The adverse cost in free energy of restricting an internal bond rotation in the binding of the peptide ligands N-succinyl-D-alanine and N-fumaryl-D-alanine to the antibiotic Ristocetin A, has been estimated to be 3.7+/-0.9 kJ mol-1 per rotor by comparison of the difference in free energies of binding measured by UV spectrometry.