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Protein residue linking in a single spectrum for magic-angle spinning NMR assignment

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Abstract

Here we introduce a new pulse sequence for resonance assignment that halves the number of data sets required for sequential linking by directly correlating sequential amide resonances in a single diagonal-free spectrum. The method is demonstrated with both microcrystalline and sedimented deuterated proteins spinning at 60 and 111 kHz, and a fully protonated microcrystalline protein spinning at 111 kHz, with as little as 0.5 mg protein sample. We find that amide signals have a low chance of ambiguous linkage, which is further improved by linking in both forward and backward directions. The spectra obtained are amenable to automated resonance assignment using general-purpose software such as UNIO-MATCH.

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Acknowledgments

We thank the members of the technical staff of the ISA for assistance with the NMR spectrometers. We acknowledge support from CNRS (Fondation pour la Chimie des Substances Naturelles) and from the People Programme of the European Union’s FP7 (FP7-PEOPLE-2012-ITN REA Grant agreement No 317127 “pNMR” and 316630 “CAS-IDP”). LBA is supported by a MC incoming fellowship (REA Grant agreement No 624918 “MEM-MAS”), and JS by an EMBO fellowship (ALTF 1506-2014) and by the Marie Curie Actions of the European Commission (LTFCOFUND2013, GA-2013-609409).

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Authors and Affiliations

  1. Centre de RMN à Très Hauts Champs, Institut des Sciences Analytiques (CNRS, ENS Lyon, UCB Lyon 1), Université de Lyon, 69100, Villeurbanne, France

    Loren B. Andreas, Jan Stanek, Tanguy Le Marchand, Andrea Bertarello, Diane Cala-De Paepe, Daniela Lalli, Magdaléna Krejčíková, Camille Doyen, Carl Öster, Lyndon Emsley, Torsten Herrmann & Guido Pintacuda

  2. Centre for Structural Biology, Central European Institute of Technology (CEITEC), Masaryk University, 625 00, Brno, Czech Republic

    Magdaléna Krejčíková

  3. Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK

    Carl Öster

  4. Bruker Biospin, 76287, Rheinstetten, Germany

    Benno Knott, Sebastian Wegner & Frank Engelke

  5. Department of Chemistry “Ugo Schiff“and Magnetic Resonance Center (CERM), University of Florence, 50019, Sesto Fiorentino, FI, Italy

    Isabella C. Felli & Roberta Pierattelli

  6. School of Chemistry, University of Wollongong, Wollongong, NSW, 2522, Australia

    Nicholas E. Dixon

  7. Department of Chemistry, Ecole Polytechnique Fédérale de Lausanne, 1015, Lausanne, Switzerland

    Lyndon Emsley

Authors
  1. Loren B. Andreas
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  2. Jan Stanek
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  3. Tanguy Le Marchand
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  5. Diane Cala-De Paepe
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  7. Magdaléna Krejčíková
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  8. Camille Doyen
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  9. Carl Öster
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  11. Sebastian Wegner
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  13. Isabella C. Felli
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  14. Roberta Pierattelli
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  15. Nicholas E. Dixon
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  16. Lyndon Emsley
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  17. Torsten Herrmann
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  18. Guido Pintacuda
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Corresponding author

Correspondence to Guido Pintacuda.

Electronic supplementary material

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10858_2015_9956_MOESM1_ESM.pdf

Sample preparation, comparison of 1H, 13C and 15N resolution and degree of overlap for inter-residue correlation, simulated 13C-13C transfer efficiency curves. Supplementary material 1 (PDF 985 kb)

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Andreas, L.B., Stanek, J., Le Marchand, T. et al. Protein residue linking in a single spectrum for magic-angle spinning NMR assignment. J Biomol NMR 62, 253–261 (2015). https://doi.org/10.1007/s10858-015-9956-1

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  • DOI: https://doi.org/10.1007/s10858-015-9956-1

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