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Journal of Biomolecular NMR

, Volume 57, Issue 1, pp 65–72 | Cite as

Resonance assignment for a particularly challenging protein based on systematic unlabeling of amino acids to complement incomplete NMR data sets

  • Peter Bellstedt
  • Thomas Seiboth
  • Sabine Häfner
  • Henriette Kutscha
  • Ramadurai Ramachandran
  • Matthias Görlach
Article

Abstract

NMR-based structure determination of a protein requires the assignment of resonances as indispensable first step. Even though heteronuclear through-bond correlation methods are available for that purpose, challenging situations arise in cases where the protein in question only yields samples of limited concentration and/or stability. Here we present a strategy based upon specific individual unlabeling of all 20 standard amino acids to complement standard NMR experiments and to achieve unambiguous backbone assignments for the fast precipitating 23 kDa catalytic domain of human aprataxin of which only incomplete standard NMR data sets could be obtained. Together with the validation of this approach utilizing the protein GB1 as a model, a comprehensive insight into metabolic interconversion ("scrambling”) of NH and CO groups in a standard Escherichia coli expression host is provided.

Keywords

Resonance assignment Unlabeling Selective isotope labeling Reverse labeling Aprataxin 

Supplementary material

10858_2013_9768_MOESM1_ESM.pdf (2.8 mb)
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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Peter Bellstedt
    • 1
  • Thomas Seiboth
    • 1
  • Sabine Häfner
    • 1
  • Henriette Kutscha
    • 1
  • Ramadurai Ramachandran
    • 1
  • Matthias Görlach
    • 1
  1. 1.Biomolecular NMR SpectroscopyLeibniz Institute for Age Research, Fritz Lipmann InstituteJenaGermany

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