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Journal of Biomolecular NMR

, Volume 14, Issue 1, pp 79–83 | Cite as

Application of amino acid type-specific 1H- and 14N-labeling in a 2H-, 15N-labeled background to a 47 kDa homodimer: Potential for NMR structure determination of large proteins

  • Mark J.S. Kelly
  • Cornelia Krieger
  • Linda J. Ball
  • Yihua Yu
  • Gerald Richter
  • Peter Schmieder
  • Adelbert Bacher
  • Hartmut Oschkinat
Article

Abstract

NMR investigations of larger macromolecules (>20 kDa) are severely hindered by rapid 1H and 13C transverse relaxation. Replacement of non-exchangeable protons with deuterium removes many efficient 1H-1H and 1H-13C relaxation pathways. The main disadvantage of deuteration is that many of the protons which would normally be the source of NOE-based distance restraints are removed. We report the development of a novel labeling strategy which is based on specific protonation and 14N-labeling of the residues phenylalanine, tyrosine, threonine, isoleucine and valine in a fully deuterated, 15N-labeled background. This allows the application of heteronuclear half-filters, 15N-editing and 1H-TOCSY experiments to select for particular magnetization transfer pathways. Results from investigations of a 47 kDa dimeric protein labeled in this way demonstrated that the method provides useful information for the structure determination of large proteins.

amino acid specific labeling 3D heteronuclear NMR deuteration heteronuclear half-filter 

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Copyright information

© Kluwer Academic Publishers 1999

Authors and Affiliations

  • Mark J.S. Kelly
    • 1
  • Cornelia Krieger
    • 2
  • Linda J. Ball
    • 1
  • Yihua Yu
    • 3
  • Gerald Richter
    • 2
  • Peter Schmieder
    • 1
  • Adelbert Bacher
    • 2
  • Hartmut Oschkinat
    • 3
  1. 1.Forschungsinstitut für Molekulare PharmakologieBerlinGermany
  2. 2.Lehrstuhl für Organische Chemie und BiochemieTechnische Universität MünchenGarchingGermany
  3. 3.EMBLHeidelbergGermany

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