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

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.
Supervisor(s):
Supervisor(s)EmailORCID
Hyde, EvaUNSPECIFIEDUNSPECIFIED
Knowles, TimothyUNSPECIFIEDUNSPECIFIED
White, ScottUNSPECIFIEDUNSPECIFIED
Licence:
College/Faculty: Colleges (2008 onwards) > College of Life & Environmental Sciences
School or Department: School of Biosciences
Funders: None/not applicable
Subjects: Q Science > QD Chemistry
Q Science > QH Natural history > QH426 Genetics
URI: http://etheses.bham.ac.uk/id/eprint/8541

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