Effects of monovalent ions were assayed using the CysLT1R crystallization construct and two mutants D2.50N and D7.49N based on it. Each receptor construct was purified, as described above for the crystallization setup, using KCl instead of NaCl in hypotonic and solubilization buffers. KCl was replaced with choline chloride during the washing step on the Talon resin. After elution, the protein solution was desalted from imidazole, concentrated to 1 mg ml−1, and diluted 50 times with the assay buffer: 10 μM CPM dye, 25 mM Hepes (pH 7.5), 10% (v/v) glycerol, 0.05% (w/v) DDM, 0.01% (w/v) CHS, containing one of the ions (Na+, K+, or Rb+) at concentrations between 0 and 150 mM. The ionic strength in all samples was compensated by choline chloride to an overall ion concentration of 150 mM. The samples were then incubated at 4°C in the dark for 15 min, and their thermal stability was analyzed using a microscale fluorescence assay as previously described (37). Briefly, fluorescence from the CPM dye (see the purification methods part) was recorded during a temperature ramp from 25° to 80°C with a 1.5°C min−1 rate using a Rotor-Gene Q real-time PCR machine (QIAGEN). Melting curves were collected for n = 3 independent experiments in triplicates. For all constructs, experiments were carried out in the presence of 50 μM zafirlukast. In the case of the CysLT1R crystallization construct, additional experiments without ligand (CysLT1R-apo) were conducted. All melting curves were fitted in GraphPad Prism 7 using the Boltzmann sigmoidal function to obtain the melting temperatures. Plots of ΔTm versus ion concentration revealed nonspecific linear effects for K+ and Rb+ in the case of CysLT1R, and for all ions in the case of D2.50N and D7.49N mutants. To estimate sodium affinity for CysLT1R crystallization construct, ΔTm versus [Na+] curves were fitted in GraphPad Prism 7 using the “One site—Total and nonspecific binding” function, with nonspecific effect taken from K+ data. In all the other cases, the ion effects on thermostability were fitted to linear functions, and the ion effect at 150 mM concentration was calculated. The results were expressed as means ± SD for three independent experiments performed in triplicates.

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