Figure 2.

A. Schematic representation of the isolated p53_(304-393) peptide (top) and segmentally ¹⁵N-labeled full-length p53 (bottom, labeled at 304-393 through intein splicing) used for NMR experiments. B. 800MHz ¹H-¹⁵N TROSY-HSQC spectra of isolated ¹⁵N/²H p53_(304-393) peptide (black) and ¹⁵N/²H segmentally labeled tetrameric p53 (¹⁵N_(304-393)-p53, red). Resonances appearing in the center of the spectra arise from the disordered NLS and CTD residues, while more dispersed cross-peaks in the spectrum of the p53_(304-393) peptide correspond to the folded TET domain. Assignments labeled in black were obtained from triple resonance spectra of p53_(304-393); assignments labeled in blue were mapped from published data for the isolated TET domain.⁵⁵ Resonances of the TET domain residues are unobservable in segmentally labeled full-length p53 and do not give observable connectivities in triple resonance spectra of the p53_(304-393) peptide. Cross peaks of residues 389 and 391-393 are split due to isomerism of Pro390. All samples were in 20mM Tris, pH7.0, 150mM NaCl, 5% D₂O, and 2mM DTT and NMR spectra were acquired at 25°C. C. Weighted average ¹H, ¹⁵N chemical shift differences between isolated p53_(304-393) peptide and the full-length segmentally labeled p53 tetramer. Resonances of residues 305, 307, and 308 are shifted due to their proximity to the intein splice site. Residues in the CTD that exhibit significant chemical shift changes between spectra of ¹⁵N_(304-393)-p53 and the p53_(304-393) peptide are labeled. Weighted average chemical shift differences were calculated using the equation: $\Delta {\delta }_{{}_{\text{avg}}}=\sqrt{\Delta {\delta }_{{}_{\mathrm{H}}}^{{}^2}+(\frac{\Delta {\delta }_{{}_{\mathrm{N}}}}{5}{)}^2}$