Interestingly, this evolutionary model has been experimentally validated using rational protein design and directed evolution. Höcker "et al." first fused two C-terminal halves of HisF, yielding HisF-CC. This construct was then stabilized by the insertion of an internal salt-bridge, yielding HisF-C*C. Further stepwise stabilization and solubilization of HisF-C*C was achieved by optimizing the half-barrel interface, generating HisF-C**C and HisF-C***C, respectively. The crystal structure of HisF-C***C revealed a 2-fold symmetric TIM barrel, validating the possibility of natural domain fusion. Moreover, Höcker created the first chimeric HisAF and HisFA TIM barrels using HisA and HisF half-barrels. These experiments led to the proposal of a novel means of diversification and evolution of TIM-barrel enzymes through the exchange of (βα)4 half-barrel domains amongst preexisting TIM barrels. In accordance with this idea, a high catalytic activity on the HisAF construct was established. Similarly, chimeric βα5-flavodoxin-like fold (CheY)/HisF TIM barrels, and a perfectly 2-fold symmetric HisF-based TIM barrel have also been created.