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Journal of Biomolecular NMR

, Volume 57, Issue 1, pp 47–55 | Cite as

Protein conformational exchange measured by 1H R1ρ relaxation dispersion of methyl groups

  • Ulrich Weininger
  • Annica T. Blissing
  • Janosch Hennig
  • Alexandra Ahlner
  • Zhihong Liu
  • Hans J. Vogel
  • Mikael Akke
  • Patrik Lundström
Article

Abstract

Activated dynamics plays a central role in protein function, where transitions between distinct conformations often underlie the switching between active and inactive states. The characteristic time scales of these transitions typically fall in the microsecond to millisecond range, which is amenable to investigations by NMR relaxation dispersion experiments. Processes at the faster end of this range are more challenging to study, because higher RF field strengths are required to achieve refocusing of the exchanging magnetization. Here we describe a rotating-frame relaxation dispersion experiment for 1H spins in methyl 13CHD2 groups, which improves the characterization of fast exchange processes. The influence of 1H–1H rotating-frame nuclear Overhauser effects (ROE) is shown to be negligible, based on a comparison of R 1ρ relaxation data acquired with tilt angles of 90° and 35°, in which the ROE is maximal and minimal, respectively, and on samples containing different 1H densities surrounding the monitored methyl groups. The method was applied to ubiquitin and the apo form of calmodulin. We find that ubiquitin does not exhibit any 1H relaxation dispersion of its methyl groups at 10 or 25 °C. By contrast, calmodulin shows significant conformational exchange of the methionine methyl groups in its C-terminal domain, as previously demonstrated by 1H and 13C CPMG experiments. The present R 1ρ experiment extends the relaxation dispersion profile towards higher refocusing frequencies, which improves the definition of the exchange correlation time, compared to previous results.

Keywords

Relaxation dispersion Conformational exchange Rotating-frame relaxation 

Notes

Acknowledgments

This research was supported by the Swedish Research Council (621-2010-4912; 822-2005-2915; 621-2012-5136), the Göran Gustafsson Foundation for Research in Natural Sciences and Medicine, and the Knut and Alice Wallenberg Foundation. UW was supported by an EMBO long-term fellowship and JH was supported by a Forum Scientium Award. HJV holds a Scientist award from Alberta Innovates Health Solutions. We thank SWEDSTRUCT for access to the 900 MHz spectrometer at the Swedish NMR Centre.

Supplementary material

10858_2013_9764_MOESM1_ESM.docx (963 kb)
Figures showing relaxation dispersion profiles for all data acquired on ubiquitin and CaM. (DOCX 963 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Ulrich Weininger
    • 1
  • Annica T. Blissing
    • 2
  • Janosch Hennig
    • 2
  • Alexandra Ahlner
    • 2
  • Zhihong Liu
    • 3
  • Hans J. Vogel
    • 3
  • Mikael Akke
    • 1
  • Patrik Lundström
    • 2
  1. 1.Department of Biophysical Chemistry, Center for Molecular Protein ScienceLund UniversityLundSweden
  2. 2.Division of Molecular Biotechnology, Department of Physics, Chemistry and BiologyLinköping UniversityLinköpingSweden
  3. 3.Department of Biological Sciences, Biochemistry Research GroupUniversity of CalgaryCalgaryCanada

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