Leonard, P.G.; (2008) Structural and biophysical characterisation of the histone-like nucleoid structuring protein from enteric bacteria. Doctoral thesis , University of London.

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Abstract

The H-NS protein is a major component of the nucleoid in enteric bacteria involved in DNA compaction and transcription regulation. The H-NS protein comprises two functional domains, an N-terminal oligomerisation domain and a C-terminal nucleic acid binding domain, separated by a flexible linker. In this thesis, the domain architecture of the protein is investigated. The residues that form the complete N-terminal oligomerisation domain of H-NS are identified and a critical role in the formation of high order oligomeric species is established for residues Pro72 to Lys82. The oligomeric state of H-NS1.74 C21S and H-NS1.83 C21S has been determined by analytical size exclusion chromatography and analytical ultracentrifugation showing that whilst H-NS C21S forms a discrete homodimer, H- NSi.83 C21S is able to oligomerise to form tetramers and much larger protein species in a concentration dependent manner. The high order oligomerisation of H-NS is shown to be disrupted by low ionic strength and this disruption has been utilised to produce protein crystals of the complete oligomerisation domain of H-NS. Point mutations at the N-terminal and C-terminal ends of the oligomerisation domain have been identified that disrupt (R15E or E73A) or enhance (R11E or R11A) the formation of high order oligomeric species. The DNA binding properties of the C-terminal nucleic acid binding domains of H-NS and its paralogue StpA have been directly compared by NMR. The H-NS and StpA nucleic acid binding domains bind to an AT-rich 20 base pairs DNA duplex with dissociation constants of 57 uM and 29 uM respectively.

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