Investigations of cluster-mediated adaptive signaling by proteins of the chemotaxis pathways using template directed assembly

by Montefusco, David J

Abstract (Summary)
An in vitro reconstitution system was developed that recapitulates the organization of a transmembrane signaling system. The system consists of a soluble fragment of a transmembrane chemotaxis aspartate receptor (Tar) of E. coli with a histidine tag (CF) anchored to lipid vesicles composed of a synthetic DOGS lipid, which presents a nitrilotriacetic acid-nickel moiety on its headgroup (DOGS-NTA-Ni2+ ). CF molecules assemble and form an array, mimicking the basic organization of the cytoplasmic domains of intact Tar. Templated CFs form ternary complexes with the histidine kinase CheA and the adapter protein CheW, activating the kinase as it does in vivo . Dynamic light scattering (DLS) was used to monitor particle size of template vesicles decorated with CF, CheA and CheW. In the absence of CheW and CheA, tip-to-tip interactions between CFs on neighboring vesicles lead to aggregation, evident as large DLS correlation times. The formation of active ternary complexes at physiological ratios of CF, CheA and CheW suppressed aggregation. Two lines of evidence are suggestive of a role for tip-to-tip interactions in ternary complexes and CheA regulation. (i)� Electron microscopic observations of soluble CheA-activating ternary complexes formed with chemotaxis receptor fragments conducted by the Stock lab revealed a barrel-like tip-to-tip structures. (ii)� Electron microscopic observations of overexpressed transmembrane receptors in the absence of CheA and CheW showed tip-to-tip interdigitation. The DLS data presented here provides evidence that formation of ternary complexes on surface-templated CF does not involve a tip-to-tip mode of ternary complex formation, and that complexes formed with CheW and CheA inhibit interdigitation. Clustering of chemotaxis receptors has long been suspected to play an important role in creating the remarkable combination of sensitivity and dynamic range displayed in chemotaxis. The surface template system is used to study clustering by diluting DOGS-NTA-Ni2+ lipid with DOPC to select the density of CF binding sites. We found that CheA activation is strongly favored by clustering, while methylation is strongly disfavored. Vesicles comprised of binary mixtures of DOGS-NTA-Ni2+ and DOPC in different ratios adjusted the density of binding sites for CF in a controlled manner, which influences the equilibrium between clustered and unclustered states.
Bibliographical Information:


School:University of Massachusetts Amherst

School Location:USA - Massachusetts

Source Type:Master's Thesis



Date of Publication:01/01/2007

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