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Journal of Biomolecular NMR

, Volume 63, Issue 2, pp 187–199 | Cite as

13CHD2–CEST NMR spectroscopy provides an avenue for studies of conformational exchange in high molecular weight proteins

  • Enrico Rennella
  • Rui Huang
  • Algirdas Velyvis
  • Lewis E. KayEmail author
Article

Abstract

An NMR experiment for quantifying slow (millisecond) time-scale exchange processes involving the interconversion between visible ground state and invisible, conformationally excited state conformers is presented. The approach exploits chemical exchange saturation transfer (CEST) and makes use of 13CHD2 methyl group probes that can be readily incorporated into otherwise highly deuterated proteins. The methodology is validated with an application to a G48A Fyn SH3 domain that exchanges between a folded conformation and a sparsely populated and transiently formed unfolded ensemble. Experiments on a number of different protein systems, including a 360 kDa half-proteasome, establish that the sensitivity of this 13CHD2 13C–CEST technique can be upwards of a factor of 5 times higher than for a previously published 13CH3 13C–CEST approach (Bouvignies and Kay in J Biomol NMR 53:303–310, 2012), suggesting that the methodology will be powerful for studies of conformational exchange in high molecular weight proteins.

Keywords

CEST Methyl-labeling 13CHD2 Chemical exchange Proteasome Sensitivity enhancement 

Notes

Acknowledgments

This work was supported by grants from the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada. L.E.K holds a Canada Research Chair in Biochemistry.

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Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Enrico Rennella
    • 1
  • Rui Huang
    • 1
  • Algirdas Velyvis
    • 1
  • Lewis E. Kay
    • 1
    • 2
    Email author
  1. 1.Departments of Molecular Genetics, Biochemistry and ChemistryThe University of TorontoTorontoCanada
  2. 2.Program in Molecular Structure and FunctionHospital for Sick ChildrenTorontoCanada

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