In vitro analysis of CNBD–TRIP8b_nano interaction. (A–D) Examples of binding of Strep-tagged TRIP8b_nano (WT and mutants) to His6-MBP-tagged human HCN2 CNBD (WT and mutants) measured by ITC. Top: Heat changes (µcal/s) during successive injections of 8 µl of the corresponding TRIP8b_nano (200 µM) into the chamber containing CNBD (20 µM). Bottom: Binding curve obtained from data displayed in the top panel. The peaks were integrated, normalized to TRIP8b_nano concentration, and plotted against the molar ratio (TRIP8b_nano/CNBD). Solid red line represents a nonlinear least-squares fit to a single-site binding model (see Materials and methods).