The biophysical characterisation of the Enterobacterial nucleoid proteins H-NS and StpA
H-NS is a major protein component of the nucleoid, found in many Gram-negative bacterial species. H-NS is involved in the modulation of expression of a wide range of genes, as well as contributing towards nucleoid structure. There are two structur ally independent domains in H-NS, one involved in protein-protein interactions (the N-terminal oligomerisation domain), and the other involved in DNA-binding (C- terminal). These are joined via a flexible linker sequence. H-NS both self-associates and interacts with other nucleoid-associated proteins to form specific oligomeric complexes that bind DNA, allowing a precise level of control in gene expression. The protein StpA, a paralogue of H-NS with 58% sequence identity, was originally identified by its RNA chaperone activity. Subsequent studies have suggested struc tural similarities between H-NS and StpA, with a degree of overlap in their function in vivo. StpA self-associates in a similar manner to H-NS, and has been shown to in teract with H-NS. Whilst small differences in the properties of H-NS and StpA have been identified, no clear distinctions have been made between the two proteins. This work investigates several key issues regarding the properties of the StpA protein. A number of biophysical techniques have been used to investigate the interaction of H-NS and StpA. The results of these experiments are consistent with a model whereby StpA self-associates via a 'head-to-taif interaction. Furthermore, StpA ex hibits a different affinity and kinetic behaviour of association in comparison to H-NS. The properties of self-association and interaction of H-NS and StpA are fully consis tent with studies that highlight the intimate relationship between the two proteins in vivo. Two independent structures of the N-terminal oligomerisation domain of H-NS have been reported. These structures were derived at different temperatures (i.e. 25 C and 35 C). To investigate the implications of these model structures on the thermoregula tory functions of H-NS, the oligomerisation properties of H-NS were investigated over a temperature range. Both the oligomerisation and DNA-binding properties of H-NS were found to vary within a physiologically relevant range of temperatures. To characterise the interaction of StpA with DNA and to allow comparison with H- NS, the solution structure of the C-terminal domain of StpA was determined, solved by NMR. The interaction of DNA with this domain was characterised using both NMR and calorimetric methods. Statement The work described in this thesis was carried out in the Department of Biochemistry and Molecular Biology, University College London between 2000 and 2004. The NMR spectra were acquired with the assistance of Dr. M. Williams, Dr. R. Harris or J. Taylor. All experiments involving H-NS 1.39 were conducted with the assistance of T. Olsson. All other work was carried out by the author. This project was funded by the Biotechnology and Biological Sciences Research Council (BBSRC). Some of the work detailed in this thesis has been published elsewhere: Esposito, D., Petrovic, A., Harris, R., Ono, S., Eccleston, J. F., Mbabaali, A., Haq, I., Higgins, C. F., Hinton, J. C, Driscoll, P. C, and Ladbury, J. E. (2002) H-NS oli gomerisation domain structure reveals the mechanism for high order self-association of the intact protein. J Mol. Biol. 324, 841-850.