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 ¹³C or ²H labeling. Here we introduce a simple strategy measuring initial ¹H relaxation rates during a ¹H TOCSY sequence like DIPSI-2, which can be appended to the beginning of any multi-dimensional NMR sequence that begins on ¹H. The TOCSY RF field compels all ¹H 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 ¹H relaxation rates correlate well with two independent NMR measures of side-chain dynamics, cross-correlated ¹³C relaxation rates in ¹³C^βH₂ methylene groups and maximum observable ³J couplings sensitive to the χ₁ side chain dihedral angle (³J_Hα,Hβ, ³J_N,Hβ, and ³J_CO,Hβ). 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 ¹H relaxation methodology should make it relatively easy to measure side chain dynamics on uniformly ¹⁵N- or ¹³C-labeled proteins, so long as chemical shift assignments are obtainable.