Objective
The research will define and characterise events involved in intracellular trafficking and membrane translocation of the toxic A-chain subunits of plant toxins mistletoe lectin MLI (MLA), ricin (RTA) and ricin agglutinin (RAA). The methods to be employed in this research include X-ray crystallography, biochemistry and immunochemistry. Initially the existing 3.7 and 3.5 structures of MLI and RCA will be extended to higher resolution. These structures will be required for X-ray analyses using mollar replacement (MR) novel of MLA and RAA Fab complexes to be derived in our bio-engineering laboratories. Similar studies will be carried out for ricin whose X-ray structure is already known to about 2.5 resolution. The Fab complexes of all three A-chains will be formed with both native proteins and structurally modified partially unfolded proteins. These X-ray analyses will depend on the generally experienced ease of cystallisation of Fab complexes. Epitope mapping and simulated structure modelling will aid interpretation of the structures and the transmembrane translocation mechanism. X-ray analyses will be undertaken by MR employing all known structural information on Fab, A-chain and epitope structures. Current hypotheses of transmembrane transport involve a partial unfolding of the A-chain which the proposals seek to stabilise through production of the Fab complexes. The structural information derived from these studies will therefore help to exlain the fine details of these important biological mechanisms. Liposome studies will be undertaken on RTA and RAA which will augment previous model studies on MLA protein and contribute to the overall picture of these intracellular events. Future applications derived from these studies will promote the design of new immuno-conjugates for use in cancer treatment and organ transplant therapy.
Topic(s)
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WC London
United Kingdom