The chemoreceptors that mediate chemotaxis by Escherichia coli are the best characterized members of a large family of bacterial sensory receptors. The prokaryotic receptors share organizational and functional features with certain eukaryotic hormone receptors. This proposal focuses on sensory transduction through chemoreceptors, with particular emphasis on transmembrane signalling. The proposed work is a combination of biochemical, genetic and structural approaches, utilizing, in many projects, the chemoreceptor Trg. Extensive site-specific mutagenesis has created a family of genes coding for cysteine-containing Trg proteins, each with a single cysteine at one of 52 positions in the two transmembrane segments of the receptor. This family will be used to (1) determine the detailed organization of the transmembrane domain, using oxidative crosslinking, accessibility to sulfhydryl reagents and assessment of the functional consequences of cysteinyl substitutions or modification of the cysteinyl residues, (2) develop procedures for detecting conformational change in the transmembrane domain in vivo and in vitro and apply the procedures to search for changes associated with transmembrane signalling, and (3) define the borders of the transmembrane domain and investigate possible movements associated with signalling. Aspects of (2) and (3) will involve collaboration with Dr. Wayne Hubbell, using electron-paramagnetic-resonance probes linked to sulfhydryl reagents. Dr. Alexander Barnakov, a collaborator who has now joined the group, obtained two-dimensional crystals of Trg suitable for structural analysis by electron microscopy. Optimization and analysis of these arrays is an important emphasis of the proposed work. The most demanding aspects of this project will be performed at the superb facility for structural analysis by electron microscopy at the Lawrence Berkeley Laboratory, where Drs. Robert Glaeser and Kenneth Downing have offered facilities and expertise. The analysis has the potential of providing substantial structural information about intact receptors, particularly the difficult transmembrane domain. There will also be continuing efforts to obtain the three-dimensional crystals suitable for x-ray diffraction. Two projects will utilize DNA manipulation and genetic analysis to investigate features of chemoreceptors crucial for intramolecular signalling, particularly in its transmembrane aspects. Fusion proteins will be used investigate functional requirements for paired residues or segments in the transmembrane domain. Effective screening methods involving assays of signalling state in vivo or a fusion proton in which signalling can be detected by production of beta-galactosidase will be used to trace the signalling pathway by mutational analysis. A final project studies the ligand-binding domain of Trg by characterization of association with polypeptide ligands and purification of the isolated domain for crystallization and subsequent x-ray diffraction. The latter project will be in collaboration with Dr. Sung-Hou Kim. This multi-faceted, multi-disciplinary approach promises to reveal new information about the structures and conformational changes that transmembrane receptors use to transduce and transmit sensory information.
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