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Journal of Biomolecular NMR

, Volume 52, Issue 3, pp 233–243 | Cite as

Estimating side-chain order in methyl-protonated, perdeuterated proteins via multiple-quantum relaxation violated coherence transfer NMR spectroscopy

  • Hechao Sun
  • Raquel Godoy-Ruiz
  • Vitali TugarinovEmail author
Article

Abstract

Relaxation violated coherence transfer NMR spectroscopy (Tugarinov et al. in J Am Chem Soc 129:1743–1750, 2007) is an established experimental tool for quantitative estimation of the amplitudes of side-chain motions in methyl-protonated, highly deuterated proteins. Relaxation violated coherence transfer experiments monitor the build-up of methyl proton multiple-quantum coherences that can be created in magnetically equivalent spin-systems as long as their transverse magnetization components relax with substantially different rates. The rate of this build-up is a reporter of the methyl-bearing side-chain mobility. Although the build-up of multiple-quantum 1H coherences is monitored in these experiments, the decay of the methyl signal during relaxation delays occurs when methyl proton magnetization is in a single-quantum state. We describe a relaxation violated coherence transfer approach where the relaxation of multiple-quantum 1H–13C methyl coherences during the relaxation delay period is quantified. The NMR experiment and the associated fitting procedure that models the time-dependence of the signal build-up, are applicable to the characterization of side-chain order in [13CH3]-methyl-labeled, highly deuterated protein systems up to ~100 kDa in molecular weight. The feasibility of extracting reliable measures of side-chain order is experimentally verified on methyl-protonated, perdeuterated samples of an 8.5-kDa ubiquitin at 10°C and an 82-kDa Malate Synthase G at 37°C.

Keywords

Relaxation violated coherence transfer NMR spectroscopy Methyl labeling on the deuterated background Methyl three-fold axis order parameters Magnetically equivalent spin-system 

Notes

Acknowledgments

The authors thank Dr. Daoning Zhang (University of Maryland) for providing the sample of {U-[2H;15N]; Ileδ1-[13CH3]; Leu,Val-[13CH3,12CD3]}-labeled human ubiquitin. The authors are grateful to Prof. Lewis E. Kay (University of Toronto, Canada) for useful and stimulating discussions.

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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Hechao Sun
    • 1
  • Raquel Godoy-Ruiz
    • 1
  • Vitali Tugarinov
    • 1
    Email author
  1. 1.Department of Chemistry and Biochemistry, Center for Biomolecular Structure and OrganizationUniversity of MarylandCollege ParkUSA

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