Skip to main content

Advertisement

Springer Nature Link
Log in

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

  • Article
  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

    We’re sorry, something doesn't seem to be working properly.

    Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Canada)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • 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 355:63–71

    Article  Google Scholar 

  • 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 39:13748–13759

    Article  Google Scholar 

  • 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 83:1205–1216

    Google Scholar 

  • 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 95:1197–1207

    Article  ADS  Google Scholar 

  • Bennett AE, Rienstra CM, Auger M, Lakshmi KV, Griffin RG (1995) Heteronuclear decoupling in rotating solids. J Chem Phys 103:6951–6958

    Article  ADS  Google Scholar 

  • 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 39:3491–3499

    Article  Google Scholar 

  • 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 95:13407–13412

    Article  ADS  Google Scholar 

  • 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 420:98–102

    Article  ADS  Google Scholar 

  • 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 39:2552–2563

    Article  Google Scholar 

  • Cornilescu G, Delaglio F, Bax A (1999) Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J Biomol NMR 13:289–302

    Article  Google Scholar 

  • 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 6:277–293

    Article  Google Scholar 

  • 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 102:15477–15482

    Article  ADS  Google Scholar 

  • 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 278:37530–37535

    Article  Google Scholar 

  • 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 127:12291–12305

    Article  Google Scholar 

  • 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 36:55–71

    Article  Google Scholar 

  • Goddard TD, Kneller DG (2006), Sparky 3.112 University of California, San Francisco

  • Hartmann SR, Hahn EL (1962) Nuclear double resonance in the rotating frame. Phys Rev 128:2042

    Article  MATH  ADS  Google Scholar 

  • Hediger S, Meier BH, Ernst RR (1995) Adiabatic passage Hartmann-Hahn cross-polarization in NMR under magic-angle sample-spinning. Chem Phys Lett 240:449–456

    Article  ADS  Google Scholar 

  • 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 102:15871–15876

    Article  ADS  Google Scholar 

  • Igumenova TI, Wand AJ, McDermott AE (2004) Assignment of the backbone resonances for microcrystalline ubiquitin. J Am Chem Soc 126:5323–5331

    Article  Google Scholar 

  • Iwai A, Yoshimoto M, Masliah E, Saitoh T (1995) Non-a-beta component of Alzheimers-disease amyloid (Nac) is amyloidogenic. Biochemistry 34:10139–10145

    Article  Google Scholar 

  • 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 48:112–117

    Article  Google Scholar 

  • 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 18:106–108

    Article  Google Scholar 

  • 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 44:2089–2092

    Article  Google Scholar 

  • 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 2:990–998

    Article  Google Scholar 

  • 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–A213

    Article  Google Scholar 

  • 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 179:206–216

    Article  ADS  Google Scholar 

  • 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 5:434–42

    Article  Google Scholar 

  • 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 113:967–974

    Article  Google Scholar 

  • Martin RW, Zilm KW (2003) Preparation of protein nanocrystals and their characterization by solid state NMR. J Magn Reson 165:162–174

    Article  ADS  Google Scholar 

  • 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 16:209–219

    Article  Google Scholar 

  • Miake H, Mizusawa H, Iwatsubo T, Hasegawa M (2002) Biochemical characterization of the core structure of alpha-synuclein filaments. J Biol Chem 277:19213–19219

    Article  Google Scholar 

  • Morcombe CR, Gaponenko V, Byrd RA, Zilm KW (2004) Diluting abundant spins by isotope edited radio frequency field assisted diffusion. J Am Chem Soc 126:7196–7197

    Article  Google Scholar 

  • Morcombe CR, Zilm KW (2003) Chemical shift referencing in MAS solid state NMR. J Magn Reson 162:479–486

    Article  ADS  Google Scholar 

  • 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 99:16742–16747

    Article  ADS  Google Scholar 

  • 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 307:262–265

    Article  ADS  Google Scholar 

  • Petkova AT, Yau WM, Tycko R (2006) Experimental constraints on quaternary structure in Alzheimer’s beta-amyloid fibrils. Biochemistry 45:498–512

