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

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.

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Acknowledgements

This research was supported by the National Institute of Health (grants GM47467 and RR00995). We are grateful to Dr M. Roehrl for the GFL peptide test sample.

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Authors and Affiliations

1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 02115, U.S.A.
   Zhen-Yu J. Sun, Dominique P. Frueh, Philipp Selenko & Gerhard Wagner
2. Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington, CT, 06030, U.S.A.
   Jeffrey C. Hoch

Authors

1. Zhen-Yu J. Sun
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2. Dominique P. Frueh
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3. Philipp Selenko
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4. Jeffrey C. Hoch
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5. Gerhard Wagner
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Correspondence toGerhard Wagner.

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Sun, ZY.J., Frueh, D.P., Selenko, P. et al. Fast Assignment of 15N-HSQC Peaks using High-Resolution 3D HNcocaNH Experiments with Non-Uniform Sampling.J Biomol NMR 33, 43–50 (2005). https://doi.org/10.1007/s10858-005-1284-4

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• Received: 23 May 2005
• Accepted: 18 July 2005
• Issue Date: September 2005
• DOI: https://doi.org/10.1007/s10858-005-1284-4

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