Proteins with multiple binding sites exhibit a complex behavior that depends on the intrinsic affinities for each site and the energetic communication between the sites. The contributions from intrinsic affinity and cooperativity are difficult to deconvolute using conventional binding experiments that lack information about the occupancies of individual sites. Here, we report the concerted use of NMR and isothermal titration calorimetry to determine the intrinsic and cooperative binding free energies for a ligand-protein complex. The NMR measurements provided the site-specific information necessary to resolve the binding parameters. Using this approach, we observed that human ileal bile acid binding protein binds two molecules of glycocholic acid with low intrinsic affinity but an extraordinarily high degree of positive cooperativity. The highly cooperative nature of the binding provides insights into the protein's biological mechanism. With ongoing improvements in sensitivity and resolution, NMR methods are becoming more amenable to dissecting the complex binding energetics of multisite systems.
|Number of pages||6|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Feb 19 2002|