Receptors that mediate chemotaxis in Escherichia coli are paradigms for the large family of bacterial sensory receptors and a larger family of two-component receptors. This proposal focuses on functional mechanisms and structures of chemoreceptors and the signaling complexes they form. This focus is consistent with the NIGMS mission of basic research that increases understanding of life processes and lays the foundation for medical advances. Studies will emphasize E. coli chemoreceptors and utilize biochemical, mutational, biophysical, structural and modeling approaches. Some approaches involve collaborative efforts with other research groups and several exploit advantages of chemoreceptors inserted in Nanodiscs, water-soluble plugs of lipid bilayer surrounded by a protein annulus, which are effective for manipulating receptors.
Specific aims address issues central to understanding chemoreceptors. 1) Structure of the fundamental receptor organizational and functional unit, the homodimer and structural changes linked to conformational signaling. Receptor structure and changes generated by ligand occupancy and/or adaptational modification will be characterized using site-directed spin labeling and electron paramagnetic resonance spectroscopy, high- resolution electron microscopy, X-ray diffraction, single-molecule Frster resonance energy transfer (FRET) and molecular modeling plus coarse-grained molecular dynamics. Results will define the structure of this crucial unit and identify fundamental mechanisms of receptor intra-molecular and transmembrane signaling. 2) Functional properties of core signaling complexes, the fundamental unit of kinase activation and control. Comprehensive characterization by functional assays will be performed on purified, kinase-activating core complexes isolated using Nanodisc-inserted receptors. Results will define the extent to which each function of signaling complexes is performed by the core unit. Functional interactions between heterologous receptors in purified core complexes will be probed, addressing basic issues of allosteric coupling and providing insights into signal integration and inter-receptor interactions. 3) Structural properties of core signaling complexes. Purified complexes will be characterized for stability by assaying activity and retention of components, for size and homogeneity by analytical ultracentrifugation and high-performance, size-exclusion chromatography, and for three-dimensional organization by advanced techniques of electron microscopy and molecular modeling. Results will provide crucial information about the structure of core signaling units. 4) Non-conventional kinetics of adaptational modification. Non-conventional kinetics of receptor methylation and demethylation will be studied using strategies of transient-state kinetics and advanced fitting software. Results will identify reaction parameters and mechanisms, providing fundamental information about mechanisms of chemoreceptor sensory adaptation. Overall, the proposed research will increase understanding of signaling by transmembrane receptors. Such signaling, present in many life processes, is relevant to human health and disease.
The ability to sense and respond to the surrounding environment is a fundamental and essential biological process shared by all living cells. We are investigating this process in bacterial cells which are easier to manipulate than more complex cells like those in humans, with the aim of discovering basic principles. Knowledge of these principles will aid the understanding and treatment of the many human health problems caused by defects in the ability of cells to sense and respond.
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