Stimulus-response coupling in all cells involves signal transduction pathways that carry information from receptors to the target molecules that effect the final responses. In bacteria, a large number of regulatory systems utilize a conserved phosphotransfer signaling strategy involving two conserved protein components, a histidine protein kinase and a response regulator. Response regulator proteins are typically composed of two domains, a conserved N-terminal regulatory domain and a variable C-terminal effector domain. The regulatory domain which catalyzes the transfer of phosphoryl groups to itself from the histidine protein kinase functions as a phosphorylation-activated switch to control the activity of the associated effector domain. Structural and functional characterization of these proteins is important for understanding the molecular basis of signal transduction. More specifically, such information may aid current pharmaceutical efforts to develop anti-microbial agents targeted against these proteins. During recent years, many aspects of the structure and biochemical activities of the conserved regulatory domain have been elucidated. However, the short lifetimes of the phosphorylated states of these domains has hindered investigation of how phosphorylation alters the conformation of the regulatory domain and how these conformational changes lead to activation of the activity of the effector domain. The proposed research focuses on addressing these questions using the muli-domain response regulators CheB, OmpR and DrrA as model proteins. There are four Specific Aims: 1. Determination of the mechanism of activation of response regulators. Non-hydrolyzable analogs of the phosphorylated proteins will be constructed by modification of unique cysteine residues. The effects of these modifications on intra- and intermolecular interactions will be characterized using activity assays, limited proteolysis, fluorescence measurements and ultimately X-ray crystallography to determine the three-dimensional structures. 2. Characterization of DNA binding by the OmpR family of transcription factors. The DNA-binding activities of the transcription factors OmpR and DrrA, representative members of the largest subfamily of response regulators, will be characterized and crystal structures of these proteins bound to DNA will be pursued. 3. Structural characterization of response regulator interactions with auxiliary proteins. Additional structural studies will focus on the interactions of response regulators with auxiliary proteins with the goal of determining whether similar molecular surfaces are used for protein-protein interactions in different response regulators. 4. Structural analysis of a histidine protein kinase. The X-ray crystal structure of the cytoplasmic region of a thermostable histidine protein kinase, T. maritima HpkA, will be pursued.
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