Figure 5.

Mapping transient R_D:RegA_C interactions by HDXMS. (A) Difference plot, plotting absolute difference in deuterons (y axis) between apo R_D and R_D:RegA_C binary complex for each pepsin fragment peptide listed from the N- to C-terminus (x axis). Points in the negative scale (shaded blue) represent a decrease in deuterium exchange upon RegA_C binding, while points in the positive scale (shaded red) represent increases in deuterium exchange upon RegA_C binding. Peptides spanning the cAMP binding pocket in CNB:A and B are highlighted (red). Each deuterium labeling time for every peptide is depicted and colored according to key. A difference of ±0.5 Da is considered significant and is shown (red dashed line). Plots were generated using the software DYNAMX. (B) (Top panel) Stacked mass spectra of peptides spanning residues 266–279 in CNB-B domain of R_D. Bimodal spectra from apo R_D are compared with bimodal spectra from the binary complex. Mass spectra are stacked in order of increasing deuterium-labeling time as shown. (Bottom panel) Deuterium exchange plot comparing apo R_D and R_D:RegA_C for the same peptide is depicted. (C) Representative mass spectra and deuterium exchange plots for two peptides spanning residues 171–183 and 184–201 are shown comparing R_D apo state and R_D:RegA_C state. Semilog deuterium exchange plots are generated with x axis in the log scale and y axis in linear scale; error bars are indicated. All plots are generated in GRAPHPAD PRISM 6.