Summary
We have used RDCs to detect methyl group motion faster than ms with special focus on the supra-τc window (ns to μs) in ubiquitin. A very wide range of motional amplitudes exist in side chains depending on solvent exposure, residue type and distance to the backbone. Considerable additional dynamics slower than the correlation time τc has been detected. On average, the amplitude of motion expressed in terms of order parameters associated with the supra-τc window contributes as much mobility as the ps-ns motion of the methyl groups measured from relaxation data. The RDC-based order parameters covering motion up to ms and the relaxation derived order parameter show a correlation coefficient of r = 0.72, suggesting that a fair portion of the additional mobility in the supra-τc region can be interpreted mostly as a “broadening” of the amplitude of motion. Amplitude-wise, we find a good match with the order parameters derived from solid-state measurements of CH dipolar couplings (Lorieau and McDermott 2006) and an even better match with those derived from a combined J-coupling and residual dipolar coupling approach (Chou et al. 2003) and supplemented with the fast motions from the backbone.
The model-free order parameters deviate quite significantly from those back-calculated from existing dynamic ensembles, probably due to the limited number of ensemble members. Still, the EROS ensemble, which was refined against the methyl group RDCs also used for this study, agrees best with the model-free order parameters. However, it will be necessary to generate better ensembles to faithfully reflect the side chain dynamics.
A rotameric analysis was performed on several amino acid side chains for which RDC-derived average spherical harmonics of two methyl groups were available. The resulting rotamer distributions were in agreement with conformations representing correlated motions about adjacent torsional angles rather than with a random collection of conformations. While such correlations of rotamer jumps in side chains are not unexpected, especially in the hydrophobic core and are regularly seen for example in phospholipid membranes (Schindler and Seelig 1975; Seelig 1977), there has been weak experimental evidence so far for such correlated motions in proteins.
The development of new sophisticated methods both in NMR and molecular dynamics will be necessary to unravel the complex behavior of side chain motions and their implication in enzyme recognition and protein folding.