Thermodynamic analyses reveal role of water release in epitope recognitionby a monoclonal antibody against the human guanylyl cyclase C receptor

The thermodynamics of a monoclonal antibody (mAb)-peptide interaction have been characterized by isothermal titration microcalorimetry, GCC:B10 mAb, g enerated against; human guanylyl cyclase C, a membrane-associated receptor and a potential marker for metastatic colon cancer, recognizes the cognate peptide epitope HIPPENIFPLE and its two contiguous mimotopes, HIPPEN and EN IFPLE, specifically and reversibly. The exothermic binding reactions betwee n 6.4 and 42 degrees C are driven by dominant favorable enthalpic contribut ions between 20 and 42 degrees C, with a large negative heat capacity (Delt a C-p) of -421 +/- 27 cal mol(-1) K-1. The unfavorable negative value of en tropy (Delta S-b(0)) at 25 degrees C, an unusual feature among protein-prot ein interactions, becomes a positive one below an inversion temperature of 20.5 degrees C, Enthalpy-entropy compensation due to solvent reorganization accounts for an essentially unchanged free energy of interaction (Delta De lta G(b)(0) congruent to 0). The role of water molecules in the recognition process was tested by coupling an osmotic stress technique with isothermal titration microcalorimetry. The results provide direct and compelling evid ence that GCC:B10 mAb recognizes the peptides HIPPENIFPLE, HIPPEN, and ENIF PLE differentially, with a concomitant release of variable and nonadditive numbers of water molecules (15, 7, and 3, respectively) hom the vicinity of the binding site.