-PPARγ LBD (expressed and purified according to ref. 5)
-Ligand A and B (synthesized according to ref. 2)
-Hepes buffer, 20 mM, pH 8 (SIGMA).
-Dimethylsulfoxide (DMSO) (SIGMA).
Things to be considered before beginning.
To obtain a complete binding isotherm the solution in the syringe should be more concentrated than that in the cell, so that at the end of experiment the molar ratio ligand/protein (or protein/ligand in the reverse experiment) is 2:1, to ensure the saturation. Considering volumes of the sample near 200 μL, a typical injection volume of 2 μL, and 20 injections, it is advisable to use a concentration of reactant in the syringe 10 times more concentrated than that in the cell.
CRITICAL STEP It is important to use the same protein expression batch in all the experiments, to be sure of the relative ratios of the direct and reverse titration. It is possible that the stoichiometry n value of the direct experiment is not a unit value, denoting that the real concentration of functional protein is different from that inserted. In this case, it is very important to use the same protein expression batch in the reverse experiment in order to correctly compare the relative n values of the two experiments.
CRITICAL STEP In the experiment with the pre-equilibrated protein, the second ligand (ligand 2)must be added in slight excess with respect to the protein to be sure that the first site is completely saturated. Let equilibrate the mix of protein and ligand for 2 h.
CRITICAL STEP Moreover, the Ka of the second ligand must be higher than that of the first ligand, to be sure that, at the concentration set for the titration, the second ligand is not diplaced from its site.
Choose the appropriate pH conditions to ensure protein stability and functionality. Choose the suitable pH buffer for the protein in order to minimize artifactual heats of buffer ionization and to mantain the protein stability (in the described experiment we used Hepes buffer that has a ΔHion of 5.7 kcal/mol) (reference).
Solve the ligand in pure DMSO if not soluble in aqueous buffers. Then use the elution buffer obtained from the protein dialysis made to reach the concentration set for the experiment.
CRITICAL STEP Calculate the final percentage of DMSO in the ligand solution and add DMSO to the protein solution in order to have the same DMSO percentage in both solutions. Consider that DMSO has a strong effect on the ITC signal. It is important that the DMSO percentage is not higher than 5% (many proteins are stable up to 2-5% of DMSO). Difference in salt or solvent composition could be cause of background heats of mixing masking the heats of the reaction of interest.
Include a reducing agent to the protein solution, such as TCEP 1mM, to avoid protein aggregation.
Solutions used in the PPARγ experiment with ligand 1 and 2. Almost 10 mg of protein expressed from 500 ml of cell culture were used for all the experiments. PPARγ was extensively dialyzed against buffer Hepes 20 mM, pH 8.0, TCEP 1mM, with Amicon Ultra Filters 10K, and the final exchange buffer was then used to dilute the ligand stock solution (50 mM in DMSO). The protein was diluted with Hepes buffer to obtain the suitable concentration for the experiment. DMSO was added to the protein solution at the same percentage of the ligand solution (below 1%). Samples were centrifuged before the experiments to eliminate possible aggregates. Protein and ligand solutions were degassed before use. The protein concentration was determined by UV280 spectroscopy (ε280=12045 M-1cm-1 for PPARγ-LBD).