Slow motions in chicken villin headpiece subdomain probed by cross-correlated NMR relaxation of amide NH bonds in successive residues.

The villin headpiece subdomain (HP36) is a widely used system for protein-folding studies. Nuclear magnetic resonance cross-correlated relaxation rates arising from correlated fluctuations of two N-H(N) dipole-dipole interactions involving successive residues were measured at two temperatures at which HP36 is at least 99% folded. The experiment revealed the presence of motions slower than overall tumbling of the molecule. Based on the theoretical analysis of the spectral densities we show that the structural and dynamic contributions to the experimental cross-correlated relaxation rate can be separated under certain conditions. As a result, dynamic cross-correlated order parameters describing slow microsecond-to-millisecond motions of N-H bonds in neighboring residues can be introduced for any extent of correlations in the fluctuations of the two bond vectors. These dynamic cross-correlated order parameters have been extracted for HP36. The comparison of their values at two different temperatures indicates that when the temperature is raised, slow motions increase in amplitude. The increased amplitude of these fluctuations may reflect the presence of processes directly preceding the unfolding of the protein.