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

摘要

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.