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Title: Methods development and radiation damage studies in macromolecular crystallography
Author: Davies, Robert
ISNI:       0000 0001 2413 2049
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2007
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Radiation damage limits the amount of time that a macromolecular crystal diffracts when exposed to X-ray irradiation. Various aspects of this problem were inv~stigated in this work along with methods development for quantitative determination of the metal content of proteins and celIs. In particular, the iron storage protein ferritin was used to extend current understanding of the changes that occur in macromolecular crystals held at 100 K during exposure to X-rays. The characteristics of the expl!nsion in unit celI volume with absorbed dose and with temperature were examined and found to be distinguishable. A comparison between the iron loaded (holo) and the iron void (apo) forms of ferritin alIowed the contribution to the damage ofthe absorbing iron core to be determined. Glutaraldehyde cross-linking was also tested as a method to increase r~diation resistance. Experiments were carried out to establish the room temperature dose limit of protein crystals and compare this with the dose limit measured at cryotemperatures. Surprisingly, it was found that there was a significant decrease in radiation damage at higher dose rates. An online microspectrophotometer at the European Synchrotron Radiation Facility was used to screen large numbers of potential radioprotectants at 100 K by monitoring the absorbance spectra of the 400 nm peak associated with a disulphide radical anion formed by X-ray cleavage of a disulphide bond. Ascorbate, quinone, TEMP and DIT were identified as effective radioprotectants. The most promising radioprotectant (ascorbate) was put into co-crystallisation and soaking trials with lysozyme to measure its protective effect against X-ray induced damage at 100 K and room temperature. For high throughput trace element detection, XRF was investigated, but calibration for proteins proved problematic. MicroPIXE was used for trace element mapping of wild type and Niemann Pick TypeC (a neurodegenerative disorder) cells to identify alterations in trace element composition.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available