Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Shape and Dynamics of Calcium-Binding Proteins 291
Fig. 2. Backbone 15 N relaxation time-constants for cbEGF32–33 measured at 11.7 T.
T 2 and T 1 data of cbEGF32 and cbEGF33 in cbEGF32–33 are shown as circles and
stars, respectively and outlying residues are labeled by residue name. The continuous
lines are calculated T 1 and T 2 values as a function of isotropic correlation time τ m and
the order parameter S 2 using the isotropic Lipari and Szabo model (see Eq. 5). Curves
with values of S 2 equalling 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 are shown with the highest and
lowest values labeled accordingly. The arrow indicates the dependence of calculated
T 1 and T 2 values on a decreasing correlation time.
affected by motions on the µs to ms time-scale (R ex). Finding a large proportion
of residues outside the theoretical T 1 and T 2 curves indicates nonideal effects,
possibly because of unspecific aggregation. Residues with increased T 1 and T 2
values, thus shifted to the upper right-hand corner, are subject to motions on
a time-scale that is smaller than the overall tumbling time of the molecule.
[ 1 H]– 15 N heteronuclear NOEs of those residues are expected to be significantly
smaller than the average.
9. Exchange contributions are estimated from model-free analysis (see Subheading
3.4., step 1) or using the quadratic field dependence of 1/T 2 – 1/(2T 1) on B 0
(see Note 5). The slope depends on the value of the chemical shift anisotropy
(CSA) and on exchange contributions, R ex (see Note 10).