The binding of cisplatin and carboplatin to hen egg white lysozyme, a model protein, has been studied using X-ray crystallography under many different crystallisation conditions (Tanley et al, 2012a; 2012b; 2013a; 2013b and Helliwell & Tanley, 2013). From this work, many new results have been obtained; (1) Two molecules of cisplatin and carboplatin are bound to HEWL in DMSO media using the co-crystallisation method. (2) Two molecules of cisplatin are bound to HEWL in aqueous media after a prolonged chemical exposure of 13months. (3) Cisplatin is stable up to 1.78MGy of X-ray radiation when bound to a protein. (4) We are the first academic group to make our raw diffraction data images freely available. (5) Carboplatin partially converts to cisplatin due to the high Cl concentration used in the co-crystallisation conditions. (6) We have also seen binding of just one molecule of carboplatin to the His-15 residue as a function of chemistry, pH and elapsed time. (7) Using NaBr crystallisation conditions, we see partial conversion of carboplatin to the trans Br form with a portion of the CBDC moiety still present. (8) Using triclinic HEWL co-crystallisation with cisplatin studied at 3 different data collection temperatures showed a more versatile binding and an overall larger summed occupancy at the Nε binding site.The human protein, heparanase is over-expressed in many different cancers and its experimental three-dimensional structure is yet to be elucidated. The work presented here includes; (1) Production of a homology model of heparanase and completion of virtual screening in order to identify potential novel small molecule inhibitors. (2) Use of this homology model as a molecular replacement search model (crystals of heparanase and their diffraction data was obtained before my PhD). Due to disorder in the crystal and resulting diffraction pattern as well as heavy metal soaks failing, the crystal structure has been difficult to obtain with this particular crystal system and molecular replacement procedure. (3) Over-expression of heparanase in insect cells and purification has been undertaken in order to produce new crystals.
|Date of Award||1 Aug 2014|
- The University of Manchester
|Supervisor||John Helliwell (Supervisor)|
Structural chemistry and structural biology of anti-cancer agents binding to proteins with reference to a model protein and to heparanase
Tanley, S. (Author). 1 Aug 2014
Student thesis: Phd