Dr. Brooks started his investigation with a standard ITC experiment (curve 1) where the primary ligand L1 is titrated into the sample cell with the receptor protein. From this experiment, he wants to get thermodynamic and structural information of the interaction. Fortunately, we count with some preliminary information that will make the analysis easier:

• Each protein is able to simultaneously bind up to two ligand molecules.
• The concentration of active protein in this experiment is not accurate. The corresponding uncertainty is about 10%. To take this in account throughout the fitting, set r(M) as a variable parameter by checking the corresponding “fit” box.
• No ligand dilution blank experiment was performed. To take this in account throughout the fitting, set Qdil as a variable parameter by checking the corresponding “fit” box.

As a second step of the investigation Dr. Brooks performed three more ITC experiments: an experiment of L2, the weak ligand, titrated into the protein sample (curve 2) and two competitive experiments where a mixture of L1 and L2 is titrated into the receptor protein solution (curve 3 and curve 4).

• The protein is also able to simultaneously bind up to two L2 molecules.
• The concentration of the active protein in these three experiments is accurate. Therefore, r(M) of curves 2,3 and 4 should be constant and equal to 1 throughout the fitting.
• This time, a dilution blank experiment was performed and subtracted from each titration experiment. Therefore, Qdil of curves 2,3 and 4 should be constant and equal to 0 throughout the fitting.