Journal of Biomolecular NMR

, Volume 54, Issue 4, pp 325–335 | Cite as

Solid state NMR of proteins at high MAS frequencies: symmetry-based mixing and simultaneous acquisition of chemical shift correlation spectra

  • Peter Bellstedt
  • Christian Herbst
  • Sabine Häfner
  • Jörg Leppert
  • Matthias Görlach
  • Ramadurai Ramachandran


We have carried out chemical shift correlation experiments with symmetry-based mixing sequences at high MAS frequencies and examined different strategies to simultaneously acquire 3D correlation spectra that are commonly required in the structural studies of proteins. The potential of numerically optimised symmetry-based mixing sequences and the simultaneous recording of chemical shift correlation spectra such as: 3D NCAC and 3D NHH with dual receivers, 3D NC′C and 3D C′NCA with sequential 13C acquisitions, 3D NHH and 3D NC′H with sequential 1H acquisitions and 3D CANH and 3D C’NH with broadband 13C–15N mixing are demonstrated using microcrystalline samples of the β1 immunoglobulin binding domain of protein G (GB1) and the chicken α-spectrin SH3 domain.


Solid state NMR Magic angle spinning Symmetry-based mixing Dual receivers Chemical shift correlation 



The FLI is a member of the Science Association’Gottfried Wilhelm Leibniz’ (WGL) and is financially supported by the Federal Government of Germany and the State of Thuringia. Also thanks to the Leibniz Graduate School on Aging and Age-Related Diseases (LGSA) for funding and support.

Supplementary material

10858_2012_9680_MOESM1_ESM.eps (2.8 mb)
Supplementary material 1 (EPS 2891 kb)
10858_2012_9680_MOESM2_ESM.eps (1.3 mb)
Supplementary material 2 (EPS 1353 kb)
10858_2012_9680_MOESM3_ESM.eps (1000 kb)
Supplementary material 3 (EPS 1000 kb)
10858_2012_9680_MOESM4_ESM.eps (10.8 mb)
Supplementary material 4 (EPS 11084 kb)
10858_2012_9680_MOESM5_ESM.eps (21.7 mb)
Supplementary material 5 (EPS 22245 kb)
10858_2012_9680_MOESM6_ESM.doc (34 kb)
Supplementary material 6 (DOC 34 kb)


