ON, Leapman RD, Delaglio F, Tycko R (2002) A structural model for Alzheimer’s beta-amyloid fibrils based on experimental constraints from solid state NMR. Proc Natl Acad Sci USA 99:16742–16747CrossRefADSGoogle Scholar
  36. Petkova AT, Leapman RD, Guo ZH, Yau WM, Mattson MP, Tycko R (2005) Self-propagating, molecular-level polymorphism in Alzheimer’s beta-amyloid fibrils. Science 307:262–265CrossRefADSGoogle Scholar
  37. Petkova AT, Yau WM, Tycko R (2006) Experimental constraints on quaternary structure in Alzheimer’s beta-amyloid fibrils. Biochemistry 45:498–512CrossRefGoogle Scholar
  38. Petkova AT, Tycko R (2004) Rotational resonance in uniformly 13C-labeled solids: effects on high-resolution magic-angle spinning NMR spectra and applications in structural studies of biomolecular systems. J Magn Reson 168:137–146CrossRefADSGoogle Scholar
  39. Pines A, Gibby MG, Waugh JS (1973) Proton-enhanced NMR of dilute spins in solids. J Chem Phys 59:569–590CrossRefADSGoogle Scholar
  40. Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, Pike B, Root H, Rubenstein J, Boyer R, Stenroos ES, Chandrasekharappa S, Athanassiadou A, Papapetropoulos T, Johnson WG, Lazzarini AM, Duvoisin RC, DiIorio G, Golbe LI, Nussbaum RL (1997) Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science 276:2045–2047CrossRefGoogle Scholar
  41. Rice DM, Wittebort RJ, Griffin RG, Meirovitch E, Stimson ER, Meinwald YC, Freed JH, Scheraga HA (1981) Rotational jumps of the tyrosine side-chain in crystalline enkephalin – 2H NMR lineshapes for aromatic ring motion in solids. J Am Chem Soc 103:7707–7710CrossRefGoogle Scholar
  42. Rice DM, Meinwald YC, Scheraga HA, Griffin RG (1987) Tyrosyl motion in peptides – 2H NMR lineshapes and spin-lattice relaxation. J Am Chem Soc 109:1636–1640CrossRefGoogle Scholar
  43. Rienstra CM, Hohwy M, Hong M, Griffin RG (2000) 2D and 3D 15N-13C-13C NMR chemical shift correlation spectroscopy of solids: Assignment of MAS spectra of peptides. J Am Chem Soc 122:10979–10990CrossRefGoogle Scholar
  44. Ritter C, Maddelein ML, Siemer AB, Luhrs T, Ernst M, Meier BH, Saupe SJ, Riek R (2005) Correlation of structural elements and infectivity of the HET-s prion. Nature 435:844–848CrossRefADSGoogle Scholar
  45. Rothwell WP, Waugh JS (1981) Transverse relaxation of dipolar coupled spin systems under Rf-Irradiation – Detecting motions in solids. J Chem Phys 74:2721–2732CrossRefADSGoogle Scholar
  46. Schaefer J, Stejskal EO (1976) 13C-NMR of polymers spinning at the magic angle. J Am Chem Soc 98:1031CrossRefGoogle Scholar
  47. Serpell LC, Berriman J, Jakes R, Goedert M, Crowther RA (2000) Fiber diffraction of synthetic alpha-synuclein filaments shows amyloid-like cross-beta conformation. Proc Natl Acad Sci USA 97:4897–4902CrossRefADSGoogle Scholar
  48. Siemer AB, Ritter C, Ernst M, Riek R, Meier BH (2005) High-resolution solid-state NMR spectroscopy of the prion protein HET-s in its amyloid conformation. Angew Chem Int Ed 44:2441–2444CrossRefGoogle Scholar
  49. Siemer AB, Arnold AA, Ritter C, Westfeld T, Ernst M, Riek R, Meier BH (2006a) Observation of highly flexible residues in amyloid fibrils of the HET-s prion. J Am Chem Soc 128:13224–13228CrossRefGoogle Scholar
  50. Siemer AB, Ritter C, Steinmetz MO, Ernst M, Riek R, Meier BH (2006b) 13C, 15N resonance assignment of parts of the HET-s prion protein in its amyloid form. J Biomol NMR 34:75–87CrossRefGoogle Scholar
  51. Spillantini MG, Schmidt ML, Lee VMY, Trojanowski JQ, Jakes R, Goedert M (1997) alpha-synuclein in Lewy bodies. Nature 388:839–840CrossRefADSGoogle Scholar
  52. Takegoshi K, Nakamura S, Terao T (2001) 13C-1H dipolar-assisted rotational resonance in magic-angle spinning NMR. Chem Phys Lett 344:631–637CrossRefADSGoogle Scholar
  53. Van Geet AL (1968) Calibration of the methanol and glycol nuclear magnetic resonance thermometers with a static thermistor probe. Anal Chem 40:2227–2229CrossRefGoogle Scholar
  54. Wagner G, DeMarco A, Wüthrich K (1976) Dynamics of the aromatic amino acid residues in the globular conformation of the basic pancreatic trypsin inhibitor (BPTI). I 1H NMR studies Biophys Struct Mech 2:139–158CrossRefGoogle Scholar
  55. Wishart DS, Bigam CG, Holm A, Hodges RS, Sykes BD (1995) 1H, 13C, and 15N random coil NMR chemical-shifts of the common amino acids .1. Investigations of nearest-neighbor effects. J Biomol NMR 5:67–81CrossRefGoogle Scholar
  56. Wishart DS, Sykes BD (1994) Chemical shifts as a tool for structure determination. Methods Enzymol 239:363–392CrossRefGoogle Scholar
  57. Zarranz JJ, Alegre J, Gomez-Esteban JC, Lezcano E, Ros R, Ampuero I, Vidal L, Hoenicka J, Rodriguez O, Atares B, Llorens V, Tortosa EG, del Ser T, Munoz DG, de Yebenes JG (2004) The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia. Ann Neurol 55:164–173CrossRefGoogle Scholar
  58. Zech SG, Wand AJ, McDermott AE (2005) Protein structure determination by high-resolution solid-state NMR spectroscopy: Application to microcrystalline ubiquitin. J Am Chem Soc 127:8618–8626CrossRefGoogle Scholar
  59. Zhao X, Eden M, Levitt MH (2001) Recoupling of heteronuclear dipolar interactions in solid-state NMR using symmetry-based pulse sequences. Chem Phys Lett 342:353–361CrossRefADSGoogle Scholar
  60. Zhou DH, Kloepper KD, Winter KA, Rienstra CM (2006) Band-selective C-13 homonuclear 3D spectroscopy for solid proteins at high field with rotor-synchronized soft pulses. J Biomol NMR 34:245–257CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Kathryn D. Kloepper
    • 1
  • Donghua H. Zhou
    • 1
  • Ying Li
    • 2
  • Kem A. Winter
    • 1
  • Julia M. George
    • 3
  • Chad M. Rienstra
    • 1
    • 2
    • 4
  1. 1.Department of ChemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  2. 2.Center for Biophysics and Computational BiologyUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  3. 3.Department of Molecular and Integrative PhysiologyUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  4. 4.Department of BiochemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA

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