A comparison of the two native CheA proteins of the bacterial signaling system of Escherichia coli

by Kott, Laila

Abstract (Summary)
The gene that codes for the chemotaxis protein A, CheA, which is an intergral part of the chemosensory system in Escherichia coli , has an internal ribosome binding site and can therefore, produce two different proteins. The full-length protein (CheAL ) is a histine kinase and is a major component in the signaling pathway. The second gene product is a protein (CheAS ) that is identical to CheAL , only missing the first 97 amino acids. The reason for the existence of this second protein is unclear and it has been the focus of little study. The main component of the signaling system in E. coli is a ternary complex of proteins consisting of a transmembrane receptor, a bridging protein (CheW) and CheA. These ternary complexes were found to cluster in the poles of the cell, which leads to high local concentrations of all three proteins. The focus of this work was to study the solution behavior of the two CheA proteins in parallel to understand how they would behave in these receptor clusters and perhaps to elucidate the function of CheA S in the cell. It was found that CheAL forms no oligomers higher than a dimer with a KDis for the monomer-dimer equilibrium of 0.49 �± 0.16 �¼M, up to a concentration of 28 mM. CheAS however, formed oligomers. The data was consistent with a monomer-dimer-tetramer-octamer model with KDis for the monomer-dimer, dimer-tetramer and tetramer-octamer equilibria being 20 nM, 300 �¼M and 4 �¼M, respectively. To better understand the interactions between CheAS dimers, CheA (long and short) mutants were constructed with disruptions in the dimerization domains. By assuming that the ratio of the free energy of the wild-type protein to the mutant would be equal for both the long and short forms, another estimate of 150 �± 140 �¼M for the KDis for the monomer-dimer equilibrium for CheAS was determined. Additionally CheA mutants of just the CheY binding domain (P2) and the phosphorylation domain (P1 for CheAL and Ps for CheAS ) were studied in the analytical ultracentrifuge to directly measure the interactions of shortened phosphorylation domain. (Abstract shortened by UMI.)
Bibliographical Information:


School:University of Massachusetts Amherst

School Location:USA - Massachusetts

Source Type:Master's Thesis



Date of Publication:01/01/2002

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