Journal of Biomolecular NMR

, Volume 45, Issue 1–2, pp 85–98 | Cite as

Partially folded equilibrium intermediate of the villin headpiece HP67 defined by 13C relaxation dispersion

  • Nichole E. O’Connell
  • Michael J. Grey
  • Yuefeng Tang
  • Pallav Kosuri
  • Vesselin Z. Miloushev
  • Daniel P. RaleighEmail author
  • Arthur G. PalmerIIIEmail author


Identification and characterization of ensembles of intermediate states remains an important objective in describing protein folding in atomic detail. The 67-residue villin headpiece, HP67, consists of an N-terminal subdomain (residues 10–42) that transiently unfolds at equilibrium under native-like conditions and a highly stable C-terminal subdomain (residues 43–76). The transition between folded and unfolded states of the N-terminal domain has been characterized previously by 15N NMR relaxation dispersion measurements (Grey et al. in J Mol Biol 355:1078, 2006). In the present work, 13C spin relaxation was used to further characterize backbone and hydrophobic core contributions to the unfolding process. Relaxation of 13Cα spins was measured using the Hahn echo technique at five static magnetic fields (11.7, 14.1, 16.4, 18.8, and 21.1 T) and the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion method at a static magnetic field of 14.1 T. Relaxation of methyl 13C spins was measured using CPMG relaxation dispersion experiments at static magnetic fields of 14.1 and 18.8 T. Results for 13C and 15N spins yielded a consistent model in which the partially unfolded intermediate state of the N-terminal subdomain maintains residual structure for residues near the unprotonated His41 imidazole ring and in the interface between the N- and C-terminal subdomains. In addition, a second faster process was detected that appears to represent local dynamics within the folded state of the molecule and is largely confined to the hydrophobic interface between the N- and C-terminal subdomains.


Carr-Purcell-Meiboom-Gill Chemical exchange Hahn echo NMR spectroscopy Protein folding 



This work was supported by National Institutes of Health (NIH) grants T32 GM008281 (N.E.O) and GM59273 (A.G.P.) and by National Science Foundation Grant MCB 0614365 (D.P.R.). A.G.P. and D.P.R. are members of the New York Structural Biology Center supported by NIH grant GM66354. We thank Jaehyun Cho (Columbia University) for helpful discussions.

Supplementary material

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Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Nichole E. O’Connell
    • 1
  • Michael J. Grey
    • 1
  • Yuefeng Tang
    • 2
  • Pallav Kosuri
    • 1
  • Vesselin Z. Miloushev
    • 1
  • Daniel P. Raleigh
    • 2
    Email author
  • Arthur G. PalmerIII
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular BiophysicsColumbia UniversityNew YorkUSA
  2. 2.Department of ChemistryState University of New York at Stony BrookStony BrookUSA

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