    Article  Google Scholar 

  • 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 168:137–146

    Article  ADS  Google Scholar 

  • Pines A, Gibby MG, Waugh JS (1973) Proton-enhanced NMR of dilute spins in solids. J Chem Phys 59:569–590

    Article  ADS  Google Scholar 

  • 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 276:2045–2047

    Article  Google Scholar 

  • 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 103:7707–7710

    Article  Google Scholar 

  • Rice DM, Meinwald YC, Scheraga HA, Griffin RG (1987) Tyrosyl motion in peptides – 2H NMR lineshapes and spin-lattice relaxation. J Am Chem Soc 109:1636–1640

    Article  Google Scholar 

  • 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 122:10979–10990

    Article  Google Scholar 

  • 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 435:844–848

    Article  ADS  Google Scholar 

  • Rothwell WP, Waugh JS (1981) Transverse relaxation of dipolar coupled spin systems under Rf-Irradiation – Detecting motions in solids. J Chem Phys 74:2721–2732

    Article  ADS  Google Scholar 

  • Schaefer J, Stejskal EO (1976) 13C-NMR of polymers spinning at the magic angle. J Am Chem Soc 98:1031

    Article  Google Scholar 

  • 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 97:4897–4902

    Article  ADS  Google Scholar 

  • 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 44:2441–2444

    Article  Google Scholar 

  • 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 128:13224–13228

    Article  Google Scholar 

  • 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 34:75–87

    Article  Google Scholar 

  • Spillantini MG, Schmidt ML, Lee VMY, Trojanowski JQ, Jakes R, Goedert M (1997) alpha-synuclein in Lewy bodies. Nature 388:839–840

    Article  ADS  Google Scholar 

  • Takegoshi K, Nakamura S, Terao T (2001) 13C-1H dipolar-assisted rotational resonance in magic-angle spinning NMR. Chem Phys Lett 344:631–637

    Article  ADS  Google Scholar 

  • Van Geet AL (1968) Calibration of the methanol and glycol nuclear magnetic resonance thermometers with a static thermistor probe. Anal Chem 40:2227–2229

    Article  Google Scholar 

  • 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 2:139–158

    Article  Google Scholar 

  • 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 5:67–81

    Article  Google Scholar 

  • Wishart DS, Sykes BD (1994) Chemical shifts as a tool for structure determination. Methods Enzymol 239:363–392

    Article  Google Scholar 

  • 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 55:164–173

    Article  Google Scholar 

  • 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 127:8618–8626

    Article  Google Scholar 

  • Zhao X, Eden M, Levitt MH (2001) Recoupling of heteronuclear dipolar interactions in solid-state NMR using symmetry-based pulse sequences. Chem Phys Lett 342:353–361

    Article  ADS  Google Scholar 

  • 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 34:245–257

    Article  Google Scholar 

Download references

Acknowledgment

We thank Wendy S. Woods for plasmid preparation, Daniel T. Ladror and Kevin L. Hartman for assistance with sample preparation, and Benjamin J. Wylie and W. Trent Franks for useful discussions. This work was supported by the National Parkinson Foundation and the National Institutes of Health (R01GM073770).

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL, 61801, USA

    Kathryn D. Kloepper, Donghua H. Zhou, Kem A. Winter & Chad M. Rienstra

  2. Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA

    Ying Li & Chad M. Rienstra

  3. Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA

    Julia M. George

  4. Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA

    Chad M. Rienstra

Authors
  1. Kathryn D. Kloepper
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Donghua H. Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ying Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kem A. Winter
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Julia M. George
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chad M. Rienstra
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Chad M. Rienstra.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kloepper, K.D., Zhou, D.H., Li, Y. et al. Temperature-dependent sensitivity enhancement of solid-state NMR spectra of α-synuclein fibrils. J Biomol NMR 39, 197–211 (2007). https://doi.org/10.1007/s10858-007-9189-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-007-9189-z

Keywords

Search

Navigation