Journal of Biomolecular NMR

, Volume 50, Issue 1, pp 13–18 | Cite as

Measuring 1HN temperature coefficients in invisible protein states by relaxation dispersion NMR spectroscopy

  • Guillaume Bouvignies
  • Pramodh Vallurupalli
  • Matthew H. J. Cordes
  • D. Flemming Hansen
  • Lewis E. Kay


A method based on the Carr-Purcell-Meiboom-Gill relaxation dispersion experiment is presented for measuring the temperature coefficients of amide proton chemical shifts of low populated ‘invisible’ protein states that exchange with a ‘visible’ ground state on the millisecond time-scale. The utility of the approach is demonstrated with an application to an I58D mutant of the Pfl6 Cro protein that undergoes exchange between the native, folded state and a cold denatured, unfolded conformational ensemble that is populated at a level of 6% at 2.5°C. A wide distribution of amide temperature coefficients is measured for the unfolded state. The distribution is centered about –5.6 ppb/K, consistent with an absence of intra-molecular hydrogen bonds, on average. However, the large range of values (standard deviation of 2.1 ppb/K) strongly supports the notion that the unfolded state of the protein is not a true random coil polypeptide chain.


CPMG relaxation dispersion Temperature coefficients Amide protons Cold denaturation 



G.B. acknowledges the European Molecular Biology Organization and the Canadian Institutes of Health Research (CHIR) for post-doctoral fellowships. D.F.H was supported by a post-doctoral fellowship from the CIHR. This work was supported by grants from the CIHR and the Natural Sciences and Engineering Research Council of Canada (LEK) and the NIH (GM066806 to M.H.J.C.). L.E.K. holds a Canada Research Chair in Biochemistry.

Supplementary material

10858_2011_9498_MOESM1_ESM.docx (783 kb)
Supplementary material 1 (DOCX 783 kb)


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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Guillaume Bouvignies
    • 1
  • Pramodh Vallurupalli
    • 1
  • Matthew H. J. Cordes
    • 2
  • D. Flemming Hansen
    • 1
    • 3
  • Lewis E. Kay
    • 1
  1. 1.Departments of Molecular Genetics, Biochemistry and ChemistryThe University of TorontoTorontoCanada
  2. 2.Department of Chemistry and BiochemistryThe University of ArizonaTucsonUSA
  3. 3.Institute of Structural and Molecular BiologyUniversity College LondonLondonUK

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