Journal of Biomolecular NMR

, Volume 33, Issue 1, pp 43–50 | Cite as

Fast Assignment of 15N-HSQC Peaks using High-Resolution 3D HNcocaNH Experiments with Non-Uniform Sampling

  • Zhen-Yu J. Sun
  • Dominique P. Frueh
  • Philipp Selenko
  • Jeffrey C. Hoch
  • Gerhard Wagner
Article

Abstract

We describe an efficient NMR triple resonance approach for fast assignment of backbone amide resonance peaks in the 15N-HSQC spectrum. The exceptionally high resolutions achieved in the 3D HncocaNH and hNcocaNH experiments together with non-uniform sampling facilitate error-free sequential connection of backbone amides. Data required for the complete backbone amide assignment of the 56-residue protein GB1 domain were obtained in 14 h. Data analysis was vastly streamlined using a ‘backbone NH walk’ method to determine sequential connectivities without the need for 13C chemical shifts comparison. Amino acid residues in the sequentially connected NH chains are classified into two groups by a simple variation of the NMR pulse sequence, and the resulting ‘ZeBra’ stripe patterns are useful for mapping these chains to the protein sequence. In addition to resolving ambiguous assignments derived from conventional backbone experiments, this approach can be employed to rapidly assign small proteins or flexible regions in larger proteins, and to transfer assignments to mutant proteins or proteins in different ligand-binding states.

Key words

HNcocaNH maximum entropy reconstruction non-uniform sampling sequential assignment triple resonance experiment 

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Copyright information

© Springer 2005

Authors and Affiliations

  • Zhen-Yu J. Sun
    • 1
  • Dominique P. Frueh
    • 1
  • Philipp Selenko
    • 1
  • Jeffrey C. Hoch
    • 2
  • Gerhard Wagner
    • 1
  1. 1.Department of Biological Chemistry and Molecular PharmacologyHarvard Medical SchoolBostonU.S.A.
  2. 2.Department of Molecular, Microbial, and Structural BiologyUniversity of Connecticut Health CenterFarmingtonU.S.A.

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