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Unusual properties of the aspartate receptor cytoplasmic fragment: Flexible structure and slow dissociation
Abstract
The transmembrane receptors which provide the information input for bacterial chemotaxis consist of a periplasmic ligand-binding domain, two transmembrane helices, and a cytoplasmic signaling domain. The cytoplasmic domain is central to the generation of intracellular signals via a phosphorylation cascade, as well as sensory adaptation through covalent modification. We are seeking insights into the mechanisms of signaling by investigating the changes which occur in a cloned 31 kDa cytoplasmic domain fragment (c-fragment) of the aspartate receptor. Point mutations in the cytoplasmic domain have been found which lead to either of two behavioral extremes of constant tumbling or constant smooth-swimming, mimicking the two signaling states of the receptor. Recent studies have revealed a monomer-oligomer equilibrium and correlated the smooth-swimming behavioral phenotype with the tendency to form oligomers. We have characterized the association/dissociation process by studying the dissociation kinetics for two oligomer-forming c-fragments using gel filtration chromatography and circular dichroism. We have also used CD to study the secondary structure of isolated monomer and oligomer forms. These studies indicate that the slow dissociation process proceeds through a highly unfolded intermediate and that the monomeric c-fragments are partially unfolded. A model is proposed to describe these results where intermolecular coiled-coil interactions in the oligomer are substituted by intramolecular coiled-coil interactions in the monomer. This change in subunit contacts may mimic the structural change involved in transmembrane signaling. We have also probed the tertiary structure of the c-fragments and found that they are globally dynamic proteins under physiological solvent conditions. Comparison of proteolysis of the c-fragment with that of the cytoplasmic domain of the intact receptor suggests that the intact receptor possesses similar flexibility. Thus the unusual flexibility of the c-fragment may persist in the intact receptor, and ligand-modulation of this flexibility may play a role in the mechanism of transmembrane signaling.
Subject Area
Biochemistry|Chemistry
Recommended Citation
Seeley, Stacy K, "Unusual properties of the aspartate receptor cytoplasmic fragment: Flexible structure and slow dissociation" (1996). Doctoral Dissertations Available from Proquest. AAI9619434.
https://scholarworks.umass.edu/dissertations/AAI9619434