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Title: Structural and biophysical characterisation of marine viruses and amino acid transporters
Author: Gillum, Ashley
ISNI:       0000 0004 7966 2807
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2018
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The field of structural biology enables us to study the fundamental architecture of biological samples in atomic detail. In this thesis, the techniques of electron cryomicroscopy and X-ray crystallography are applied to structural and biophysical investigations of marine viruses and membrane transporters respectively. In part I, two marine viruses - a freshwater lake bacteriophage known as FLiP and a helical archaeal virus known as APBV1 - are studied in order to gain insights into their evolutionary origins. The 4Å reconstruction of FLiP is presented, which reveals an icosahedral capsid encapsulating an intact lipid bilayer. The presence of an internal membrane makes FLiP unique among single-stranded DNA viruses. The major capsid protein exhibits an upright double b-barrel fold, which is iconic of the PRD1-adenovirus structural lineage of double-stranded DNA viruses, indicating that FLiP should be classified as a new member of this lineage. Secondly, the structure of APBV1 at nearatomic resolution is reported, revealing the evolutionary adaptions undertaken for survival in a hyperthermophilic environment. The formation of extensive hydrophobic interfaces via the tight packing of the major virion glycoprotein contributes to the extreme thermostability of the capsid, whilst the dsDNA genome is packaged as a lefthanded superhelix within the capsid. In part II, the fundamental mechanisms of membrane transport by eukaryotic amino acid transporters is investigated. Transporters belonging to the human SLC36 family, which play a potential role in nutrient sensing, were screened for high expression yield and protein stability. Comprehensive screening of homologous proteins resulted in purification of proteins belonging to the vacuolar basic amino acid transporter family of Saccharomyces cerevisiae for crystallisation trails and transport assays. Vba4 was successfully crystallised but the resolution obtained by X-ray diffraction prevented structure determination. In addition, the substrate specificity and mechanism of transport were investigated using differential scanning fluorimetry, microscale thermophoresis and liposome-based functional assays.
Supervisor: Newstead, Simon ; Huiskonen, Juha Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry ; Structural Biology