Figure 3

Binding of Asp-Phe (left) and D(pS)DNDL(pS)LEDF (right) to N(31-252). (A) Contour plots of backbone amide resonances of 0.15 mM ¹⁵N-labeled N(31-252) obtained from two-dimensional [¹⁵N, ¹H] TROSY spectra recorded at a range of ligand concentrations. Numbered resonances correspond to the graphs in panel B. Dotted arrows denote the putative directions of shift for resonances 1 and 7. The missing resonances of 1 and 7 in the presence of 0.1 mM D(pS)DNDL(pS)LEDF indicate the binding interaction is in the intermediate exchange regime [36]. (B) Chemical shift perturbations Δδ(N, H)_obs of selected amide resonances of ¹⁵N-labeled N(31-252) as a function of ligand concentration. The symbols represent the experimental data and the lines are global best-fit theoretical curves for a simple binding isotherm. The best-fit values ± standard error of the fit of K_ds were 2.6 ± 0.3 mM for Asp-Phe (left) and 74 ± 4 μM for D(pS)DNDL(pS)LEDF (right).