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HN-NCA heteronuclear TOCSY-NH experiment for 1HN and 15N sequential correlations in (13C, 15N) labelled intrinsically disordered proteins

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Abstract

A simple triple resonance NMR experiment that leads to the correlation of the backbone amide resonances of each amino acid residue ‘i’ with that of residues ‘i−1’ and ‘i+1’ in (13C, 15N) labelled intrinsically disordered proteins (IDPs) is presented. The experimental scheme, {HN-NCA heteronuclear TOCSY-NH}, exploits the favourable relaxation properties of IDPs and the presence of 1 J CαN and 2 J CαN couplings to transfer the 15N x magnetisation from amino acid residue ‘i’ to adjacent residues via the application of a band-selective 15N–13Cα heteronuclear cross-polarisation sequence of ~100 ms duration. Employing non-uniform sampling in the indirect dimensions, the efficacy of the approach has been demonstrated by the acquisition of 3D HNN chemical shift correlation spectra of α-synuclein. The experimental performance of the RF pulse sequence has been compared with that of the conventional INEPT-based HN(CA)NH pulse scheme. As the availability of data from both the HCCNH and HNN experiments will make it possible to use the information extracted from one experiment to simplify the analysis of the data of the other and lead to a robust approach for unambiguous backbone and side-chain resonance assignments, a time-saving strategy for the simultaneous collection of HCCNH and HNN data is also described.

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Acknowledgments

The FLI is a member of the Science Association ‘Gottfried Wilhelm Leibniz’ (WGL) and is financially supported by the Federal Government of Germany and the State of Thuringia. We are deeply grateful to Dr. Lisa D. Cabrita and Prof. Dr. John Christodoulou (Insitute of Structural and Molecular Biology, University College London and Birkbeck College, London, United Kingdom) for providing us with the plasmid and purification protocol for the α-synuclein sample used in this study. Financial support by the Deutsche Forschungsgemeinschaft (DFG) within FOR 1738 (to O.O. and N.G.) is gratefully acknowledged.

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  1. Christian Herbst

    Present address: Department of Physics, Faculty of Science, Ubon Ratchathani University, Ubon Ratchathani, 34190, Thailand

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  1. Research Group Biomolecular NMR Spectroscopy, Leibniz Institute for Age Research, Fritz Lipmann Institute, Beutenbergstr. 11, 07745, Jena, Germany

    Christoph Wiedemann, Nishit Goradia, Sabine Häfner, Christian Herbst, Matthias Görlach, Oliver Ohlenschläger & Ramadurai Ramachandran

  2. Institute of Biochemistry/Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120, Halle/Salle, Germany

    Christoph Wiedemann

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Wiedemann, C., Goradia, N., Häfner, S. et al. HN-NCA heteronuclear TOCSY-NH experiment for 1HN and 15N sequential correlations in (13C, 15N) labelled intrinsically disordered proteins. J Biomol NMR 63, 201–212 (2015). https://doi.org/10.1007/s10858-015-9976-x

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