Ae. Meekhof et Smv. Freund, Separating the contributions to N-15 transverse relaxation in a fibronectin type III domain, J BIOM NMR, 14(1), 1999, pp. 13-22
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).