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Site-specific analysis of heteronuclear Overhauser effects in microcrystalline proteins

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Abstract

Relaxation parameters such as longitudinal relaxation are susceptible to artifacts such as spin diffusion, and can be affected by paramagnetic impurities as e.g. oxygen, which make a quantitative interpretation difficult. We present here the site-specific measurement of [1H]13C and [1H]15N heteronuclear rates in an immobilized protein. For methyls, a strong effect is expected due to the three-fold rotation of the methyl group. Quantification of the [1H]13C heteronuclear NOE in combination with 13C-R 1 can yield a more accurate analysis of side chain motional parameters. The observation of significant [1H]15N heteronuclear NOEs for certain backbone amides, as well as for specific asparagine/glutamine sidechain amides is consistent with MD simulations. The measurement of site-specific heteronuclear NOEs is enabled by the use of highly deuterated microcrystalline protein samples in which spin diffusion is reduced in comparison to protonated samples.

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Acknowledgments

This research was supported by the Helmholtz-Gemeinschaft, the Leibniz-Gemeinschaft and the DFG (Re1435, SFB1035). We are grateful to the Center for Integrated Protein Science Munich (CIPS-M) for financial support.

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Author notes

  1. Juan Miguel Lopez del Amo

    Present address: CIC Energigne, Albert Einstein 48, 0150, Miñano, Spain

  2. Vipin Agarwal

    Present address: ETH Zurich, Wolfgang-Pauli-Str. 10, 8093, Zuerich, Switzerland

  3. Yi Xue

    Present address: Department of Biochemistry, Duke University Medical Center, Durham, NC, 27710, USA

Authors and Affiliations

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

    Juan Miguel Lopez del Amo, Riddhiman Sarkar, Justin Porter, Sam Asami, Martin Rübbelke, Oliver F. Lange & Bernd Reif

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

    Juan Miguel Lopez del Amo, Riddhiman Sarkar, Justin Porter, Sam Asami, Martin Rübbelke, Oliver F. Lange & Bernd Reif

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

    Juan Miguel Lopez del Amo, Vipin Agarwal, Uwe Fink & Bernd Reif

  4. Department of Chemistry, Purdue University, West Lafayette, IN, 47907-2084, USA

    Yi Xue

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  1. Juan Miguel Lopez del Amo
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Correspondence to Bernd Reif.

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Juan Miguel Lopez del Amo and Vipin Agarwal have contributed equally to this work.

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Amo, J.M.L.d., Agarwal, V., Sarkar, R. et al. Site-specific analysis of heteronuclear Overhauser effects in microcrystalline proteins. J Biomol NMR 59, 241–249 (2014). https://doi.org/10.1007/s10858-014-9843-1

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