Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.606282
Title: Magnetic resonance studies of proteins and model systems
Author: Bowen, Alice M.
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2012
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Abstract:
This thesis discusses, employs and develops further applications of both Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR) methodology in the study of a variety of biological systems. Chemically Induced Dynamic Nuclear Polarization (CIDKP) is used to study the surface accessibility of Tryptophan and Tyrosine residues in the Equine Lysozyme-Oleic Acid complex (ELOA). It is believed that these amino acids may play a vital role in the interaction of this partially structured complex with cell walls leading to the apoptosis of the cell. Double Electron-Electron Resonance (DEER) is an EPR technique that can be used to study the distance and, in some cases, the orientation between two paramagnetic centres on a nano-meter length scale. Attaching spin labels, such as Methane Thiosulphonate Spin Label (MTSL), to proteins, or by using spin active cofactors in proteins as innate spin labels, allows the measurement of distances within proteins or their complexes. In this thesis, model copper systems were initially studied in order to gain a better understanding of the orientational information obtainable from such spectra. The methodology developed from this project was applied to the protein-protein complexes of ferredoxin reductase-ferredoxin and ferredoxin-P450, three proteins that form a vital electron transport chain, leading to the catalytic hydroxylation of many small molecules. The structure of the docked P450-ferredoxin complex is the first experimentally determined structure of its type and thus is of considerable biochemical interest. A.s part of the structural determination of the ferredoxin complexes, it was necessary to measure the relative orientation of the g-matrix with respect to the molecular structure. This was achieved using a combination of orientationally selective proton Electron Nuclear Double Resonance (EKDOR) and HYperfine Sublevel CORrElation (HYSCORE) spectroscopy. The expansion of the DEER technique to measure an ever-increasing variety of systems has shown the limitations of the pulse sequences most frequently employed at present, particularly in relation to the measurement of many of the metal centres, which could be used as innate spin probes in protein systems. Methodological work has also been undertaken to examine possible solutions to circumvent some of these problems, namely low modulation depths and fast relaxation times. This has resulted in the development of two modifications to the existing techniques: Dead-time free three-pulse DEER and Repeated Excitations IN DEER (REINDEER). These techniques have been used to study of both synthetic model systems and also spin labelled proteins.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.606282  DOI: Not available
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