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Optimal degree of protonation for 1H detection of aliphatic sites in randomly deuterated proteins as a function of the MAS frequency

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Abstract

The 1H dipolar network, which is the major obstacle for applying proton detection in the solid-state, can be reduced by deuteration, employing the RAP (Reduced Adjoining Protonation) labeling scheme, which yields random protonation at non-exchangeable sites. We present here a systematic study on the optimal degree of random sidechain protonation in RAP samples as a function of the MAS (magic angle spinning) frequency. In particular, we compare 1H sensitivity and linewidth of a microcrystalline protein, the SH3 domain of chicken α-spectrin, for samples, prepared with 5–25 % H2O in the E. coli growth medium, in the MAS frequency range of 20–60 kHz. At an external field of 19.96 T (850 MHz), we find that using a proton concentration between 15 and 25 % in the M9 medium yields the best compromise in terms of sensitivity and resolution, with an achievable average 1H linewidth on the order of 40–50 Hz. Comparing sensitivities at a MAS frequency of 60 versus 20 kHz, a gain in sensitivity by a factor of 4–4.5 is observed in INEPT-based 1H detected 1D 1H,13C correlation experiments. In total, we find that spectra recorded with a 1.3 mm rotor at 60 kHz have almost the same sensitivity as spectra recorded with a fully packed 3.2 mm rotor at 20 kHz, even though ~20× less material is employed. The improved sensitivity is attributed to 1H line narrowing due to fast MAS and to the increased efficiency of the 1.3 mm coil.

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Acknowledgments

This research was supported by the Leibniz- and the Helmholtz-Gemeinschaft, the DFG (Re1435, SFB449, SFB740) and the Bio-NMR project (European Commission’s Framework Program 7, project number: 261863). We are grateful to the Center for Integrated Protein Science Munich (CIPS-M) for financial support and to Bruker BioSpin for providing measurement time, especially to S. Wegner and G. Althoff for technical support. P.S. acknowledges funding from the French Research Agency (Contract ANR-10-PDOC-011-01 ProtDynByNMR).

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Authors and Affiliations

  1. Helmholtz-Zentrum München (HMGU), Deutsches Forschungszentrum für Gesundheit und Umwelt (HMGU), Ingolstädter Landstr. 1, 85764, Neuherberg, Germany

    Sam Asami & Bernd Reif

  2. Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Straße 10, 13125, Berlin, Germany

    Sam Asami & Bernd Reif

  3. Eidgenössische Technische Hochschule Zürich (ETH Zürich), Wolfgang-Pauli-Str. 10, 8093, Zürich, Switzerland

    Kathrin Szekely, Paul Schanda & Beat H. Meier

  4. Institut de Biologie Structurale Jean-Pierre Ebel, CEA-CNRS-UJF, 41, rue Jules Horowitz, 38027, Grenoble, France

    Paul Schanda

  5. Department of Chemistry, Munich Center for Integrated Protein Science at Department Chemie (CIPS-M), Technische Universität München (TUM), Lichtenbergstr. 4, 85747, Garching, Germany

    Bernd Reif

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  1. Sam Asami
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  2. Kathrin Szekely
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Correspondence to Bernd Reif.

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Asami, S., Szekely, K., Schanda, P. et al. Optimal degree of protonation for 1H detection of aliphatic sites in randomly deuterated proteins as a function of the MAS frequency. J Biomol NMR 54, 155–168 (2012). https://doi.org/10.1007/s10858-012-9659-9

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