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Rational approaches to ligand design suffer from the fact that our knowledge of the factors determine affinity and specificity of biomolecular interactions is still very rudimentary despite enormous methodological advances in structure determination techniques. Thermodynamic studies show that the affinity is not simply governed by structural features alone, but result as a complex interplay of structure and dynamics. Therefore, lead discovery requires both structural and thermodynamic studies in parallel. Isothermal titration calorimetry (ITC) provides reliable experimental data on the overall thermodynamic parameters that determine the biomolecular recognition process. In a congeneric series of low molecular-weight ligands, the binding properties with respect to trypsin and thrombin has been studied by ITC and crystallography. Crystal structure analysis supports the thermodynamic findings. Based on the crystal structures, it is possible to explain selectivity variations between trypsin and thrombin. The racemic pair of two ligands, obtained from synthesis, can be studied in terms of individual signals in one ITC measurements resulting in a modulated titration curve. In a comparative measurement, the separated enantiomers were studied and show Ki values deviating by a factor of about 1000. Advantage of the ITC experiment is the simultaneous determination of both Ki values in one experiment without separating the enantiomers. Changes of protonation states of the ligands have been discovered upon binding in agreement with the results observed in a previous studies. They result from induced pKa shifts depending on the local environment of the ligand and protein functional groups brought into close contact upon complex formation ( induced dielectric fit ). Such changes in protonation states produce additional heat effects that must be considered before any conclusive factorisation into enthalpic (DH) and entropic (DS) binding contributions can be drawn. After such corrections, trends in both contributions can be interpreted in structural terms with respect to the hydrogen-bond inventory or residual ligand motions.  U. Obst, P. Betschmann, C. Lerner, P. Seiler, F. Diederich, Helv. Chim. Acta 2000, 83, 855 909.  F. Dullweber, M. T. Stubbs, D. Musil, J. Stürzebecher, G. Klebe, JMB 2001, 313, 593 614.