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Nuclear overhauser spectroscopy of chiral CHD methylene groups

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Abstract

Nuclear magnetic resonance spectroscopy (NMR) can provide a great deal of information about structure and dynamics of biomolecules. The quality of an NMR structure strongly depends on the number of experimental observables and on their accurate conversion into geometric restraints. When distance restraints are derived from nuclear Overhauser effect spectroscopy (NOESY), stereo-specific assignments of prochiral atoms can contribute significantly to the accuracy of NMR structures of proteins and nucleic acids. Here we introduce a series of NOESY-based pulse sequences that can assist in the assignment of chiral CHD methylene protons in random fractionally deuterated proteins. Partial deuteration suppresses spin-diffusion between the two protons of CH2 groups that normally impedes the distinction of cross-relaxation networks for these two protons in NOESY spectra. Three and four-dimensional spectra allow one to distinguish cross-relaxation pathways involving either of the two methylene protons so that one can obtain stereospecific assignments. In addition, the analysis provides a large number of stereospecific distance restraints. Non-uniform sampling was used to ensure optimal signal resolution in 4D spectra and reduce ambiguities of the assignments. Automatic assignment procedures were modified for efficient and accurate stereospecific assignments during automated structure calculations based on 3D spectra. The protocol was applied to calcium-loaded calbindin D9k. A large number of stereospecific assignments lead to a significant improvement of the accuracy of the structure.

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Acknowledgments

We thank Mikael Akke (Lund University) for a sample of partially deuterated calbindin D9k, as well as Dominique Frueh (Johns Hopkins University) and Lewis Kay (University of Toronto) for fruitful suggestions. This research was supported by the Polish budget funds for science in 2013–2014 (Project IP2012 057872 awarded to J.S.) Access to the Research Infrastructure at University of Warsaw was financed by the European Commission’s FP7 (Contract 228461, EAST-NMR). Financial support of the Bio-NMR Project No. 261863 is gratefully acknowledged.

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Authors and Affiliations

  1. Département de chimie, Ecole Normale Supérieure – PSL Research University, 24 rue Lhomond, 75005, Paris, France

    Rafal Augustyniak, Henri Colaux, Geoffrey Bodenhausen & Fabien Ferrage

  2. Sorbonne Universités, UPMC Université Paris 6, 4 Place Jussieu, 75005, Paris, France

    Rafal Augustyniak, Henri Colaux, Geoffrey Bodenhausen & Fabien Ferrage

  3. UMR 7203 LBM, CNRS, 75005, Paris, France

    Rafal Augustyniak, Henri Colaux, Geoffrey Bodenhausen & Fabien Ferrage

  4. Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093, Warsaw, Poland

    Jan Stanek & Wiktor Koźmiński

  5. Ecole Polytechnique Fédérale de Lausanne, Institut des Sciences et Ingénierie Chimiques, BCH, 1015, Lausanne, Switzerland

    Geoffrey Bodenhausen

  6. Institut des Sciences Analytiques, Centre de RMN à Très Hauts Champs, Université de Lyon/UMR 5280 CNRS/ENS Lyon/UCB Lyon 1, 5 rue de la Doua, 69100, Villeurbanne, France

    Torsten Herrmann

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  1. Rafal Augustyniak
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  2. Jan Stanek
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Augustyniak, R., Stanek, J., Colaux, H. et al. Nuclear overhauser spectroscopy of chiral CHD methylene groups. J Biomol NMR 64, 27–37 (2016). https://doi.org/10.1007/s10858-015-0002-0

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