Fig. 7: ITC validation of the hydrophobic interactions between D-helix and C-helix.
From: A high affinity switch for cAMP in the HCN pacemaker channels
a Structure of D and E helices (in yellow) along with the C-helix, shown in gray. Side chains of residues involved in the hydrophobic interactions are shown as sticks. Van der Waals surfaces are displayed as spheres (PDB:6UQF). b Examples of ITC thermogram obtained by titrating purified human HCN2 CNBD L670G/L671G with and without the mutation D698A in E-helix with cAMP (see Fig. 4). Top panels show heat changes (μcal/sec) during successive injections of cAMP. Bottom panels show binding curves obtained from data displayed in the upper panel. The peaks were integrated, normalized to cAMP concentration, and plotted against the molar ratio (cAMP/CNBD). Solid red line represents a nonlinear least-squares fit to a single-site binding model yielding, in the present examples, equilibrium dissociation constant (KD) and stoichiometry (N) values as shown. Mean KD, N values and statistical analysis are reported in Supplementary Table 3. c Mean KD values ± SEM from measurements shown in panel B. Values for CNBD ΔC-term and ΔDE are replotted as black and red bars, without data points, from Fig. 2e.
