Crystal Structure Investigations of Small Heat Shock Proteins and Serine Palmitoyltransferase

Abstract

The content of this thesis concerns the pursuit of protein structures from cradle to grave. This process is illustrated though the progress made on two separate proteins. The first is the serine palmitoyltransferase from Toxoplasma gondii (TgSPT). This protein is of interest as sphingolipid biosynthesis in apicomplexan parasites is currently the subject of study as a possible drug target, most notably in Plasmodium. We begin by looking at the techniques of bioinformatics used to analyse the amino acid sequence of this protein, and by calculation and comparison with other known protein sequences we are able to begin to identify the different domains of the protein and also to begin to identify their respective functions. Bioinformatic techniques are then further used in order to design constructs to be moved forward for overproduction in vivo. A number of different constructs showing significant soluble expression of various truncations of TgSPT are reported, and various techniques relating to overexpression an purification are considered, including high-throughput screening work undertaken at the Oxford Protein Production Facility. The second protein looked at is the small heat shock protein from Methanococcus jannaschii (MjHSP). This protein is used as a more tractable analogue for the human α-B-crystallin. We look here at the wild type protein as well as a mutant mimicking the disease-causing R120G mutation in α-B-crystallin, R107G, and also two different truncation mutants, mimicking the Q151X truncation. The processes relating to crystal growth and optimisation, to data collection, and to data analysis and structure solution are then considered. Finally, a redetermined structure of wild type MjHSP at significantly increased resolution, and the structure of the R107G mutant are reported.