Lenton, Samuel (2017) Neutrons reveal the structure and dynamics of osteopontin phosphopeptides and nanocluster formation. Doctoral thesis, Keele University.

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In bovine milk the highly phosphorylated cleavage fragment of osteopontin (OPN1-149) is capable of stabilising high concentrations of calcium phosphate ions by formation of calcium phosphate nanoclusters (CPN). Here OPN1-149 isolated from bovine milk is characterised by a range of biophysical techniques as adopting an intrinsically disordered conformation in solution. The transient ensemble of conformations sampled by OPN1-149 is characterised by small angle X-ray scattering (SAXS) in combination with the ensemble optimisation method. Formation of CPN by OPN1-149 is characterised using dynamic light scattering and small angle neutron scattering (SANS). The dynamics of the protein and the effects of sequestration on the molecular fluctuations are quantified on the nanosecond-angstrom resolution by elastic incoherent neutron scattering. The molecular fluctuations of the free phosphopeptide are found to represent a highly flexible protein. Upon sequestration of calcium phosphate, stiffening of OPN1-149 is reflected in molecular fluctuations more closely resembling those characteristic of globular proteins. The quantification of the effects provides a handle for future comparisons and classification of molecular fluctuations. The formed OPN1 149 calcium phosphate nanoclusters are characterised according to a core shell model using SANS and static light scattering. The structure of the core is probed using neutron and X-ray diffraction. The results bring insight into the modulation of the activity of OPN1-149 and phosphopeptides with a role in the control of biomineralisation.
A recombinant human fragment (hOPN1-157) equivalent to OPN1 149 is produced and characterised in order to extend the work further towards the human regulatory system. The effects of phosphorylation, a key requirement for sequestration of calcium phosphate, on the structure and dynamics are determined using SAXS and elastic incoherent neutron scattering. The results indicate that phosphorylation of the protein has limited impact on both the structure and dynamics of hOPN1-157.

Item Type: Thesis (Doctoral)
Subjects: Q Science > Q Science (General)
Divisions: Faculty of Natural Sciences > School of Physical and Geographical Sciences
Depositing User: Lisa Bailey
Date Deposited: 17 Feb 2017 15:05
Last Modified: 28 Apr 2022 16:03
URI: https://eprints.keele.ac.uk/id/eprint/2916

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