FIGURE 3.

NMR-based identification of the YscP binding surface on YscU_C. A, overlay of the two-dimensional ¹H-¹⁵N HSQC spectra of free ¹⁵N-enriched YscU_C (100 μm) before (blue) and after addition of YscP (400 μm) (red). The spectra were acquired in PBS buffer containing 1 mm TCEP at 37 °C. Solvent-exposed residues that exhibit significant chemical shift perturbations upon YscP addition are marked with arrows. B, compounded chemical shift perturbations (ppm) between free ¹⁵N-labeled YscU_C and ¹⁵N-labeled YscU_C in complex with unlabeled YscP were calculated using Equation 1 and plotted against the YscU_C primary sequence. The threshold value used to define a significant chemical shift perturbation is indicated by the red line, and residues exhibiting significant shifts are represented by green dots except for solvent-accessible amino acid residues with significant chemical shift perturbations (Leu²⁸⁰, Ala³²⁸, Glu³³², Ala³³⁵, and Glu³³⁶), which are represented by red dots. C, display of the YscP binding interface on YscU_C. The structure of YscU_C illustrates the positions of the residues with significant chemical shifts (green) and the solvent-exposed residues with significant chemical shift perturbations (indicated as red ball and sticks). D, determination of the K_d value for the YscP-YscU_C interaction obtained by using Equation 2 to fit the changes in the chemical shifts of the methyl groups of YscU_C induced by YscP addition. Plots for three peaks in the methyl region are shown in black, red, and green, respectively. The error bars represent S.D.