Structural studies of the DNA partitioning protein KorB from the plasmid RK2

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Gautam, Anmol (2018). Structural studies of the DNA partitioning protein KorB from the plasmid RK2. University of Birmingham. Ph.D.

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Abstract

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.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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