Chloride Binding to CLC-ec1 Mutant

Cl^– binding to the A399C/A432C mutant of CLC–ec1 was carried out as described^{36 (link),38 (link)} using a nanoITC instrument (TA Instruments). For these experiments, the final purification step of the protein was purified over a gel filtration column pre-equilibrated in 100 mM Na-K-Tartrate, 20 mM Hepes, 50 μM DMNG, pH 7.5 (Buffer B₀) and concentrated to 50-195 μM. The A399C/A432C mutant was not stable in the absence of Cl^– upon incubation with Hg²⁺. Therefore, in this case the Hg²⁺ reaction was carried out in Buffer B. Excess Cl^– and Hg²⁺ were successively removed by running the protein over a gel filtration column pre-equilibrated in buffer B₀. The injection syringe was filled with buffer B₀ with 50 mM KCl added, to achieve final Molar Ratios of 100-250. Each experiment consisted of 30-48 injections of 1 μl of the ligand solution at 3-4 min intervals into the experimental chamber kept under constant stirring at 350 rpm and at 25.0±0.1 °C. All solutions were filtered and degassed prior to use. The ITC data was fit to a single site Wiseman isotherm as described^{36 (link),38 (link)} using the NanoAnalyze program from TA instruments.