FIGURE 5.

Disruption of YscP binding by the A335N replacement in YscU_C. A, overlay of two-dimensional ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled YscU_C^WT (blue) and YscU_C^A335N (red) at concentrations of 100 μm. The buffer consisted of PBS and 1 mm TCEP at 37 °C. Solvent-exposed residues that exhibit significant chemical shift perturbations upon mutation are indicated with arrows. B, compounded chemical shift perturbations (ppm) between ¹⁵N-labeled YscU_C^WT and ¹⁵N-labeled YscU_C^A335N calculated by Equation 1 and plotted against the YscU residue number. The threshold value used to define significant chemical shift changes is indicated by the red line. The residues with the largest chemical shift perturbations (Ser²³⁶, Leu²⁸², Glu³³⁶, Leu³³⁸, and Arg³³⁹) cluster around the mutation site and are shown in blue; position 335 is shown in red on the YscU_C crystal structure in C. D, overlay of two-dimensional ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled YscU_C^A335N at a concentration of 100 μm alone before (blue) and after addition of unlabeled YscP (red) at a molar ratio of 1:4 in PBS.