The continuing project investigates the broad class of two-component signaling pathways, recently found to be widespread eukaryotes and ubiquitous in prokaryotes. Two-component 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 pathways components will have significant impacts on signaling biology and pharmaceutical development. The present research program focuses on two central elements of the chemotaxis pathway of Escherichia coli and Salmonella typhimurium: the transmembrane asperate receptor and the cytoplasmic CheA kinase it regulates. The goals address four fundamental questions. First, how does attractant binding to a chemoreceptor generate a transmembrane signal? Second, how does a receptor adapt to a constant background stimulus? Third, how do these distinct attractant and adaptations signals modulate the activity of a receptor-bound kinase? Fourth, how do the components of the pathway assemble into a cooperative, multi-protein signaling complex? Novel approaches utilizing site-directed cysteine chemistry and spectroscopy are being utilized to address these questions. The progress reports describes the chemical determination of a low- resolution structure for the cytoplasmic domain of the aspartate receptor, and the identification of regions of the cytoplasmic domain critical for CheA kinase regulation. In addition, a highly dynamic region of the cytoplasmic domain is described, and working models for the mechanism of receptor-regulated kinase regulation are proposed. The specific aims utilize site-directed cysteine chemistry, fluorescence and EPR spectroscopy to further investigate the mechanism by which signals are transmitted through the receptor to the kinase, and the effects of these signals on kinase structure, dynamics and activity. The geometry of the assemble receptor-kinase signaling complex is also probed. Overall, the broad goal of these studies is to understand the mechanisms of transmembrane signaling, receptor adaptation, and kinase regulation in a fully assembled, multi-protein signaling complex.