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Title: Chemical synthesis, recombinant expression and structural characterisation of complement protein modules
Author: O'Leary, Joanne Marie
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2000
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This thesis compares methods of producing protein for structural analysis by NMR spectroscopy and describes the structural characterisation of the N-terminal CP-module of membrane cofactor protein (MCP-1). CP-modules are autonomously folding domains composed of approximately sixty amino acids. They are defined by a consensus sequence including four cysteines, and have been identified in over forty different proteins. The proteins discussed in this thesis, MCP and factor H, are members of the regulators of complement activation family. Each comprises a contiguous array of CP-modules which serve as the binding sites for complement components and various other ligands. The structures of CP-modules have been determined previously by NMR and X-ray crystallography. Whilst individual modules are structurally similar, the nature of the interface between modules is highly variable. The high-resolution structure determination of MCP-1 forms part of an on-going study to determine the structure of the extracellular portion of MCP. MCP-1 is of particular interest as it comprises part of the measles virus receptor. It is anticipated that understanding the structural characteristics of this module in solution may assist in determining the nature of the interaction with the virus. Synthetic and recombinant methods have been assessed for the production of CP-modules in sufficient yield and purity for structure determination by NMR. Two CP-modules, MCP-1 and the fifteenth module of factor H (fH~15), were synthesised using standard Fmoc methodology. fH~15 was used as a model since the 3D-solution structure of this stable and soluble module has been determined by NMR. The purification and renaturation strategy utilised a number of chromatographic techniques and was monitored by mass spectrometry and NMR. However, it was not possible to purify the synthetic modules to homogeneity in sufficient yield for structural analysis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available