Structural and biochemical studies of cold shock domain containing proteins

• Main Author: Morgan, Hugh P.
• Published: University of Edinburgh 2008
• Subjects: 572.6
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.659586

This thesis describes a novel DNA microarray method for determining the sequence specificity of single-stranded nucleic acid binding proteins (SNABPs). In <i>Salmonella typhimurium </i>six homologous CSPs (<i>St</i>CspA, <i>St</i>CspB, <i>St</i>CspC, <i>St</i>CspD, <i>St</i>CspE, <i>St</i>CspH) have been identified, although their functions are yet to be clearly elucidated. The novel microarray assay revealed two different types of ss DNA binding preferences for either purine or pyrimidine rich sequences. CspD bound purine (guanine) rich sequences, with the consensus binding sequence, 5’-ACGGgg-3’. CspA, CspB, CspC, and CspE bound pyrimidine (thymine) rich sequences, with an identical consensus core binding sequence. 5’-TCTTT-3’. The kinetics and thermodynamics of CSP/ss DNA interactions were examined for <i>St</i>CspE and <i>St</i>CspD, using the surface plasmon resonance method and isothermal titration calorimetry, which complemented the initial results determined by the novel microarray method. In addition, the X-ray crystal structure of <i>St</i>CspE was determined at 1.1 Å resolution and refined to R = 0.203. A computer generated model of <i>St</i>CspD was also created. The consensus ss DNA binding sequences for <i>St</i>CspE and <i>St</i>CspD (5’GTCTTTT-3’ and 5’-ACGGGG-3’, respectively), were docked onto the structures to reveal key molecular interactions, which accounted for the observed ss DNA sequence specificities. This work reveals key differences in selective ss DNA binding, existing within a small highly conserved family of CSPs, thus reflecting potential differences in function. Classification of SNABPs in this manner may provide a means of elucidating their cellular function and identifying gene networks regulated by specific SNABPs.