A beautiful example of how entropy influences positive cooperativity is in the dimeric enzyme human thymidylate synthase (hTS) . Careful calorimetric measurements at multiple protein concentrations demonstrated positive cooperativity between sites, with the second binding event requiring ~1.5kcal/mol less energy than the first. This difference is driven primarily by entropy. Both crystallography and sensitive NMR measurements revealed no protein structural changes upon ligand binding. However, NMR measurements of side chain order parameters suggest that the first binding event greatly reduces conformational entropy throughout the protein, including at the second binding site. This ordering leads to virtually no change in protein conformational entropy after the second binding event, which is consistent with calorimetry indicating that the second binding event is more favored entropically (Figure ). In addition, NMR relaxation dispersion and Chemical Exchange Saturation Transfer (CEST) experiments revealed a reduction in the exchange of some lowly populated states (~1%) upon ligand saturation at both sites. However, this population change is insufficient to explain the large swing in entropy. The loss of slow dynamics and dramatic change in side chain dynamics can be reconciled through population shuffling, where the fast dynamics undergo reorganization due to the influence of slower movements . Remarkably, the entropy-driven positive cooperativity is highly specific to hTS's substrate dUMP. Even though the substrate (dUMP) and product (dTMP) differ by only a single methyl group, no positive cooperativity is observed by calorimetry for dTMP binding. Again, the NMR data are consistent with this observation, showing no difference in side chain order parameters between hTS-TMP and apo hTS. These observations link the thermodynamics of cooperativity to the pre-ordering of side chain conformations across the protein.