Studies of extremophilic single-stranded DNA-binding proteins

• Main Author: Mack, Lynsey A.
• Published: University of Edinburgh 2005
• Subjects: 579
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.654268

In this study, 6 bacterial SSBs are investigated which have been obtained from 4 different <i>Shewanella</i> strains, from <i>Aquifex aeolicus</i> and from <i>E. coli.</i> The 4 <i>Shewanella</i> SSBs have been taken from strains isolated at different depths of the ocean, from sea-level down to 8600m. These organisms therefore differ in their growth pressure optima. By comparing the characteristics of each of these proteins, differences will lead to clues which relate to the pressure differences. In order to highlight the different adaptations of these proteins, the thermophilic SSB from <i>Aquifex aeolicus</i> and the mesophilic SSB from <i>E. coli </i>were used as benchmarks to the piezophilic <i>Shewanella </i>SSBs. Circular dichroism was used to determine proportions of secondary structure present in each SSB and these were compared to the values obtained from previous crystallography work on the <i>E. coli, </i>in order to get some preliminary details about each structure. Further biophysical work was carried out using ITC and DSC which provided thermodynamic data regarding the binding between ssDNA and SSB, and also probed the denaturation temperatures of each protein. Exhaustive crystallisation trials were carried out on each <i>Shewanella</i> SSB but unfortunately did not produce any crystals of sufficient quality. As AqSSB had previously been crystallised, the structure determination is described in this study. To complement the binding data, a crystal of AqSSB in complex with ssDNA was produced and its structure determined. The structure showed great similarities with the several previously published structures of EcoSSB. Therefore this study is focussed on SSB proteins from bacteria isolated form very different habitats. By comparing their various structural and biophysical properties, further clues as to how piezophilic proteins are able to survive extreme pressures may be gained.