FIGURE 4.

ITC measurement of otoferlin C2F domain calcium binding. Affinity-purified wild-type GST-C2F otoferlin protein and a fusion protein representing an otoferlin pachanga mutant (see text) were cleaved with PreScission protease (GE Healthcare) to remove the GST tag. Individual 5-μl volumes of a 5–10 mm CaCl₂ solution prepared in ITC buffer were incrementally injected into an ITC chamber (Microcal ITC200) containing a 50–100 μm concentration of the purified C2F domain solution at 25 °C, and the heat change was recorded. Negative controls were either C2F samples injected with ITC buffer alone or ITC buffer injected with 10 mm CaCl₂. A, raw data obtained in the titration of C2F domain by calcium. The CaCl₂ solution was injected 50 times, and the response was recorded. The binding reaction was endothermic. Fitted data for the integrated heat change on injection of CaCl₂ were plotted after subtraction from control values. B, curve fitting for A performed with Microcal Origin software using a two-calcium binding model. One main binding site was predicted with a K_D of 267 μm. A second predicted site was in the mm range, which likely reflects nonspecific heat changes. C and D, raw data and corresponding curve fitting, respectively, obtained for the C2F pachanga point mutation (Asp to Gly). Experimental conditions were identical to those for the wild-type C2F. Data indicate a very small endothermic response with little reduction in the titration, suggesting either no binding or low affinity binding. Minimal changes in integrated heat did not allow determination of affinity values. E and F, raw data for control experiments. E, increments of ITC buffer alone (no added CaCl₂) added to a 50 μm solution of wild-type C2F. No heat response was associated with the injections. F, ITC buffer alone incrementally injected with ITC buffer containing 10 mm CaCl₂. A negligible endothermic response was observed for each injection, probably indicating the heat of calcium dilution.