Bergqvist, Simon; (2002) The role of ions in an archaeal protein-DNA interaction. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Pyrococcus woesei (Pw) is a hyperthermophilic archaeal species, that thrives at temperatures in excess of 100°C. This study investigates the involvement of ions in the adaptation of the TATA binding protein of Pw to DNA binding, under the conditions of high intracellular salt concentration, commonly found in such species. It had previously been shown, (O'Brien, R., DeDecker, B., Fleming, K., Sigler, P. B., & Ladbury, J. E., (1998) J Mol Biol 279, 117-125.) that despite the similarity of primary and secondary structure, the TBP from Pw binds thermodynamically in a fundamentally different way than its mesophilic counterparts. This unusual character has been defined as halophilic because the formation of the PwTBP-DNA complex involves a net uptake of ions, resulting in an increased affinity with increasing salt concentration. It is the opposite effect to that normally observed in protein-DNA interactions. From sequence and structural insight, site-directed mutagenesis of the protein has been used to substitute residues in the PwTBP-DNA binding site in an attempt identify regions in the complex where ion uptake occurs. This has led to the identification of four glutamate residues, in regions of the TBP-DNA binding surface, which produce a quantifiable change in ion uptake. The combined effect of all four mutations results in a net release of ions and the reversal of the halophilic binding character. Mutation of the residues to change halophilic behaviour did not compromise the stability of the protein- DNA complex, but in fact appears to increase the temperature of optimal stability. This effect has been attributed to the disruption of a limited network of restricted water molecules in the DNA complex of the wild type PwTBP. In addition to these findings, the importance of the effect of water release on complex formation has been confirmed by osmotic stress experiments and the involvement of proton uptake coupled to binding has been established. This work was supported by the Medical Research Council.

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