Structural studies of the SRA domain of NP95 and enhancing the stability of membrane proteins

• Main Author: Jackson, A. F.
• Published: University of Cambridge 2009
• Subjects: 572
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.605004

Regions of heterochromatin, characterised by histone H3 lysine 9 methylation, are often co-incident with sites of DNA methylation and the mechanisms by which these epigenetic often co-incident with sites of DNA methylation and the mechanisms by which these epigenetic marks are replicated and maintained, and the feedback between them, is a key area of study. The SRA (SET and RING finger associated) domain is found in a number of proteins implicated in the maintenance of heterochromatin and more recently in the recognition of DNA methylation. Here we attempted to gain insight into the functional role of the SRA domain by undertaking a structural study of it. In this thesis the purification and partial characterisation of the SRA domain of the murine protein NP95 is described. The SRA domain was successfully crystallised and a complete native dataset was recorded. The selenomethionine labelled derivative was also crystallised but crystal twinning prevented structure determination by experimental phasing. The difficulties encountered due to twinning and approaches taken to overcome it are described. The structure was eventually solved by molecular replacement and its role in the maintenance of DNA methylation is discussed. Membrane protein structures are under representated in the PDB (Protein Data Bank) with only around 100 having been determined. Structure determination requires the removal of the protein from its native membrane into an artificial detergent environment. Membrane proteins are, however, often highly unstable when removed from their native membrane and mutagenesis is one possible approach to enhance their stability. The final chapter in this thesis describes the steps taken towards the development of an assay to identify membrane protein mutants with enhanced stability using the β₂-adrenergic receptor as a model.