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.

To construct a cyclin-luciferase fusion protein (pSP cyc-luc), the N-terminal sequence of Xenopus laevis cyclin B1, including amino acids 2-97, was amplified by PCR, digested with BstEII, and ligated into the pSP-lucNF expression vector (Promega).The resulting vector was sequence verified. The fusion protein was expressed by coupled in vitro transcription and translation in reticulocyte lysate using the SP6-TNT Coupled Reticulocyte Lysate System (Promega) and flash frozen in liquid nitrogen until the time of use. The parental pSP-lucNF vector was used to express unmodified luciferase.A vector for expression of cyclin-luciferase in E. coli (pET cyc-luc) was also constructed; this protein behaved identically in all assays to the protein expressed in reticulocyte lysate, but could be made in higher quantities necessary for screening. pSP cyc-luc was digested with HindIII and XhoI. The resulting 1949 bp fragment containing the cyclin B1-luciferase sequence was ligated into the pET 28b expression vector