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Non-equilibrium hydrogen exchange for determination of H-bond strength and water accessibility in solid proteins

Abstract

We demonstrate measurement of non-equilibrium backbone amide hydrogen–deuterium exchange rates (HDX) for solid proteins. The target of this study are the slowly exchanging residues in solid samples, which are associated with stable secondary-structural elements of proteins. These hydrogen exchange processes escape methods measuring equilibrium exchange rates of faster processes. The method was applied to a micro-crystalline preparation of the SH3 domain of chicken α-spectrin. Therefore, from a 100% back-exchanged micro-crystalline protein preparation, the supernatant buffer was exchanged by a partially deuterated buffer to reach a final protonation level of approximately 20% before packing the sample in a 1.3 mm rotor. Tracking of the HN peak intensities for 2 weeks reports on site-specific hydrogen bond strength and also likely reflects water accessibility in a qualitative manner. H/D exchange can be directly determined for hydrogen-bonded amides using 1H detection under fast magic angle spinning. This approach complements existing methods and provides the means to elucidate interesting site-specific characteristics for protein functionality in the solid state.

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Acknowledgements

RL acknowledges support from the Deutsche Forschungsgemeinschaft (SFBs 803, project A04, and 749, as well as the Emmy Noether program), the Verband der Chemischen Industrie (VCI) in terms of a Liebig junior group fellowship, and the Excellence Cluster CIPSM. We thank Anne Diehl and Kristina Rehbein (FMP Berlin) for their kind support regarding protein purification.

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Correspondence to Rasmus Linser.

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Grohe, K., Movellan, K.T., Vasa, S.K. et al. Non-equilibrium hydrogen exchange for determination of H-bond strength and water accessibility in solid proteins. J Biomol NMR 68, 7–17 (2017). https://doi.org/10.1007/s10858-017-0110-0

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  • DOI: https://doi.org/10.1007/s10858-017-0110-0

Keywords

  • Solid-state NMR
  • H-bond determination
  • Water accessibility
  • Protein structure
  • Proton detection
  • MAS