The Crane group studies mechanisms of signal transduction with the overall goal of understanding behavior at the molecular level. This understanding will be achieved by defining the structure and dynamics of key macromolecular complexes that coordinate gene expression and transmembrane signaling in two systems that rely on highly cooperative interactions to respond to light, redox and chemical environment. The first, bacterial chemotaxis, concerns the motion of prokaryotic cells toward external stimulants. Chemotaxis is a paradigm for understanding transmembrane communication, intracellular information transfer, and motility. Importantly, many human pathogens that cause diseases such as cholera, gastric cancer and lyme rely on chemotaxis to establish infection. The sensory apparatus underlying chemotaxis, hereafter called ?the chemosome?, displays amazing sensitivity, dynamic range and a rudimentary molecular memory. In the chemosome, receptors, histidine kinases (CheA) and coupling proteins assemble into a specific architecture, whose details are just emerging. This proposal continues efforts to understand chemosome assembly, chemoreceptor conformational signaling, and ultimately, CheA regulation through restructuring of the receptor arrays. Chemosome output modulates Nature's consummate nanomachine ? the flagella motor. The ultrastructure of the switch complex within the motor will be defined to understand torque generation, direction switching and response to chemosome signals. The second system, eukaryotic circadian clocks, comprises 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, mania), cell growth deregulation (cancer) and metabolic defects (diabetes and obesity). This project proposes structural and mechanistic investigations of the key repressor and light-setting activities common to clocks in higher organisms. Biophysical studies will be conducted on the circadian proteins of fungi (Neurospora crassa) and flies (Drosophila melanogaster). Both model organisms provide genetic systems and behavioral assays to probe the biological relevance of mechanistic insights. A complimentary set of 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. For PDS, new strategies for incorporating spin probes based on nitroxides, flavins, nucleotides, and metal ions will be developed and deployed. Overall, this program aims to provide a molecular understanding for sensing and response in bacterial chemotaxis and eukaryotic circadian rhythms through the synergistic application of biophysical methods.
Bacterial chemotaxis enables pathogens such as Vibrio cholerae (cholera), Helicobacter pylori (ulcers and gastric cancer), Treponema pallidum (syphilis) and Borrelia burgderfori (lyme disease) to invade tissues and evade the immune system. In contrast, eukaryotic circadian clocks impact nearly every aspect of behavior and their dysfunction contributes to mental illness (sleep disorders, depression, mania, schizophrenia), cancer, and metabolic disease (diabetes and obesity). Defining molecular mechanisms of these two functionally diverse signaling systems will provide the basic understanding to guide new strategies for the treatment of infectious disease and mental disorders.
