abstract: Viral protein U (Vpu) is a type-III integral membrane protein encoded by the Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays vital roles in down-regulation of CD4 receptors in T cells and also in the budding of virions. But, there remain key structure/function questions regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis and thus, it makes for an attractive target to study the structural attributes of this protein by elucidating a structural model with X-ray crystallography. This study describes a multi-pronged approach of heterologous over-expression of Vpu. The strategies of purification and biophysical/ biochemical characterization of the different versions of the protein to evaluate their potential for crystallization are also detailed. Furthermore, various strategies employed for the crystallization of Vpu by both in surfo and in cubo techniques, and the challenges faced towards the structural studies of this membrane protein by characterization with solution Nuclear magnetic resonance (NMR) spectroscopy are also described. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2016

G protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that perform transmembrane signal transduction. Due to their pivotal role in a wide range of essential physiological functions GPCRs represent a high proportion of all drug targets. High resolution X-ray structures of GPCRs are however underrepresented in the Protein Data Bank. This is due to their instability in detergent, low expression levels and the presence of misfolded receptors in many heterologous expression systems. The objective of this project was to engineer the angiotensin II type 1 receptor (AT1R), a human GPCR, to make it suitable for structural studies. It was determined that detergentsolubilised AT1R was thermostable with antagonist bound with an apparent Tm of ~45°C, which was sufficiently stable for purification without further thermostabilisation by rational mutagenesis. Two expression systems were then evaluated for large-scale production of AT1R, namely baculovirus-mediated expression in insect cells and mammalian expression in HEK293 cells. Radioligand binding assays showed that only the mammalian system produced sufficient quantities of active AT1R for structural studies. Expression in the mammalian system was further optimised to approximately 6 mg/L. An AT1R-GFP fusion was created to examine membrane localisation using confocal laser scanning microscopy, to assay expression levels, to select highly expressing monoclonal cell lines using fluorescence activated flow cytometry and to develop a fluorescence size-exclusion chromatographybased assay to examine the suitability of 12 different ligands for co-crystallization. AT1R was also engineered to facilitate crystallisation, including C-terminal truncations to remove predicted disordered regions and bacteriophage T4-lysozyme being added to the third intracellular loop to provide additional points of contact for crystallisation, which increased the apparent Tm by approximately 10°C. All modified versions of AT1R were assessed for expression, stability and monodispersity. Additionally a rapid western blotting based assay was developed for the detection of unfolded membrane proteins, which will have wide applicability in the field.