Experimentally, membranes and their lipid and protein components, are extremely challenging to work with. They are the main interface between the biological cell contents and the exterior medium and this makes them important targets when looking at human disease in pathogenic species. However membrane proteins require innovative and specialized strategies and techniques to produce, purify, characterize, and, in the case of structural biology, to crystallize for structure determination. This project aims to shed light on one particular protein export system present in the human pathogen Pseudomonas aeruginosa while further developing the in meso crystallization method, the data collection and data processing strategies. We will perform structure-function studies on the alkaline protease system present in pseudomonal and related species. This system is one of the important virulent factors used by these pathogens to create a favourable environment for continued infection. Increasing our knowledge on this system will be important to improve the life of patients with cystic fibrosis and those affected by bacterial refractory keratitis. We aim to clone, express, purify and crystallize three membrane proteins involved in the alkaline protease expression system by exploiting the knowledge and experience that resides within the Membrane Structural and Functional Biological. Become proficient and independent in the execution of these highly specialized techniques as applied to membrane proteins. Contribute to the methodology development from in meso crystallogenesis and crystal harvesting to diffraction data collection and processing using multiple small crystals (<80 microns). Train group members in data collection and processing, and structure determination. Solve the structure of one novel membrane protein involved in virulence in Pseudomonas aeruginosa. Biochemically and biophysically characterise the target protein.
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