Separating the contributions to 15N transverse relaxation in a fibronectin type III domain

In proteins, dynamic mobility is an important feature of structure, stability, and biomolecular recognition. Uniquely sensitive to motion throughout the milli- to picosecond range, rates of transverse relaxation, R-2, are commonly obtained for the characterization of chemical exchange, and the construction of motional models that attempt to separate overall and internal mobility. We have performed an in-depth study of transverse relaxation rates of backbone N-15 nuclei in TNfn3(1-90), the third fibronectin type III domain from human tenascin. By combining the results of spin-echo (CPMG) and off-resonance T-1 rho experiments, we present R-2 rates at effective field strengths of 2 to 40 krad/s, obtaining a full spectrum of 16 independent R-2 data points for most residues. Collecting such a large number of replicate measurements provides insight into intrinsic uncertainties. The median standard deviation in R-2 for non-exchanging residues is 0.31, indicating that isolated measurements may not be sufficiently accurate for a precise interpretation of motional models. Chemical exchange events on a timescale of 570 mu s were observed in a cluster of residues at the C terminus. Rates of exchange for five other residues were faster than the sampled range of frequencies and could not be determined. Averaged 'exchange free' transverse relaxation rates, R-2(0), were used to calculate the diffusion tensor for rotational motion. Despite a highly asymmetric moment of inertia, the narrow angular dispersion of N-H vectors within the beta sandwich proves insufficient to define deviations from isotropic rotation. Loop residues provide exclusive evidence for axially symmetric diffusion (D-par/D-per = 1.55).