Figure 2

HDX-MS analysis of NCX_Mj.

(A) Sequences of the α-repeats. Ion-coordinating residues are in red. Helix-breaking residues are in blue. (B) The heat map at 1,200 sec is overlaid on the crystal structure of NCX_Mj (PDB 3V5U) for the apo, Na⁺ -bound and Ca²⁺ -bound forms. The color key indicates the HDX level. The numberings indicate regions with deuterium information (not taking into account the N-terminus amino acids of peptides without amide hydrogens). (C) Deuterium uptake plots for regions 48–52 and 231–237 and for peptides encompassing ion-coordinating residues. Data are presented as the mean ± SD (n = 3). Plots of regions 48–52 and 231–237 were calculated from the difference of deuteration between two overlapping peptides (47–59 & 53–59 and 215–230 & 215–237, respectively). (D) Mass spectra of peptide 215–230 in the presence of Ca²⁺. Note the characteristic EX1 kinetics bimodal isotope distribution and the intensity of the one at high masses increasing with time relative to the one at a low masses.