Figure 2. Two conserved residues of PBC contribute to cAMP binding affinity but do not alter channel opening. (A) A ribbon diagram showing the position of R591 and T592, which are deep in the phosphate binding region of the CNBD and were individually substituted with alanine. PDB accession no. 1Q50. Their side chains form hydrogen bonds with the phosphate oxygen, shown in green. (B) Plots of heat produced upon progressive injections of cAMP to 200 μM of R591A and T592A 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 graphs of high-affinity (left) and low-affinity (middle) binding values and thermodynamics (right) that arise from the fitting of the heat values with a two-site independent binding model for the binding of cAMP to the two mutant C-termini (as in B) and the wild-type HCN2 C-terminus. Values represent means ± SEM. Each mean was determined from independent ITC binding experiments. Values for binding affinity and stoichiometry are also given in Table 1. (D) Concentration–response data and theoretical curves for the shift in activation gating produced by cAMP. The solid red lines are produced by the mathematical model using the experimental binding affinities obtained by ITC for the wild-type protein and fitted to the concentration–response data for the wild-type channel (red squares). The solid blue lines are produced by the mathematical model using the experimental binding affinities obtained for cAMP binding to the mutant proteins but using the gating parameters that were obtained from the fit of the model to the wild-type data. Note how well the theoretical blue curves fit the experimental concentration–response data for the mutant channels (blue squares). The concentration–response data were reproduced from Zhou and Siegelbaum (2007).
Figure 2.

Two conserved residues of PBC contribute to cAMP binding affinity but do not alter channel opening. (A) A ribbon diagram showing the position of R591 and T592, which are deep in the phosphate binding region of the CNBD and were individually substituted with alanine. PDB accession no. 1Q50. Their side chains form hydrogen bonds with the phosphate oxygen, shown in green. (B) Plots of heat produced upon progressive injections of cAMP to 200 μM of R591A and T592A 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 graphs of high-affinity (left) and low-affinity (middle) binding values and thermodynamics (right) that arise from the fitting of the heat values with a two-site independent binding model for the binding of cAMP to the two mutant C-termini (as in B) and the wild-type HCN2 C-terminus. Values represent means ± SEM. Each mean was determined from independent ITC binding experiments. Values for binding affinity and stoichiometry are also given in Table 1. (D) Concentration–response data and theoretical curves for the shift in activation gating produced by cAMP. The solid red lines are produced by the mathematical model using the experimental binding affinities obtained by ITC for the wild-type protein and fitted to the concentration–response data for the wild-type channel (red squares). The solid blue lines are produced by the mathematical model using the experimental binding affinities obtained for cAMP binding to the mutant proteins but using the gating parameters that were obtained from the fit of the model to the wild-type data. Note how well the theoretical blue curves fit the experimental concentration–response data for the mutant channels (blue squares). The concentration–response data were reproduced from Zhou and Siegelbaum (2007).

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