The light-oxygen-voltage (LOV)-histidine kinase from Brucella, a virulence factor suitable for a sustained blue-light response: Unlocking the signaling state.

摘要

My dissertation begins with the discovery of the blue-light-activated autophosphorylation of four different in vitro expressed bacterial LOV-histidine kinases. In the case of Brucella, we show associated effects on virulence (Chapter 2). Characterization of the blue-light-activated two-component phosphoryl-transfer system from Brucella is presented in Chapter 3, in which a bacterial two-hybrid assay identified the cognate response regulator. In addition, in order to understand the unique stability of the bacterial LOV domain of the Brucella sensory kinase, I carried out UV-Vis analysis of the isolated BM-LOV domain clone and showed that blocking of the adduct decay is not caused by interactions with the kinase domain. I attempted to photolyse the cysteinyl-flavin covalent adduct and showed that the system is stable to this perturbation (Chapter 4). In addition, I identified specific amino acid residues that regulate the thermal stability of the covalent adduct in BM-LOV. Finally, I modeled the intramolecular interactions of these residues. Based on this analysis, I rationally designed two point mutations, T105C and E107Q, which are expected to disrupt those tertiary interactions, and consequently to unlock the adduct decay. I confirmed this experimentally showing that the two rationally designed point mutations undergo normal photocycles allowing the spotaneous recovery of the ground-state species in the dark (Chapter 5).