Chatterjee, Abhishek (2012) Biophysical Characterization of NleD: An effector protein from Escherichia coli O157:H7. Masters thesis, Concordia University.
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Pathogenic Escherichia coli strains (O157:H7) are one of the main causes behind the lethal E. coli outbreaks in North America, UK and Japan. NleD, a zinc-dependent endopeptidase, is one of the several effector proteins secreted by these E. coli strains. After being injected directly into the host cell cytoplasm, NleD regulates the activity of specific MAP kinases (p38α kinase and JNK) leading to the inhibition of the host inflammatory response-signaling network. The current thesis is focused on the biophysical properties of NleD. NleD was identified as a monomer based on the results obtained from size exclusion chromatography, dynamic light scattering and analytical ultracentrifugation. The protein was prone to proteolytic cleavage at its C-terminal region as determined by mass spectrometry. Different approaches to improve the stability of purified NleD were also identified. NleD was found to have enhanced stability at lower temperatures and in the presence of trehalose. Using bioinformatic analysis as a tool, we were able to predict putative similarity between the active sites of botulinum neurotoxin and E. coli O157:H7 NleD. Preliminary characterization of the proteolytic activity of wild-type and variant forms of NleD was performed using p38α kinase as a substrate. Residues important for catalysis were identified. Our approach for improving the stability of the enzyme might facilitate its crystallization for structural work, while further mutagenesis studies may help identify the role of active site residues involved in catalysis.
|Divisions:||Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry|
|Item Type:||Thesis (Masters)|
|Degree Name:||M. Sc.|
|Date:||14 September 2012|
|Thesis Supervisor(s):||Ekiel, Irena and Turnbull, Joanne|
|Deposited By:||ABHISHEK CHATTERJEE|
|Deposited On:||30 Oct 2012 14:38|
|Last Modified:||15 Sep 2014 01:38|
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