Methylation of the CheA autokinase in the bacterial chemotaxis signal transduction pathway.
Item
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Title
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Methylation of the CheA autokinase in the bacterial chemotaxis signal transduction pathway.
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Identifier
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AAI9707074
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identifier
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9707074
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Creator
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Chang, Feng-Ching.
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Contributor
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Adviser: Simon A. Simms
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Date
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1996
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry | Biology, Microbiology
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Abstract
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Responses of Escherichia coli to chemical stimuli are mediated by regulating the direction of flagellar rotation and are the end result of a cascading series of regulatory reactions. Two types of reversible protein modification reactions are involved, methylation and phosphorylation. The reversible methylation of a family of sensory transducer proteins has been correlated with adaptation. The phosphorylation is involved in response to repellents stimulation.;Computer analysis of the GenBank database indicated the presence of a 27 amino acid region from Leu{dollar}\sp{lcub}169{rcub}{dollar} to Lys{dollar}\sp{lcub}195{rcub}{dollar} in CheA which is highly homologous to the methylation region, termed K1 site, of the chemoreceptor, Tar. This analysis also showed that CheA contains an identical glutamic acid that is methylated in Tar by CheR methyltransferase. These findings prompted me to investigate possible methylation of CheA by CheR. My results showed that purified CheR could not methylate ovalbumin, BSA, or a peptide of isoaspartic acid but it methylated the two forms of CheA, CheA{dollar}\sb{lcub}\rm L{rcub}{dollar} and CheA{dollar}\sb{lcub}\rm S{rcub}{dollar}, in vitro, in the presence of AdoMet. The methylated CheA protein, CheA{dollar}\sb{lcub}\rm L{rcub}{dollar}, whose molecular weight is 73 kDa, has autophosphorylation activity. The methylation rate of CheA was found to be very sensitive to the ionic strength of the medium.;In an effort to investigate the nature of this reaction, the methylation behavior of CheR on Tar, phosphorylated CheA and unphosphorylated CheA were compared. The results showed that both phosphorylated CheA and unphospholylated CheA were specifically methylated by CheR but at a slower rate than that of Tar. CheB methylesterase which demethylates membrane receptor-transducer proteins failed to remove the methyl group in methylated CheA in vitro.;Protease digestion and site-specific mutagenesis were used to further confirm the methylation site of CheA. Taken together, these results suggested that CheA could be methylated by CheR and that both Glu{dollar}\sp{lcub}182{rcub}{dollar} and Glu{dollar}\sp{lcub}183{rcub}{dollar} in CheA were involved in CheA methylation.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
