The ability to predict protein-ligand binding affinities is a difficult and elusive goal in the field of molecular recognition. Models exist to predict binding energetics; however, they are not always capable of considering the incidental events in ligand-binding due to the tendency of the Gibbs free energy (ΔG°) to lack a correlation with enthalpy (ΔH°), entropy (ΔS°), or both. Binding studies of various pYEEI-derived peptidomimetic ligands to the Src SH2 domain were evaluated to investigate the effects of structural changes on protein-ligand binding energetics. The effect of preorganizing the pYEEI ligand into its binding conformation was analyzed by substituting the isoleucine residue with a conformationally constrained amino acid analog as well as a flexible analog. Isothermal titration calorimetry studies were performed to assess the effects of ligand structure on protein-ligand binding energetics.