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

In proteins, dynamic mobility is an important feature of structure, stabili ty, and biomolecular recognition. Uniquely sensitive to motion throughout t he milli- to picosecond range, rates of transverse relaxation, R-2, are com monly obtained for the characterization of chemical exchange, and the const ruction of motional models that attempt to separate overall and internal mo bility. 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 doma in from human tenascin. By combining the results of spin-echo (CPMG) and of f-resonance T-1 rho experiments, we present R-2 rates at effective field st rengths 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 stan dard deviation in R-2 for non-exchanging residues is 0.31, indicating that isolated measurements may not be sufficiently accurate for a precise interp retation 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 ex change for five other residues were faster than the sampled range of freque ncies and could not be determined. Averaged 'exchange free' transverse rela xation rates, R-2(0), were used to calculate the diffusion tensor for rotat ional motion. Despite a highly asymmetric moment of inertia, the narrow ang ular dispersion of N-H vectors within the beta sandwich proves insufficient to define deviations from isotropic rotation. Loop residues provide exclus ive evidence for axially symmetric diffusion (D-par/D-per = 1.55).