The partitioning of low-copy number broad host range plasmids depend on a centromere binding protein (CBP) that binds to a centromere-like site on plasmid. For RK2 plasmid, the CBP is a 358 residue, multi-domain protein, KorB. KorB contains an N-terminal domain (NTD), a central DNA-binding domain (DBD), and a C-terminal dimerisation (CTD) domain and the protein binds to the O\(_B\) site on the plasmid as a dimer. Structures of central DBD and CTD have been elucidated whilst limited information is available about the N-terminal of the KorB. In this study, NTD KorB protein was expressed and purified. Size exclusion chromatography and analytical ultracentrifugation data confirm that the NTD KorB behaves as a monomer in solution. Using solution-state NMR spectroscopy data, majority of the backbone and side-chain resonances for the NTD KorB are assigned and an ensemble structure of the protein is calculated. The flexibility of the NTD KorB is studied with coarse-grain Molecular Dynamics simulation package, AWSEM. The N-terminal of the NTD KorB is mostly unstructured whilst two α-helices towards the C-terminal of the protein exhibit limited motion. Two C-terminal KorB deletion mutants capable of binding the OB DNA (CΔ100 KorB and NΔ31CΔ100 KorB) were purified and characterised using circular dichroism (CD) and mass spectrometry. DNA-binding properties of these two deletion mutants are compared to the KorB wild-type (WT) using circular dichroism, fluorescence anisotropy and microscale thermophoresis measurements, indicating weaker binding of the deletion mutants with respect to the KorB WT to its centromere-like site (O\(_B\)). Considering the current state of structural information about the KorB and homologous proteins, a DNA partitioning model for the KorB is proposed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:760416 |
| Date | January 2018 |
| Creators | Gautam, Anmol |
| Publisher | University of Birmingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.bham.ac.uk//id/eprint/8541/ |
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