FIG. 5.

Titration of ¹⁵N-PX with N_TAIL. (A) Superimposition of seven ¹H-¹⁵N HSQC spectra at relative ratios of N_TAIL to PX that are indicated on the top right of the figure. This zoom illustrates PX resonances that shift (bold), broaden (boxed), or disappear (italic) upon N_TAIL binding, which is typical for intermediate exchange. (B) Backbone amide chemical shift variations between the free and bound forms of PX. Chemical shift changes larger than the average chemical shift change plus 1 standard deviation (0.065 ppm) were considered significant and are represented by the orange (>0.065 ppm) and red (>0.13 ppm) bars. Some residues from the His tag (<474) are missing in the spectrum; all of the other gaps represent proline residues. Green bars correspond to residues showing extreme line broadening. (C) Ribbon representation of the lowest-energy structure of the PX C-subdomain (Protein Data Bank accession no. 1R4G), color coded according to the chemical shift changes shown in panel B. Balls represent the backbone amide groups that are affected by the presence of N_TAIL. (D) Relative chemical shift changes for V559 versus the ratio between ¹⁵N-PX and N_TAIL concentrations. The intersection at a ratio of 1:1 implies that one PX molecule binds to one N_TAIL molecule.