  1. Agarwal V, Linser R, Fink U, Faelber K, Reif B (2010) Identification of hydroxyl protons, determination of their exchange dynamics and characterisation of hydrogen bonding in a microcrystallin protein. J Am Chem Soc 132:3187–3195CrossRefGoogle Scholar
  2. Akbey U, Lange S, Franks WT, Linser R, Rehbein K, Diehl A, Rossum BJ, Reif B, Oschkinat O (2010) Optimum levels of exchangeable protons in perdeuterated proteins for proton detection in MAS NMR spectroscopy. J Biomol NMR 46:67–73CrossRefGoogle Scholar
  3. Astrof NS, Lyon CE, Griffin RG (2001) Triple resonance solid state NMR experiments with reduced dimensionality evolution periods. J Magn Reson 152:303–307ADSCrossRefGoogle Scholar
  4. Bennett AE, Rienstra CM, Griffiths JM, Zhen W, Lansbury PT, Griffin RG (1998) Homonuclear radio frequency-driven recoupling in rotating solids. J Chem Phys 108:9463–9479ADSCrossRefGoogle Scholar
  5. Brinkmann A, Levitt M (2001) Symmetry principles in the nuclear magnetic resonance of spinning solids: heteronuclear recoupling by generalized Hartmann-Hahn sequences. J Chem Phys 115:357–384ADSCrossRefGoogle Scholar
  6. Brinkmann A, Schmedt auf der Günne J, Levitt MH (2002) Homonuclear zero-quantum recoupling in fast magic-angle spinning nuclear magnetic resonance. J Magn Reson 156:79–96ADSCrossRefGoogle Scholar
  7. Castellani F, van Rossum BJ, Diehl A, Rehbein K, Oschkinat H (2003) Determination of solid-state NMR structures of proteins by means of three-dimensional 15 N–13C–13C dipolar correlation spectroscopy and chemical shift analysis. Biochemistry 42:11476–11483CrossRefGoogle Scholar
  8. Chen L, Kaiser JM, Lai J, Polenova T, Yang J, Rienstra CM, Mueller LJ (2007) J-based 2D homonuclear and heteronuclear correlation in solid-state proteins. Magn Reson Chem 45:S84–S92CrossRefGoogle Scholar
  9. 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 β1 immunoglobulin binding domain of protein G (GB1): 15N and 13C chemical shift assignments and conformational Analysis. J Am Chem Soc 127:12291–12305CrossRefGoogle Scholar
  10. Franks WT, Kloepper KD, Wylie BJ, Rienstra CM (2007) Four-dimensional heteronuclear correlation experiments for chemical shift assignments of solid proteins. J Biomol NMR 39:107–131CrossRefGoogle Scholar
  11. Frericks Schmidt HL, Sperling LJ, Gao YG, Wylie BJ, Boettcher JM, Wilson SR, Rienstra CM (2007) Crystal polymorphism of protein GB1 examined by solid-state NMR spectroscopy and X-ray diffraction. J Phys Chem B 111:14362–14369CrossRefGoogle Scholar
  12. Gopinath T, Veglia G (2012a) Dual acquisition magic-angle solid-state NMR-spectroscopy: simultaneous acquisition of multidimensional spectra of biomacromolecules. Angew Chem Int Ed 51:1–6CrossRefGoogle Scholar
  13. Gopinath T, Veglia G (2012b) 3D DUMAS: simultaneous acquisition of three-dimensional magic angle spinning solid-state NMR experiments of proteins. J Magn Reson 220:79–84ADSCrossRefGoogle Scholar
  14. Habenstein B, Wasmer C, Bousset L, Sourigues Y, Schutz 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 51:235–243CrossRefGoogle Scholar
  15. Herbst C, Riedel K, Ihle Y, Leppert J, Ohlenschläger O, Görlach M, Ramachandran R (2008) MAS solid state NMR of RNAs with multiple receivers. J Biomol NMR 41:121–125CrossRefGoogle Scholar
  16. Herbst C, Herbst J, Kirschstein A, Leppert J, Ohlenschläger O, Görlach M, Ramachandran R (2009a) Design of high-power, broadband 180° pulses and mixing sequences for fast MAS solid state chemical shift correlation NMR spectroscopy. J Biomol NMR 43:51–61CrossRefGoogle Scholar
  17. Herbst C, Herbst J, Kirschstein A, Leppert J, Ohlenschläger O, Görlach M, Ramachandran R (2009b) Recoupling and decoupling of nuclear spin interactions at high MAS frequencies: numerical design of CNnv symmetry-based RF pulse schemes. J Biomol NMR 44:175–184CrossRefGoogle Scholar
  18. Herbst C, Herbst J, Leppert J, Ohlenschläger O, Görlach M, Ramachandran R (2009c) Numerical design of RNnv symmetry-based RF pulse schemes for recoupling and decoupling of nuclear spin interactions at high MAS frequencies. J Biomol NMR 44:235–244CrossRefGoogle Scholar
  19. Herbst C, Herbst J, Leppert J, Ohlenschläger O, Görlach M, Ramachandran R (2010) Broadband 15N–13C dipolar recoupling via symmetry-based RF pulse schemes at high MAS frequencies. J Biomol NMR 47:7–17CrossRefGoogle Scholar
  20. Herbst C, Herbst J, Leppert J, Ohlenschläger O, Görlach M, Ramachandran R (2011) Chemical shift correlation at high MAS frequencies employing low-power symmetry-based mixing schemes. J Biomol NMR 50:277–284CrossRefGoogle Scholar
  21. Hou G, Yan S, Sun S, Han Y, Byeon IL, Ahn J, Concel J, Samoson A, Gronenborn AM, Polenova T (2010) Spin diffusion driven by R-symmetry sequences: applications to homonuclear correlation spectroscopy in MAS NMR of biological and organic solids. J Am Chem Soc 133:3943–3953CrossRefGoogle Scholar
