Proton TOCSY NMR relaxation rates quantitate protein side chain mobility in the Pin1 WW domain.

Protein side chain dynamics play a vital role in many biological processes, but differentiating mobile from rigid side chains remains a technical challenge in structural biology. Solution NMR spectroscopy is ideally suited for this but suffers from limited signal-to-noise, signal overlap, and a need for fractional C or H labeling. Here we introduce a simple strategy measuring initial H relaxation rates during a H TOCSY sequence like DIPSI-2, which can be appended to the beginning of any multi-dimensional NMR sequence that begins on H. The TOCSY RF field compels all H atoms to behave similarly under the influence of strong coupling and rotating frame cross-relaxation, so that differences in relaxation rates are due primarily to side chain mobility. We apply the scheme to a thermostable mutant Pin1 WW domain and demonstrate that the observed H relaxation rates correlate well with two independent NMR measures of side-chain dynamics, cross-correlated C relaxation rates in CH methylene groups and maximum observable J couplings sensitive to the χ side chain dihedral angle (J, J, and J). The most restricted side chains belong to Trp26 and Asn40, which are closely packed to constitute the folding center of the WW domain. None of the other conserved aromatic residues is as immobile as the first tryptophan side chain of the WW domain. The proposed H relaxation methodology should make it relatively easy to measure side chain dynamics on uniformly N- or C-labeled proteins, so long as chemical shift assignments are obtainable.