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Journal of Biomolecular NMR

, Volume 59, Issue 4, pp 241–249 | Cite as

Site-specific analysis of heteronuclear Overhauser effects in microcrystalline proteins

  • Juan Miguel Lopez del Amo
  • Vipin Agarwal
  • Riddhiman Sarkar
  • Justin Porter
  • Sam Asami
  • Martin Rübbelke
  • Uwe Fink
  • Yi Xue
  • Oliver F. Lange
  • Bernd Reif
Article

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.

Keywords

MAS solid-state NMR Deuteration Protein dynamics Spin relaxation 

Notes

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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Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Juan Miguel Lopez del Amo
    • 1
    • 2
    • 3
    • 5
  • Vipin Agarwal
    • 3
    • 6
  • Riddhiman Sarkar
    • 1
    • 2
  • Justin Porter
    • 1
    • 2
  • Sam Asami
    • 1
    • 2
  • Martin Rübbelke
    • 1
    • 2
  • Uwe Fink
    • 3
  • Yi Xue
    • 4
    • 7
  • Oliver F. Lange
    • 1
    • 2
  • Bernd Reif
    • 1
    • 2
    • 3
  1. 1.Munich Center for Integrated Protein Science (CIPS-M) at Department ChemieTechnische Universität München (TUM)GarchingGermany
  2. 2.Helmholtz-Zentrum München (HMGU)Deutsches Forschungszentrum für Gesundheit und UmweltNeuherbergGermany
  3. 3.Leibniz-Institut für Molekulare Pharmakologie (FMP)BerlinGermany
  4. 4.Department of ChemistryPurdue UniversityWest LafayetteUSA
  5. 5.CIC EnergigneMiñanoSpain
  6. 6.ETH ZurichZuerichSwitzerland
  7. 7.Department of BiochemistryDuke University Medical CenterDurhamUSA

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