Biochemical characterization of HCN2 CNBD and TRIP8b_nano proteins. (A) Representative SEC profiles of human HCN2 CNBD WT (black), K666E (red), R650E (blue), and N547D/A548D (green) mutants. (B) Representative SEC profiles of TRIP8b_nano WT (black) and D252N (orange) mutant. Both CNBD and TRIP8b_nano mutant proteins show a symmetrical and monodisperse peak comparable to their respective WT proteins, indicating that mutations did not alter the biochemical features of the proteins. Peak fractions used for the ITC assay are delimited by the gray dotted lines. mAU, milli-arbitrary units.