  22. Huang KY, Siemer AB, McDermott AE (2011) Homonuclear mixing sequences for perdeuterated proteins. J Magn Reson 208:122–127ADSCrossRefGoogle Scholar
  23. Kehlet C, Bjerring M, Sivertsen AC, Kristensen T, Enghild JJ, Glaser SJ, Khaneja N, Nielsen NC (2007) Optimal control based NCO and NCA experiments for spectral assignments in biological solid-state NMR spectroscopy. J Magn Reson 188:216–230ADSCrossRefGoogle Scholar
  24. Knight MJ, Webber AL, Pell AJ, Guerry P, Barbet-Massin E, Bertini I, Felli IC, Gonnelli L, Pierattelli R, Emsley L, Lesage A, Herrmann T, Pintacuda G (2011) Fast resonance assignment and fold determination of human superoxide dismutase by high-resolution proton-detected solid-state MAS NMR spectroscopy. Angew Chem Int Ed 50:11697–11701CrossRefGoogle Scholar
  25. Leppert J, Heise B, Ohlenschläger O, Görlach M, Ramachandran R (2003) Broadband RFDR with adiabatic inversion pulses. J Biomol NMR 26:13–24CrossRefGoogle Scholar
  26. Levitt MH (2002) Symmetry-based pulse sequences in magic-angle spinning solid-state NMR. In: Grant DM, Harris RK (eds) Encyclopedia of nuclear magnetic resonance. Wiley, ChichesterGoogle Scholar
  27. Lewandowski JR, Dumaz JN, Akbey U, Lange S, Emsley L, Oschkinat H (2011) Enhanced resolution and coherence lifetimes in the solid-state NMR spectroscopy of perdeuteraded proteins under ultrafast magic-angle spinning. J Phys Chem Lett 2:2205–2211CrossRefGoogle Scholar
  28. Linser R (2011) Side-chain to backbone correlations from solid-state NMR of perdeuterated proteins through combined excitation and long-range magnetization transfers. J Biomol NMR 51:221–226CrossRefGoogle Scholar
  29. Linser R (2012) Backbone assignment of perdeuterated proteins using long-range H/C transfers. J Biomol NMR 52:151–158CrossRefGoogle Scholar
  30. Linser R, Bardiaux B, Higman V, Fink U, Reif B (2011) Structure calculation from unambiguous long-range amide and methyl 1H–1H distance restraints for a microcrystalline protein with MAS solid-state NMR spectroscopy. J Am Chem Soc 133:5905–5912CrossRefGoogle Scholar
  31. Loening NM, Bjerring M, Nielsen NC, Oschkinat H (2012) A comparison of NCO and NCA transfer methods for biological solid-state NMR spectroscopy. J Magn Reson 214:81–90ADSCrossRefGoogle Scholar
  32. Nielsen AB, Bjerring M, Nielsen JT, Nielsen NC (2009) Symmetry-based dipolar recoupling by optimal control: band-selective experiments for assignment of solid-state NMR spectra of proteins. J Chem Phys 131:025101ADSCrossRefGoogle Scholar
  33. Paulson EK, Morcombe CR, Gaponenko V, Dancheck B, Byrd RA, Zilm KW (2003) High-sensitivity observation of dipolar exchange and NOEs between exchangeable protons in proteins by solid-state NMR spectroscopy. J Am Chem Soc 125:14222–14223CrossRefGoogle Scholar
  34. Schuetz A, Wasmer C, Habenstein B, Verel R, Greenwald J, Reik R, Böckmann A, Meier BH (2010) Protocols for the sequential solid-state NMR spectroscopic assignments of a uniformly labeled 25 kDa protein:HET-s(1–227). ChemBioChem 11:1543–1551CrossRefGoogle Scholar
  35. Shi L, Kawamura I, Jung KH, Brown LS, Ladizhansky V (2011) Conformation of a seven- helical transmembrane photosensor in the lipid environment. Angew Chem Int Ed 50:1302–1305CrossRefGoogle Scholar
  36. Sperling LJ, Berthold DA, Sasser TL, Jeisy-Scott V, Rienstra CM (2010) Assignments strategies for large proteins by magic-angle spinning NMR: the 21-kDa disulfide-bond forming enzyme Dsba. J Mol Biol 399:268–282CrossRefGoogle Scholar
  37. States DJ, Haberkorn RA, Ruben DJ (1982) A two-dimensional nuclear Overhauser experiment with pure absorption phase in four quadrants. J Magn Reson 48:286–292Google Scholar
  38. Takegoshi K, Nakamura S, Terao T (2001) 13C–1H dipolar-assisted rotational resonance in magic-angle spinning NMR. Chem Phys Lett 344:631–637ADSCrossRefGoogle Scholar
  39. Ward ME, Shi L, Lake E, Krishnamurthy S, Hutchins H, Brown LS, Ladizhansky V (2011) Proton-detected solid-State NMR reveals intramembrane polar networks in a seven-helical transmembrane protein proteorhodopsin. J Am Chem Soc 133:17434–17443CrossRefGoogle Scholar
  40. Zhou DH, Rienstra CM (2008) High-performance solvent suppression for proton detected solid- state NMR. J Magn Reson 192:167–172ADSCrossRefGoogle Scholar
  41. Zhou DH, Shea JJ, Nieuwkoop AJ, Franks WT, Wylie BJ, Mullen C, Sandoz D, Rienstra CM (2007) Solid-State Protein-Structure Determination with Proton-Detected Triple-Resonance 3D Magic-Angle-Spinning NMR Spectroscopy. Angew Chem Int Ed 46:8380–8383CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Peter Bellstedt
    • 1
  • Christian Herbst
    • 2
  • Sabine Häfner
    • 1
  • Jörg Leppert
    • 1
  • Matthias Görlach
    • 1
  • Ramadurai Ramachandran
    • 1
  1. 1.Biomolecular NMR spectroscopy, Leibniz Institute for Age ResearchFritz Lipmann InstituteJenaGermany
  2. 2.Department of Physics, Faculty of ScienceUbon Ratchathani UniversityUbon RatchathaniThailand

Personalised recommendations