Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.560299
Title: Fibril formation by human transferrin
Author: Booyjzsen, Claire
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2011
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Abstract:
There is a well-established connection between anomalous protein aggregates and disease, exemplified by the amyloidoses. Many neurological disorders such as Al heimer’s and Parkinson’s disease also show increased concentrations of iron deposits along with protein fibres. The increased metal concentrations observed in these instances have yet to be fully understood or explained. The research in this thesis is concerned with the aggregation of the protein transferrin, especially on surfaces. Human serum transferrin is an ~80 kDa iron-transporting glycoprotein found at 35 μM in the blood. It is also the body’s generic metal transporter, not only of the “natural” metals iron and manganese but also of metals used to detect or treat disease such as gallium and ruthenium. It has recently been reported from this laboratory that some batches of human serum transferrin can form fibrils on surfaces. This appeared to occur preferentially at lower salt and protein concentrations. The fibres exhibited a distinctive subunit structure with a consistent width of ca. 200 nm, and dark periodic striations along the length of the fibres apparently due to deposition of ferric (oxy)hydroxo mineral, lepidocrocite. The ability of human serum transferrin and recombinant (largely deglycosylated) transferrin to form fibres on surfaces has been investigated. Batches which formed fibres appeared to have normal primary, secondary and tertiary structures, and iron-binding properties, as determined by UV-visible and circular dichroism, spectroscopy, isothermal titration calorimetry, and ion-mobility-mass spectrometry. Dye binding experiments in solution suggested that classical amyloid fibres are not formed in solution. However, investigations of gas-phase conformations of transferrin from fibre-forming solutions, by ion mobility mass spectrometry, revealed an intrinsic transferrin dimer and higher order structures. These were separated by chromatography. Dimeric and monomeric transferrin were imaged on surfaces using transmission electron microscopy and atomic force microscopy. Only the dimer yielded structured aggregates, circular subunits composed of transferrin fibres. Dynamic light scattering and polyacrylamide gel electrophoresis further confirmed the presence of higher order structures in solution. Hence dimerisation of transferrin appears to trigger the initiation formation of fibrils possibly by pre-ordering of the protein in solution. Further atomic force microscopy analysis of deposited transferrin on mica surfaces by atomic force microscopy revealed the deformation (flattening) of the protein perhaps indicating structural flexibility that may be important for fibril formation. Some long, thin fibrils with distinct curvature were detectable by AFM. This appears to support the hypothesis that many proteins can exhibit fibril-like behaviour under specific conditions. If transferrin aggregation can occur when the protein is deposited on natural surfaces in the body, these findings may have important implications for certain physiological disorders including neurological conditions and lead to new treatments.
Supervisor: Not available Sponsor: University of Warwick
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.560299  DOI: Not available
Keywords: QP Physiology
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