| Summary: | This thesis presents an investigation into a 9th-10th type III fibronectin (FN) domain pair (FIII9-10) focused on the characterisation and functional activity of the protein. Initial work attempted to establish whether there was a binding interaction between FIII9-10 and IGFBP-5. Surface plasmon resonance failed to demonstrate an interaction and so thereafter the programme examined the use of FIII9-10 as a protein scaffold for cell adhesion. Surface-induced unfolding of proteins results in a loss of function. To improve the conformational stability of FIII9-10, Pro-Pro pairs were introduced. A previously developed multimeric FIII9'10 α5β1 ligand was also studied. FIII9-10 cDNA constructs were expressed in E. coli. Purity, fold and molecular weight were confirmed using SDS-PAGE, circular dichroism (CD) and mass spectrometry. Changes in conformational stability generated by the mutations were assessed by equilibrium chemical denaturation. An automated CD system was used to follow the secondary structure changes generated by thermal denaturation. Adsorption induced structural changes were investigated using a novel 'solid state' CD technique for a range of surface energies. Neutron reflectometry was employed to estimate the surface coverage and packing arrangement of the FIII9-10 mutants. Calculation of the Gibbs free energy demonstrated a two-fold increase in stability compared to wild type with a 9-11°C increase in the Tm. The mutated proteins showed enhanced stability when adsorbed to silica, but lost structure as surface hydrophobicity increased. This loss was less than that for the wild-type. ELISA studies verified that the mutation of the FIII9-10 did not compromise the receptor binding affinity. Immunofluorescence microscopy of baby hamster kidney on protein coated substrates displayed improved cell adhesion and spreading. The enhanced structural stability of the FIII9-10 mutants showed promise for use in influencing and controlling the interactions between medical implants and host tissue, mimicking the role of the extracellular matrix and improving biocompatibility.
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