Structural and functional characterisation of Escherichia coli stringent starvation protein A (EcSspA)

• Main Author: Matenchi, Kamogelo
• Format: Others
• Language: en
• Published: 2019
• Online Access: https://hdl.handle.net/10539/26694

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg in fulfilment of the requirements for the degree of Master of Science, 2018 === Escherichia coli Stringent starvation protein A (EcSspA) is an RNA polymerase associated transcription factor required for the activation of phage P1 late genes and plays an important role in stationary phase induced acid tolerance of Escherichia coli. The crystal structure of the Yersinia pestis orthologue of SspA (YpSspA) shows that the protein assumes a glutathione S-transferase (GST) fold and contains both the N and C-terminus GST-like domains. However, little is known about the structure of EcSspA and the functional roles of the GST domains within the protein. This study focuses on the characterisation of EcSspA in terms of its secondary, tertiary and quaternary structure, and functional characterisation through an enzyme assay and ligand binding studies. Secondary structure analysis by far-UV circular dichroism spectroscopy indicates that EcSspA is a predominantly alpha helical protein. Intrinsic tryptophan fluorescence has shown that EcSspA tryptophan residues are less solvent accessible and predominantly hidden within the interior of the protein. Like the YpSspA orthologue and other GST-like proteins, EcSspA is a homodimeric protein with a structure similar to that of YpSspA as shown by homology modelling. Using both the standard GSH/CDNB conjugation assay and a glutathione binding test it was confirmed that EcSspA does not have GST activity and does not bind GSH. Binding studies with the amphipathic ligand 8-anilino-1-naphthalene sulfonate (ANS) using fluorescence spectroscopy and isothermal titration calorimetry (ITC) indicated for the first time that EcSspA possesses ligandin function and that the interaction is exothermic. Molecular docking of ANS to the homology model of EcSspA indicates the binding of a single molecule of ANS to occur at the dimer interface of EcSspA involving weak interactions from hydrogen bonding and electrostatic interactions. The results of this study show that some functional and structural properties of eukaryotic GSTs are conserved in the bacterial EcSspA homologue. === XL2019