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Title: Structural characterisation of ligand, redox and catalytic states in haem enzymes using in crystallo spectroscopies and serial crystallography
Author: Moreno-Chicano, Tadeo
ISNI:       0000 0004 7962 8174
Awarding Body: University of Essex
Current Institution: University of Essex
Date of Award: 2019
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X-ray crystallography is a powerful tool for the study of biomolecules and has determined for instance the structures of numerous enzymes in order to unveil their conformation, interactions with substrates and ligands, and better understand their function. However catalytic processes carried out by enzymes can be of great complexity, involving several steps and intermediates prior to arriving to the final reaction product. Therefore, they are difficult to structurally characterise, since one of the limitations of X-ray crystallography is that provides static 'snapshots' of the average conformation within the crystal. Another important limitation is established by radiation damage, which results from the interaction of the X-ray beam with the protein crystal, and results in photo-reduced species which are not fully representative of the state of the measured protein. This is particularly relevant for metalloproteins such as haem proteins, since their metal centres are prone to reduction upon irradiation. In this research project, I aimed to overcome these limitations to study four haem enzymes, DtpA, DtpAa, DHP and NOD; and their catalytic cycles, trying to obtain intact species of relevant functional states. To this aim I have used complementary tools such as single-crystal spectroscopies, and the emerging methodologies of serial crystallography, applied at XFEL and synchrotron sources. Spectroscopic information of the crystals provided by in crystallo spectroscopies was used to validate the structures obtained by X-ray crystallography, identify generated species, and assign them within the enzyme catalytic mechanism (e.g. peroxidase cycle). Serial crystallography methodologies were used to measure room-temperature damage-free structures at XFEL sources of our protein targets, such as the resting state Fe(III). The application of serial crystallography at synchrotron sources, delivered low-dose structures of the haem enzymes, and allowed to perform dose-series measurements with the intention to follow radiation-induced processes at redox metal sites of haem enzymes.
Supervisor: Not available Sponsor: Leverhulme Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Q Science (General) ; QH301 Biology