Smyth, Clare Patricia; (1999) Oligomerisation of the chromatin-structuring protein, H-NS. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

H-NS is a major component of the bacterial nucleoid, involved in packaging DNA and in modulating gene expression. The mechanism by which this is achieved remains unclear, but is likely to depend on the oligomerisation properties of the protein. A number of different techniques have been applied, including circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR), analytical ultracentrifugation (AUC), and gel filtration to examine the self-associated states and thermal stability of H-NSC20S from S. typhi in concentrations ranging from the low μM to mM range. The results of these techniques are consistent with a model in which H-NSC20S is composed of two domains, a N-terminal domain required for the formation of large homomolecular complexes and a structurally independent C-terminal domain. Data on a 64 residue, C-terminally truncated form of H-NSC20S demonstrates that these residues self-associate to form a trimeric complex, which is mainly α-helical in nature and likely to be in a coiled-coil conformation. The self-association properties depend upon the size of the truncated form, since a polypeptide truncated at residue 89 results shows higher-order self-association. The oligomerisation properties of H-NSC20S are localised to the N-terminal domain, and remain functionally distinct from the C-terminal 48 residues. The C-terminus is an independent domain that exists as a monomer in isolation and functions as a DNA-binding site. The N- and C-terminal domains are joined by a flexible linker, allowing them to function independently within the context of the full-length protein. H-NSC20S exhibits an oligomerisation behaviour similar to that the 1-89 polypeptide; self-association involves the formation of higher-order complexes. Models for these unusual oligomerisation properties are presented, and the possible ways in which this might influence H-NS function are discussed.

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