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An extended combinatorial 15N, 13Cα, and \( ^{13} {\text{C}}^{\prime } \) labeling approach to protein backbone resonance assignment

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Abstract

Solution NMR studies of α-helical membrane proteins are often complicated by severe spectral crowding. In addition, hydrophobic environments like detergent micelles, isotropic bicelles or nanodiscs lead to considerably reduced molecular tumbling rates which translates into line-broadening and low sensitivity. Both difficulties can be addressed by selective isotope labeling methods. In this publication, we propose a combinatorial protocol that utilizes four different classes of labeled amino acids, in which the three backbone heteronuclei (amide nitrogen, α-carbon and carbonyl carbon) are enriched in 15N or 13C isotopes individually as well as simultaneously. This results in eight different combinations of dipeptides giving rise to cross peaks in 1H–15N correlated spectra. Their differentiation is achieved by recording a series of HN-detected 2D triple-resonance spectra. The utility of this new scheme is demonstrated with a homodimeric 142-residue membrane protein in DHPC micelles. Restricting the number of selectively labeled samples to three allowed the identification of the amino-acid type for 77 % and provided sequential information for 47 % of its residues. This enabled us to complete the backbone resonance assignment of the uniformly labeled protein merely with the help of a 3D HNCA spectrum, which can be collected with reasonable sensitivity even for relatively large, non-deuterated proteins.

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Acknowledgments

This work was supported by the state of Hesse (Center for Biomolecular Magnetic Resonance), the German Research Foundation (SFB 807), NIH (U54GM087519) and the Cluster of Excellence Frankfurt (Macromolecular Complexes).

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The authors declare that they have no conflict of interest. This article does not contain any studies with human participants or animals performed by any of the authors.

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  1. Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany

    Frank Löhr & Volker Dötsch

  2. Institute of Biochemistry, Goethe University, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany

    Franz Tumulka, Christoph Bock & Rupert Abele

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  1. Frank Löhr
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Correspondence to Rupert Abele or Volker Dötsch.

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Löhr, F., Tumulka, F., Bock, C. et al. An extended combinatorial 15N, 13Cα, and \( ^{13} {\text{C}}^{\prime } \) labeling approach to protein backbone resonance assignment. J Biomol NMR 62, 263–279 (2015). https://doi.org/10.1007/s10858-015-9941-8

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