Molecular mechanisms of signaling systems responsive to light, redox and chemical environment

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The Crane group studies signal transduction systems that respond to or involve photochemistry and redox chemistry. Our overall goal is to understand the behaviors of bacterial chemotaxis and eukaryotic circadian rhythms at the level of molecular reactivity through the study of macromolecular complexes that underlie transmembrane signaling, motility, and gene expression. Chemotaxis has long served as a key system for studying transmembrane signaling, intracellular information transfer, and cell locomotion. Furthermore, many human pathogens that cause diseases, such as cholera, gastric cancer, and Lyme, rely on chemotaxis to establish and sustain infection. The sensory apparatus of chemotaxis displays remarkable sensitivity, dynamic range, and molecular memory. Chemoreceptors, histidine kinases (CheA), and coupling proteins assemble into large molecular arrays, wherein long-range cooperative interactions among components produce highly specific responses that adapt to changing conditions. This proposal continues efforts to understand receptor:kinase assembly, chemoreceptor conformational signaling, and ultimately, CheA regulation. CheA output modulates Nature's consummate nanomachine – the flagella motor. The architecture of the switch complex within the motor will be refined to better understand torque generation and direction switching. A particular focus will be the pathogenic spirochetes, which exhibit asymmetric flagella rotation at their respective cell ends. The second system, circadian clocks, comprises of cell-autonomous timing devices that pace metabolism to the diurnal cycle. Clocks are composed of transcriptional-translational feedback loops (TTFLs) within which repressor proteins inhibit the transcriptional activators of their own genes. Light entrains the clock phase by stimulating photosensors that impinge directly on the TTFLs. In humans, aberrant clock function causes mental illness (sleep disorders, depression, and mania), cell growth deregulation (cancer), and metabolic defects (diabetes and obesity). This project proposes structural and mechanistic investigations of the key light sensor and repressor activities in representative clocks from fungi (Neurospora crassa) and flies (Drosophila melanogaster). A complimentary set of biophysical techniques, including X-ray crystallography, small-angle X-ray scattering, optical spectroscopy, cryo-electron microscopy, and pulse dipolar ESR spectroscopy (PDS), will be applied to accomplish these goals. Biochemical reconstitution that leverages protein engineering to procure key entities will be combined with cellular assays and organismal studies in order to correlate physical properties with biological function. For PDS, new methods for incorporating spin probes that are based on nitroxides, flavins, nucleotides, and metal ions will be deployed for measuring structure and dynamics both in vitro and in vivo. Computational design and molecular dynamics will be used to test and consolidate models. Overall, this program aims to provide a molecular-level understanding for sensing and response through the synergistic application of chemical and biophysical methods.