In a crystallographic fragment screen in the ATP-site of Protein Kinase A (PKA), benzoic acid was identified as a binder to its hinge region. The interaction pattern (see Figure ) of benzoic acid complexing PKA suggests that the protonated form of benzoic acid binds to PKA. This would only occur if the pKa value of the carboxylic acid group of benzoic acid, which ranges from 4.0 to 4.2 in its unbound state in aqueous solution (as noted in ), shows a significant shift in pKa value upon binding to the protein PKA, resulting in a protonation event during the complexation. Such a protonation event can be detected by isothermal titration calorimetry (ITC), for which two different approaches exist for low-affinity binders such as fragments: low c value titration and displacement titration (see Materials and methods).