Journal of Biomolecular NMR

, Volume 44, Issue 4, pp 245–260 | Cite as

Assigning large proteins in the solid state: a MAS NMR resonance assignment strategy using selectively and extensively 13C-labelled proteins

  • Victoria A. Higman
  • Jeremy Flinders
  • Matthias Hiller
  • Stefan Jehle
  • Stefan Markovic
  • Sebastian Fiedler
  • Barth-Jan van Rossum
  • Hartmut OschkinatEmail author


In recent years, solid-state magic-angle spinning nuclear magnetic resonance spectroscopy (MAS NMR) has been growing into an important technique to study the structure of membrane proteins, amyloid fibrils and other protein preparations which do not form crystals or are insoluble. Currently, a key bottleneck is the assignment process due to the absence of the resolving power of proton chemical shifts. Particularly for large proteins (approximately >150 residues) it is difficult to obtain a full set of resonance assignments. In order to address this problem, we present an assignment method based upon samples prepared using [1,3-13C]- and [2-13C]-glycerol as the sole carbon source in the bacterial growth medium (so-called selectively and extensively labelled protein). Such samples give rise to higher quality spectra than uniformly [13C]-labelled protein samples, and have previously been used to obtain long-range restraints for use in structure calculations. Our method exploits the characteristic cross-peak patterns observed for the different amino acid types in 13C-13C correlation and 3D NCACX and NCOCX spectra. An in-depth analysis of the patterns and how they can be used to aid assignment is presented, using spectra of the chicken α-spectrin SH3 domain (62 residues), αB-crystallin (175 residues) and outer membrane protein G (OmpG, 281 residues) as examples. Using this procedure, over 90% of the Cα, Cβ, C′ and N resonances in the core domain of αB-crystallin and around 73% in the flanking domains could be assigned (excluding 24 residues at the extreme termini of the protein).


Solid-state NMR Membrane proteins Protein structure determination Isotopic labelling Resonance assignment 



This work was supported by the Deutsche Forschungsgemeinschaft (SFB449). J.F. gratefully acknowledges funding from the National Science Foundation International Research Fellowship Program (Award #0402114).

Supplementary material

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Supplementary material 1 (PDF 6602 kb)


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Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Victoria A. Higman
    • 1
  • Jeremy Flinders
    • 1
    • 2
  • Matthias Hiller
    • 1
  • Stefan Jehle
    • 1
  • Stefan Markovic
    • 1
  • Sebastian Fiedler
    • 1
  • Barth-Jan van Rossum
    • 1
  • Hartmut Oschkinat
    • 1
    Email author
  1. 1.Leibniz-Institut für Molekulare PharmakologieBerlinGermany
  2. 2.Structural Biology DepartmentGenentech, Inc.South San FranciscoUSA

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