(260g) Probing Into the Mechanism for Bacterial Biosensor for Nuclear Receptor Ligand

Our lab has developed series of bacterial biosensor for the screening of the nuclear receptor ligands. The basic feature of these biosensors lies in the insertion of the ligand binding domain (LBD) of a nuclear receptor into between an engineered intein linked to maltose binding protein (MBP) and a thymidylate synthase (TS) reporter enzyme. TS deficient E. coli transformed with these fusion constructs exhibited enhanced growth phenotype in response to the presence of agonist, while it showed inhibited growth upon the antagonist binding. Up to now the mechanism for the sensitivity behavior of these biosensors remained illusive. Here we have attempted to disclose the mechanism by using site-directed mutations and domain depletion. N440A mutation and G422D mutation demonstrated that the TS activation resulted from the ligand causing allosteric structural variation rather than the release of active TS by intein. Deletion of the intein domain caused an overgrowth phenotype in the absence of ligands, and abolished sensor behavior. On the other hand, MBP domain removal blocked the cell growth completely. Interestingly, the removal of both intein domain and MBP domain allowed a slight response to the ligands, but also resulted in the overgrowth phenotype. These observations indicate that the intein plays a critical role in the signal transmission from LBD to TS. We also found that the spacer connecting LBD and intein participated in the formation of sensor signal. It was hypothesized that the introduction of flexible linkers might be able to improve the sensitivity of the sensor to the ligands by destabilizing the switch domain of LBD and then stabilizing it upon ligand binding. The incubation temperature significantly influenced the growth phenotype and sensor behavior. It was deduced that there might be an equilibrium between the active conformation and inactive conformation of LBD that can be shifted by temperature variation and ligand-binding. The proposed mechanism provided a clue for the rational design of biosensor based on a fusion protein.