Creation of Novel Allosteric Regulation of Proteins for Synthetic Biology Based on a New Concept of Thermodynamic Model

A novel strategy to engineer ligand-induced allosteric regulation is proposed based on a new concept of thermodynamic model of protein dynamics with the aim to create novel allosteric regulation of proteins, especially regulated by non-natural ligands. In this model, allosteric process upon ligand binding is considered as the energetic development of a thermodynamic system proceeding from an initial ensemble to a final ensemble. The key feature of the thermodynamic model is that the allosteric process is divided into two sub-processes: conformational change and molecular binding and the allosteric process upon ligand binding is regarded as the overall effects of the two sub-processes. As a consequence of the separation of the allosteric process, the ligand-induced allosteric regulation can be examined from different perspectives. To prove the concept, aspartokinase III (AK3) from E. coli was used as a as a model system. After construction of the thermodynamic model for E. coli AK3, the thermodynamic model was first used to guide the design for enhancement of the allosteric effect of AK3 by its natural effector. Then the new concept was employed to establish new allosteric effects for both the natural and the non-natural ligands. For this purpose, an allosteric determinant was identified and followed by different strategies of focused protein design. Experimental results showed that the natural ligand was successfully altered from an inhibitor to an activator and both inhibition and activation effects have been established for the non-natural ligand guided by the same allosteric determinant. These results indicate that the new concept is powerful in constructing novel switchable biomolecules with potential applications in synthetic biology.