Other C-helix residues make variable contributions to cAMP binding to the tetrameric HCN2 C-terminus. (A) A ribbon diagram showing the position of I630, D631, L633, and D634, which are located in the C-helix at the distal region of the CNBD. PDB accession no. 1Q50. (B) Bar graphs of heat produced upon progressive injections of cAMP to 200 μM of the mutant or wild-type HCN2 C-terminus, measured by ITC. The inflections in the top plot arise from injections of cAMP, where each inverted peak shows the heat difference between the sample and reference compartment. The peaks decrease in magnitude as binding sites become saturated. The lower plot shows values determined by integration of the area under the peaks from the upper plot versus the ratio of injected ligand to protein. The solid line through the values represents a two-site independent binding site model, which yielded values for affinity and energetics (ΔG, ΔH, and ΔS). (C) Bar graph of thermodynamics parameters that arise from the fitting of the heat values with a two-site independent binding model for the binding of cAMP to the mutants (as in B) and the wild-type HCN2 C-terminus. (D) Bar graphs of binding affinities that arise from the fitting of the heat values with two-independent binding site model for the binding of cAMP to the mutants (as in B) and the wild-type HCN2 C-terminus. Values for binding affinity are shown in Table 1. Values in C and D represent means ± SEM. Each mean was determined from independent ITC binding experiments.