Figure 6.

NMR titration experiments using the ¹⁵N-labeled SB-HMG fragment. (A) Chemical-shift changes in the titration of nonphosphorylated AID fragment (AID) to ¹⁵N-labeled SB-HMG; AID/SB-HMG molar ratios were 0.0 (black), 0.3 (red), 0.7 (blue), 1.0 (orange), 1.5 (pink), 2.0 (green), and 3.0 (yellow). (B) The corresponding spectral changes observed for phosphorylated AID (pAID); pAID/SB-HMG ratios were 0.00 (black), 0.07 (red), 0.21 (blue), 0.49 (orange), 0.70 (pink), and 1.04 (green). (C and D) Histograms of the chemical-shift differences in SB-HMG upon binding with AID (C) and pAID (D). Chemical-shift differences are plotted against residue numbers of SB-HMG. Red bars indicate that the chemical-shift differences are over the average plus one standard deviation. Short bars in cyan and purple along the x axis represent prolines and the residues for which assignment information was missing. Chemical-shift changes are shown for the representative residues in SB-HMG in the titrations with AID (E) and pAID (F). The numerically determined dissociation constant, K_D, and number of binding sites, n, for the AID and pAID titration experiments were K_D = 9.7 ± 0.3 μM, n = 2.0 ± 0.1 and K_D = 1.01 ± 0.02 μM, n = 3.9 ± 0.1, respectively. Values for K_D and n were determined by global fitting using the residues marked by red bars in C and D (see Supporting Material).