This continuing project investigates the broad class of two-component signaling pathways widespread in eukaryotes and ubiquitous in prokaryotes. Two-component pathways play especially critical roles in bacteria, where they control most normal cellular processes and most pathological processes including wound detection during infection, activation of virulence, and antibiotic resistance. The receptors and signaling proteins which comprise these ancient pathways are conserved across species and are attractive targets for broad-spectrum antibiotics. Thus, a basic mechanistic understanding of the pathway components will have significant impacts on signaling biology and pharmaceutical development. The present project focuses on the best characterized two-component signaling pathway: the chemosensory pathway of bacterial chemotaxis. The soluble components of this pathway assemble onto the transmembrane receptor to yield a large, membrane-bound sensory complex. The sensory complex is well-suited for molecular analysis, but much remains to be learned about its structure and its mechanism of signal transduction. The Progress Report describes new insights into fundamental features of the complex, including (i) the mechanism of receptor transmembrane signaling, (ii) the mechanism of receptor adaptation, (iii) the mechanism of signal transduction through the receptor cytoplasmic domain, and (iv) the architecture of the sensory complex. The four Specific Aims of continuing studies address two broad questions. First, how are transmembrane and adaptation signals transmitted through the receptor to the kinase? Second, how do receptor signals control the on-off switching of the kinase? Novel approaches utilizing site-directed cysteine chemistry and spectroscopy are being used to answer these questions in the fully functional, membrane- bound sensory complex. Overall, the broad goal of these studies is to understand the mechanisms of receptor transmembrane signaling, receptor adaptation, and kinase regulation in the native environment that includes the membrane and other pathway components.