Journal
JOURNAL OF BIOMOLECULAR NMR
Volume 45, Issue 1-2, Pages 85-98Publisher
SPRINGER
DOI: 10.1007/s10858-009-9340-0
Keywords
Carr-Purcell-Meiboom-Gill; Chemical exchange; Hahn echo; NMR spectroscopy; Protein folding
Categories
Funding
- National Institutes of Health (NIH) [T32 GM008281, GM59273, GM66354]
- National Science Foundation [MCB 0614365]
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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 N-15 NMR relaxation dispersion measurements (Grey et al. in J Mol Biol 355:1078, 2006). In the present work, C-13 spin relaxation was used to further characterize backbone and hydrophobic core contributions to the unfolding process. Relaxation of C-13(alpha) 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 C-13 spins was measured using CPMG relaxation dispersion experiments at static magnetic fields of 14.1 and 18.8 T. Results for C-13 and N-15 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.
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