This continuing project investigates the broad class of histidine kinase signaling pathways, recently found to be widespread in eukaryotes and ubiquitious in prokaryotes. Histidine kinase pathways play especially critical roles in bacteria, where they control diverse cellular functions including cell division, antibiotic resistance, activation of virulence, and wound detection during infection. The receptors and signaling proteins which comprise these ancient pathways are conserved across species and are considered to be attractive targets for broad-spectrum antibiotics. Thus, a basic mechanistic understanding of the pathway components will have significant medical, as well as scientific, impact. The present research program probes the activation and dynamics of selected pathway components in order to address three fundamental questions in signaling biology. First, how does a transmembrane signal modulate the activity of a receptor-bound kinase? Second, what is the role of thermal backbone dynamics in the on-off switches of receptors and signaling proteins? Third, how does phosphorylation activate a soluble kinase? Novel approaches utilizing site-directed cysteine chemistry and 19 F NMR have proven extremely useful in studies addressing these questions.
The specific aims i nvestigate the conformational dynamics of three carefully chosen pathway components. The E. Coli aspartate receptor is representative of a large family of prokaryotic and eukaryotic transmembrane receptors that regulate histidine kinases. The transmembrane signal of this receptor has been described in molecular detail, and the next phase of research will focus on the receptor domain responsible for kinase control. Cysteine and disulfide scanning will be used to probe the conformational change which regulates kinase activity, and to identify special disulfide bonds that lock the kinase ~on~ or ~off~. The E. Coli galactose binding protein represents a large family of soluble receptors that activate histidine kinase pathways. This receptor has proven to be ideally suited for novel studies of long-range backbone dynamics within a stable, folded protein. Disulfide trapping studies will map out the effects of receptor activation on the thermal dynamics of surface alpha-helices, and will examine the function of these dynamical changes. The oncogenic MAP kinase ERK2 typifies a large family of soluble eukaryotic kinases activated by phosphorylation, including downstream components of eukaryotic histidine kinase pathways. 19F NMR and other solution methods will probe the effects of phosphorylation on both the conformation and dynamics of the activation loop that directly controls kinase activity.
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